Evidence for ‘dawn’ and ‘dusk’ oscillators in the Nasonia photoperiodic clock

Females of Nasonia vitripennis were maintained in light cycles from 12 to 72 hr in length, with 4 to 28 hr photoperiods, and their offspring examined for larval diapause. This ‘resonance’ technique revealed periodic maxima of diapause induction, about 24 hr apart. The ‘ascending slopes’ of these maxima appeared to obtain their principal time cue from dusk and the ‘descending slopes’ from dawn. This suggests that two independent—dawn and dusk—oscillators are involved in the Nasonia photoperiodic clock. The results are interpreted in terms of ‘internal coincidence’.N. vitripennis was shown to be able to distinguish between 12 and 18 hr of red light (>600 nm) in the photoperiodic sense. A ‘positive’ resonance experiment using such a red light was also performed. This shows that the spectral sensitivity of the pigment coupling the circadian system to the environmental light cycle extends into the red end of the spectrum.     

External coincidence and photoperiodic time measurement in the spider mite Tetranychus urticae

The incidence of diapause in the spider mite Tetranychus urticae was predicted for various photoperiodic regimes, according to the external coincidence model of photoperiodic time measurement. A phase response curve was constructed for the hypothetical photoperiodic oscillator in these mites: entrainment of this photoperiodic oscillator to a variety of ‘complete’ and ‘skeleton’ photoperiods was calculated using a transformation method for circadian rhythms. The external coincidence model proved adequate to describe experimental results with T. urticae in ‘complete’ photoperiods (T = 24 hr), symmetrical ‘skeleton’ photoperiods (T = 24 hr), asymmetrical ‘skeleton’ photoperiods (T = 24 hr) (night-interruption experiments), and ‘resonance’ experiments, in which the light component of a light/dark cycle was held constant at 8 hr and the dark component was varied over a wide range in successive experiments, providing cycles with period lengths up to 92 hr. The external coincidence model proved inadequate to explain results obtained in a ‘T-experiment’ with T. urticae comprising 1 hr pulses of light in a cycle of LD1:17.5 (T = 18.5 hr) with the first pulse of the train starting at different circadian phases. The validity and limitations of the external coincidence model as an explanation of photoperiodic time measurement in T. urticae are discussed in view of the above results.     

Photoperiodic induction of the pupal diapause in the tobacco hornworm, Manduca sexta

A study was made of photoperiodic induction of the facultative pupal diapause in the tobacco hornworm, Manduca sexta, reared on artificial diet in the laboratory. The species entered a prolonged diapause when the egg and larval feeding stages were reared in daily photoperiods of 13·5 hr or less. Diapause was induced in all insects at photoperiods ranging from 1 to 13 hr, and part of the population entered diapause at only 15 to 30 min of light per day. Photoperiods of 14 hr or more and continous darkness prevented diapause. Duration of diapause varied with the inductive photoperiod in which the hornworms were reared during the sensitive period. Insects reared in longer diapause-inducing photoperiods within a range of 12 to 13·25 hr remained in diapause longer than those reared in shorter photoperiods. There was no difference in the rate of larval development of hornworms reared in diapause-inducing vs diapause-preventing photoperiods. Temperatures of 26 to 30°C were most favourable for the photoperiodic induction of diapause; at 21°C, the critical photoperiod and incidence of diapause were decreased. Diapause induction was suppressed by low (18°C) and higher (33°C) temperatures. The number of inductive 12L:12D (light = 12 hr; dark = 12 hr) cycles required to induce diapause ranged from as few as 5 for some insects to as many as 12 for others when the post-inductive régimen was continuous light, but with insects previously held in continuous dark, as few as 2 12L:12D cycles during the last 2 days of larval feeding induced diapause in 38 per cent of the population. Only 3 to 4 cycles of 15L:9D during the final larval instar reversed inductive effects of 14 to 15 12L:12D cycles. Photoperiodic sensitivity extended from the late embryo to the end of larval feeding but showed considerable fluctuation during development with maximum sensitivity occurring just before egg hatch and during larval growth.Light breaks applied at different times during the dark period of 12L:12D cycles generated different response curves, depending on the number of cycles in which light breaks were repeated. When repeated for 6 cycles, a unimodal response curve was obtained; 10 cycles produced a bimodal curve and light breaks given for 18 cycles throughout the sensitive period averted diapause regardless of time of night applied. It is suggested that diapause is regulated by a photo- and thermolabile substance that accumulates during long nights (11 hr or more) and acts during the early pupal stage to inhibit the translocation and release of development-promoting neurosecretion from the brain.     

Photoperiodic induction of pupal diapause in Sarcophaga argyrostoma: Temperature effects on circadian resonance

Larval cultures of the flesh-fly Sarcophaga argyrostoma maintained in circadian ‘resonance’ experiments produced a high incidence of pupal diapause when the period of the light cycle was close to (T) 24, 48 or 72 hr, but a low incidence of diapause at T 36, 60 or 84 hr. Cultures pre-programmed for diapause by exposing pregnant females to long nights indicated the induction of non-diapause development at T 36, 60 and 84, whereas cultures pre-programmed for diapause-free development by exposing females to continuous light indicated the induction of diapause at T 24, 48 and 72.Raising the temperature reduced the heights of the diapause peaks whereas lowering the temperature raised them. With progeny from long-night-reared flies the lowest temperature tested (18°C) produced a result indistinguishable from an ‘hour-glass’ response, warning that ‘negative’ resonance experiments may merely indicate non-permissive conditions for demonstrating the involvement of circadian rhythmicity in insect photoperiodism.The results of the ‘resonance’ experiments and the effects of temperature are interpreted in terms of a multioscillator ‘external coincidence-photoperiodic counter’ model for the clock.     

Photoperiodic time measurement in the aphid Megoura viciae

Megoura produces parthenogenetic virginoparae in long day conditions, gamic oviparae in short days. The nature of this photoperiodic response has been analysed by rearing parent apterae in a wide range of circadian and non-circadian light cycles. By varying the light and dark components independently in a two-component cycle it has been established that the time measuring function is associated primarily with the dark period. There is no evidence that an endogenous circadian oscillation is implicated: thus (a) the ‘short day’ response is abolished by ‘night interruptions’ positioned in the early or late night. But this bimodal response pattern remains unchanged when the duration of the ‘main’ photoperiod is varied from ca. 6 hr to at least 25·5 hr. The stability of the maxima within the scotophase is inconsistent with the ‘coincidence’ models of photoperiodic timing that have been proposed. It is suggested that the essential timing process operates on the hour-glass principle, beginning anew with the onset of each period of darkness; (b) night interruption experiments employing very long (up to 72 hr) scanned dark periods yielded response maxima explicable in terms of the hour-glass hypothesis but did not reveal any circadian relationship between the maxima.The ‘dark reaction’ comprises a sequence of four stages, definable by the effects of light. Stage 1, extending from dark hr 0 to ca. 2·5, is fully photoreversible: at the next dark period the entire timing sequence is repeated up to the 9·5 hr critical night length. Towards the end of stage 1 reversibility is gradually lost and after a light interruption the reaction is resumed from a later time