Circadian Rhythms (Insects)

Circadian rhythms are daily oscillations in physiology, metabolism, or behavior that persist (freerun) in organisms that have been isolated from periodic fluctuations in the environment. These rhythms are under the control of innate regulatory systems that are based on internal oscillators (or pacemakers) whose periods approximate those of the naturally recurring 24-hour environmental cycles. The oscillators are subject to control by a limited number of these environmental cycles which synchronize or entrain the period to exactly 24 h and establish specific phase relationships between the rhythms and the external world (Fig. 1 ) . Light cycles are virtually universally effective in the entrainment of circadian rhythms, and in insects, daily cycles of temperature are also effective.
Event recording of the wheel running activity of a cockroach, Leucophaea maderae. Data for successive days are placed one below the other in chronological order. The bar at the top of the record indicates the light cycle to which the animal was exposed during the first 14 days of the recording. The animal was then placed in constant darkness (DD) and its endogenously generated, free-running circadian rhythm was expressed for the remainder of the record with a period of about 23.5 h.
FIGURE 1 Event recording of the wheel running activity of a cockroach, Leucophaea maderae. Data for successive days are placed one below the other in chronological order. The bar at the top of the record indicates the light cycle to which the animal was exposed during the first 14 days of the recording. The animal was then placed in constant darkness (DD) and its endogenously generated, free-running circadian rhythm was expressed for the remainder of the record with a period of about 23.5 h.
The study of both function and mechanism of circadian systems of insects has a long and productive history. The heuristic model that generally forms the starting point for these studies is illustrated in Fig. 2. There are four essential elements—a pacemaker or oscillator that generates the primary timing signal, photoreceptors for entrain-ment, and two coupling pathways, one that mediates the flow of entrainment information from the photoreceptor to the pacemaker, and another that couples the pacemaker to the effector mechanisms that it controls. The model identifies several basic questions. What is the functional importance of the system? Can the anatomical location of circadian clocks that drive specific rhythms be identified? What are the molecular mechanisms that generate the endogenous circadian oscillation? What are the pathways and mechanisms by which inputs to the pacemaking system regulate its phase and period? Finally, what are the neural and endocrine signals by which the pacemaking system regulates the various processes under its control?
Functionally defined model of the circadian system. An entrainment pathway that consists of a photoreceptor and coupling mechanism (input) synchronizes a self-sustaining oscillator (pacemaker) to the external light/dark cycle. The output of the pacemaker regulates the timing of various processes (e.g., activity) via coupling to the effector mechanisms.
FIGURE 2 Functionally defined model of the circadian system. An entrainment pathway that consists of a photoreceptor and coupling mechanism (input) synchronizes a self-sustaining oscillator (pacemaker) to the external light/dark cycle. The output of the pacemaker regulates the timing of various processes (e.g., activity) via coupling to the effector mechanisms.


FUNCTION OF THE CIRCADIAN SYSTEM

In insects, the circadian system is responsible for imposing daily rhythmicity on a wide variety of processes including locomotor activity, mating, oviposition, egg hatching, pupation and pupal eclosion, phe-romone release, retinal sensitivity to light, olfactory sensitivity, and even learning and memory. This list is by no means exhaustive, and more extensive tables can be found in Saunders (1982). It is generally accepted that the functional importance of this control is to restrict processes that are best undertaken at particular phase of the environmental cycle to a particular time of day. It has also been suggested that a secondary role of the circadian system is to provide for internal temporal organization, coordinating the timing of various processes within the individual.
In addition to its role in generating daily rhythms, a circadian clock has been shown to be involved in photoperiodic time measurement for seasonal regulation of reproduction, development, and diapause in many insects. In honey bees (Apis mellifera), it is also involved in time measurement necessary for time compensated sun orientation and in Zeitgedachtnis which is the ability to return at the appropriate time to a food source that is only available at particular times of day.
Thus the circadian system functions as a biological clock that is capable of providing the individual with information on the time of day and with the ability to measure lapse of time. Entrainment by signals from the immediate environment ensures the clock is set to local time.

MOLECULAR BASIS OF CIRCADIAN RHYTHMS

There has been remarkable progress in the past 20 years in identifying the molecular basis of circadian clocks in a variety of organisms. In animals, much of this progress has been due to pioneering work in the fruitfly. In 1971 the first clock gene, the period gene, was discovered in a mutagenesis screen in Drosophila melanogaster) A decade later the gene was cloned paving the way for studies of the gene’s regulation. This work led to the discovery of several other genes that appear to be part of the clock mechanism, including those involved in entrainment.

The Molecular Basis of the Clock

Four genes, the transcriptional regulators period (per), timeless (tim), cycle (cyc), and clock (clk), have been shown to be critical components for generating the basic circadian oscillation. Of the four, three, per, tim, and clk, are rhythmically expressed, and circadian oscillations in both mRNA and protein levels are well documented. The fourth gene, cyc, is
Simplified model of the transcription/translation negative feedback loop of the Drosophila circadian clock. CLK-CYC het-erodimers bind to E-boxes of nuclear DNA and activate transcription of per and tim. As PER protein is produced it is phosphorylated (e.g., by DBT) which leads to its degradation. However, if TIM is present it can bind to, and stabilize, phosphorylated PER, which remains bound to DBT. The Tim-Per-Dbt complexes are further phosphorylated which promotes their transport into the nucleus. These complexes then bind to CLK-CYC, inhibiting per and tim transcription. As PER and TIM levels decline (degradation not shown), the inhibition is removed and the cycle begins again. Phase shifts and entrainment by light occur through a pathway in which activation of CRY by light leads to TIM degradation. Solid lines with arrow, sequential steps in the feedback loop; blocked line, inhibitory interaction; wavy line, per and tim mRNA; dashed lines, degradation; P, protein phosphorylation (after Hardin, 2005).
FIGURE 3 Simplified model of the transcription/translation negative feedback loop of the Drosophila circadian clock. CLK-CYC het-erodimers bind to E-boxes of nuclear DNA and activate transcription of per and tim. As PER protein is produced it is phosphorylated (e.g., by DBT) which leads to its degradation. However, if TIM is present it can bind to, and stabilize, phosphorylated PER, which remains bound to DBT. The Tim-Per-Dbt complexes are further phosphorylated which promotes their transport into the nucleus. These complexes then bind to CLK-CYC, inhibiting per and tim transcription. As PER and TIM levels decline (degradation not shown), the inhibition is removed and the cycle begins again. Phase shifts and entrainment by light occur through a pathway in which activation of CRY by light leads to TIM degradation. Solid lines with arrow, sequential steps in the feedback loop; blocked line, inhibitory interaction; wavy line, per and tim mRNA; dashed lines, degradation; P, protein phosphorylation (after Hardin, 2005).
present at relatively constant levels throughout the day. Both CLK and CYC proteins are transcription factors that utilize bHLH domains to bind to E boxes, and both contain protein-protein interaction domains (PAS domains) that mediate the association of the two proteins leading to the formation of heterodimers. The fundamental mechanism for generating the oscillation involves two, interlocked transcription/translation feedback loops—one that regulates rhythmic expression of per and tim, and a second that regulates rhythmic expression of clk. The per/tim loop is the most critical and is illustrated in Fig. 3. A heterodimer composed of CLK and CYC binds to promoters of per and tim leading to an increase in transcription of these two genes that continues throughout the day. Levels of mRNA for the two genes peak in the early night. Protein products of these two genes increase as well, but peak levels of protein are delayed by several hours, peaking after the middle of the subjective night. PER and TIM then form a heterodimer via interaction through PAS domains. The heterodimer moves to the nucleus and functions as the negative element in the feedback loop, acting on the positive regulators CLK and CYC to suppress their activation of the per/ tim promoters. This leads to a decline in the per and tim mRNA levels that continues throughout the night. The degradation of PER and TIM allows the cycle to start over. The CLK-CYC heterodimer also regulates the expression of two transcription factors Vri and Pdp1 E that form the second feedback loop by regulating Clk transcription.
The time delay between mRNA synthesis and the accumulation of PER and TIM protein is likely to be critical element in the generation of the oscillation. PER is unstable in the absence of TIM whereas the dimerization with TIM stabilizes PER and promotes nuclear entry. Several kinases (e.g., double time—DDBT) and phosphatases regulate the phosphorylation state of PER and TIM that, in turn, appears to be involved in regulation of PER degradation as well as nuclear entry of the PER-TIM heterodimer.
The extent to which the molecular mechanisms detailed for Drosophila are applicable to other insects is not yet completely resolved. However, there has been considerable progress in identifying homologous proteins in other insects. Further, homologous genes have also been studied in both fish and mammals and while there are differences in detail, the basic framework of the oscillator seems to have persisted through the evolutionary process giving one some confidence that the story will be broadly applicable in insects as well.

Mechanism of Entrainment

In Drosophila, light acts to cause rapid decrease in the levels of TIM, and because TIM stabilizes PER, PER levels also decline. In the late day and early night when levels of these proteins are increasing, their destruction delays the progress of the oscillation while in the late night and early day, PER and TIM levels are decreasing and hastening their demise advances the oscillation. Interestingly, genetic ablation of the eyes or mutations in the visual phototransduction pathway, although reducing sensitivity of the circadian clock to light, does not block its entrainment. The altered sensitivity to light observed with mutations that affect the visual system indicate that an opsin-based photoreceptor can contribute to entrainment of the circadian rhythm of locomotor activity, but the persistence of entrainment in these mutants implicates an extraretinal photoreceptor. Action spectra for entrainment have suggested a flavin-based photoreceptor.
Cryptochrome (CRY), is a member of a family of flavoproteins, which includes photolyases and plant blue-light receptors. A mutant allele of the cry gene disrupts normal light responses of the locomotor activity rhythm while flies over-expressing CRY are hypersensitive to light pulses. Furthermore, in the periphery, CRY is required for light-dependent TIM degradation. These results suggest that CRY is a central element in the phototransduction pathway for entrainment.

Output of the Molecular Clock

The general supposition is that the clock ultimately regulates rhythms through the regulation of gene expression. This view is supported by the observation that there are several clock-controlled genes (CCGs) in Drosophila. However, at this point there is little information available on how CCGs are linked to overtly expressed physiological or behavioral rhythms, and this is an area of research that is likely to receive increased attention as researchers work to further elucidate the molecular details of the circadian system.

PHYSIOLOGICAL BASIS OF CIRCADIAN ORGANIZATION

Studies on the anatomical and physiological organization of cir-cadian systems in insects have largely focused behavioral rhythms (locomotor activity or eclosion) and their control by the nervous system. The goal of these studies has been to identify tissues and cells that comprise the functional defined components of the circadian system that are illustrated in Fig. 2.

Circadian Oscillations Are Generated by Discrete, Localized Populations of Cells

Compelling evidence that the brain is the site of generation of circadian timing signals for rhythms in behavior has been obtained in several species with much of the early work involving studies on the locomotor activity rhythm of the cockroach. It was first discovered in 1968 that surgical removal of both optic lobes or disconnecting them from the rest of the brain by section of the optic tracts abolished the activity rhythm of the cockroach Leucophaea maderae . Results of lesion studies on other cockroach species, several species of crickets, and on beetles have also suggested that the optic lobes might contain the pacemaker. Compelling evidence arose from the observation that it is possible to transplant optic lobes between cockroaches whose activity rhythms had quite different freerunning periods. Animals that received transplanted optic lobes recovered rhythmicity in a few weeks after regeneration of the optic tracts, and the pre-opera-tive period of the donor and the post-operative period of the host were strongly correlated. Other studies involving small electrolytic lesions indicated that the cells responsible for generating the circa-dian signal have their somata and/or processes in the proximal half of the optic lobe, likely in a group of cells located ventrally near the medulla.
In contrast to cockroaches, crickets, and beetles, in a variety of other insects, the optic lobes do not appear to be required for rhythmicity and the pacemaker appears instead to reside in the cerebral lobes (mid-brain). In a classic series of experiments by James Truman and colleagues, it was shown that the circadian pacemaker which controls the timing of eclosion in two silk moth species, Hyalophora cecropia and Antheraea pernyi. is located in the cerebral lobes of the brain. The times of day when eclosion occurs are different for the two species. When the insects are maintained in LD 17:7, H. cecropia emerges shortly after lights-on whereas
A. pernyi emerges just before lights-off. Removal of the brain did not prevent eclosion, but did disrupt its timing. However, if the brain was re-implanted in the abdomen, normal rhythmicity was restored in both entrained and freerunning conditions. When brains were transplanted between species, individuals exhibited normal species-specific eclosion behavior, but the phase of the rhythm was characteristic of the donor and not the host. The demonstration that the transplanted brains restored rhythmicity and determined the phase of the rhythm left little doubt that the circadian pacemaker that regulates the timing of the eclosion rhythm is located in the brains of these moths. The fact that the pacemaker was located in the cerebral lobes and not the optic lobes was demonstrated subdividing the brain prior to transplantation. It was found that the optic lobes were unnecessary and that transplantation of the cerebral lobes alone was sufficient to restore rhythmicity.
Similarly, in a variety of dipterans including the fruitfly, the housefly, the blowfly, and the mosquito regions of the nervous system controlling locomotor activity rhythms have been dissected with both surgical and genetic lesions; and in each case the pacemak-ing oscillation appears to be generated in the cerebral lobes. In the fruitfly, Drosophila melanogaster, extensive behavioral genetic evidence demonstrates a crucial role for the period (per) gene in the circadian pacemaker controlling locomotor activity and eclosion rhythms. The per gene is widely, and in some cell types rhythmically, expressed in the fly including the head, thorax, and abdomen—thus its spatial expression pattern in wild-type flies provided no definitive localization of the central pacemaker for regulating these behaviors.. However, the expression pattern has been altered by numerous genetic and molecular manipulations and it has been possible to determine the identity of the pacemaker cells in Drosophila by correlating per expression in specific cell types with the presence or absence of behavioral rhythmicity. The results suggest that there are approximately 150 “clock” neurons in the fly’s brain that can be divided into two major groups, the lateral neurons (LNs) and the dorsal neurons (DNs). The LNs appear to be most critical for persistence of the behavioral circadian rhythms of locomotor activity and eclosion in constant conditions, whereas members of the DN group have more subtle effects on the pattern of activity.
The potential for further cellular identification of pacemaker neurons in insects was provided by an observation that in cockroaches and crickets optic lobe neurons that fulfilled the predicted anatomical criteria to be pacemaker cells were labeled by an antibody to crustacean pigmentdispersing hormone (PDH). When anti-PDH was applied to Drosophila brains it labeled a ventral subset (LNv) of the per-expressing lateral neurons that were identified as pacemaker neurons in genetic studies. Taken together, the results indicated that the PDH-immunoreactive neurons are strong candidates for pacemaker neurons in insects and raised the possibility that the insect version of crustacean PDH (called Pigment Dispersing Factor or PDF) is an important temporal signaling molecule. Results from both cockroaches and Drosophila have indicated that PDF may play an important role in communication among oscillating components in circadian system. The G-protein coupled receptor for PDF has recently been identified which should help in the evaluating the role of PDF in circadian organization.
Interestingly, the numbers and projection patterns of PDF neurons in cockroaches and crickets are strikingly similar to the LNv of Drosophila, suggesting that they are functionally homologous. The most salient difference in the morphology of these neurons is in the locations of their somata: between the lobula and medulla of the optic lobes in cockroaches and crickets, as opposed to the location between the medulla of the optic lobes and the lateral margin of the cerebral lobes in fruit flies. This difference may be sufficient to account for the fact that the lesion and transplant studies suggested different anatomical organizations for pacemaker structures of the central nervous system in different insects.

Circadian Pacemakers Are Also Found in Tissues Outside the Nervous System in Insects

The localization of circadian pacemakers that regulate behavioral rhythms to the brain raised the question of whether other rhythms are controlled by the same clock. In crickets, beetles, and cockroaches, studies indicate the pacemaker regulating the daily rhythm in retinal sensitivity to light as measured by the electroretinogram (ERG) amplitude is located in the optic lobe and suggest that the same pacemaker controls both the ERG amplitude and activity rhythms. However, in other cases rhythms have been found to be regulated by pacemakers outside the nervous system. These include rhythmic secretion of cuticular layers in newly molted cockroaches, the release of sperm from the testis into the seminal ducts in gypsy moths (Lymantria dis-par), and the timing of ecdysteroid release from the prothoracic gland of the saturniid moth Samia cynthia. In each of these examples, the rhythms were shown to persist in vitro in the absence of neural pace-making structures. Finally, genetic manipulations have shown that a rhythm in olfactory sensitivity in the Drosophila antenna is regulated by a pacemaker in olfactory receptors in the antenna and is completely independent of the circadian pacemaking system in the brain.
These results indicate that the distribution of circadian pacemak-ing centers may be widespread in insects. In support of this view, one recent study by Plautz and colleagues in Drosophila in which the per promotor was coupled to the coding sequence for luciferase indicated that rhythmic promoter activity could be detected in a wide variety of tissues including the wing, leg, proboscis, antennae maintained in isolation in tissue culture.
The fact that the circadian system in the individual may be composed of several widely distributed oscillators raises the question of whether or not there is communication between component oscillators. In general the answer is uncertain. Work on cockroaches has shown that the bilaterally distributed oscillators in the two optic lobes are connected to one another (mutually coupled) and suggested that the coupling was relatively strong. In contrast, in both the beetle, Blaps gigas, and crickets the data indicated that coupling between optic lobe pacemakers is either absent or weak. Coupling relationships among other oscillators have not yet been systematically explored; however, in Drosophila there is evidence that coupling between circadian oscillator neurons in the brain is important for locomotor activity rhythms and that regulation of the timing of eclosion dependents on interactions between lateral neuron and pro-thoracic gland oscillators.

Photoreceptors for Entrainment

Extraretinal photoreceptors are typically involved in entrainment of behavioral rhythms. The classical example is the silkmoth where it was shown that the photoreceptor for entrainment of the eclosion rhythm of the silkmoth resides in the brain. Brains were removed from a population of pupae and were either replaced in the head region or were transplanted to the abdomen. The pupae were then placed in holes in a partition which separated two chambers in which the light dark cycles were out of phase. Whether the pupae entrained the light cycle to which the anterior end of the pupae was exposed,or entrained the light cycle at the posterior end, corresponded to the location of the brain.
Additional evidence for extraretinally mediated entrainment of pacemakers that are located in the nervous system has been obtained in a variety of other insects including other lepidopterans, dipterans, and orthopterans. Notably in Drosophila, isolated tissues that can sustain an oscillation in per transcription in vitro can also be entrained by light in vitro. This likely occurs via the action of CRY as discussed earlier, since CRY is expressed in cells that generate oscillations throughout the body in the fruitfly. Thus it appears that both the mechanism for generating the oscillation as well as the mechanism for entrainment can be contained within the cell.
There are several other instances where there is convincing evidence for circadian photoreceptors outside the nervous system. In the case of the moth testis, since the rhythm measured i n vitro responds to light, some cells in the testis-seminal duct complex must be photosensitive. Similarly, in the moth S. cynthia, the photore-ceptor for entrainment of the pacemaker in the prothoracic glands appears to be in the gland itself.
Although the compound eyes may not be necessary for entrain-ment, they may nevertheless participate. In Drosophila, for example, genetic lesions to the eyes or the phototransduction pathway can alter the entrainment pattern. Furthermore, there are at least two cases, the cockroach and the cricket, in which the compound eyes appear to be the exclusive photoreceptors for entrainment because sectioning the optic nerves between the eyes and the optic lobe or painting over the compound eyes eliminated entrainment of the locomotor activity rhythm to light cycles.

Signals to Communicate Timing Information

Another important issue is how circadian oscillators impose periodicity on the various physiological and behavioral processes they control. A priori, several alternative mechanisms are plausible. Timing information within the individual could be represented by the level of a circulating hormone, impulse frequency in specific neural circuits, changes in general levels of neural excitability through neuro-modulation, or as the weight of the available evidence suggests, by some combination of these mechanisms.
A large number of studies suggest secretion of a variety of insect hormones including ecdysone, prothoracicotropic hormone, and eclosion hormone is under the control of the circadian system during development. The experiments involving the transplantation of the silkmoth brain, described earlier, provide the clearest demonstration of a hormonal link in the control of behavior by the circadian system. The signal for the eclosion behavior is the eclosion hormone which is produced in neurosecretory cells located in a region near the mid-line of the brain, the pars intercerebralis, and released via the neu-rohemal organs, the corpora cardiaca. As Zitnan and colleagues have shown, the release of the hormone triggers the release of two other peptide hormones, pre-ecdysis triggering hormone (PETH) and ecd-ysis triggering hormone (ETH). PETH and ETH act on the CNS to initiate a stereotyped sequence of behavior that ultimately results in the emergence of the adult moth from the pupal case.
The role of humoral factors in the regulation of adult behaviors in insects (e.g., locomotor activity) is less clear. In cockroaches and crickets the timing signal that originates in the optic lobe is transmitted to the brain via the optic tracts and transmission from the brain to the activity centers in the thorax requires the connectives of the ventral nerve cord be intact. Nerve impulse activity is rhythmic in both the optic tracts and cervical connective.
In summary, the mechanism by which circadian phase information is transmitted to behavioral effectors in insects is generally not well understood. The emerging picture is that temporal regulation of behavior involves a modulation of excitability in the central nervous system. Axonal connections between the brain and the lower elements of the central nervous system are clearly required for the maintenance of some behavioral rhythms (e.g., cockroach locomotor activity) whereas others appear to rely heavily on hormonal mechanisms (moth eclosion). An important step in understanding how temporal information is transmitted will be the identification of the signal molecules involved.

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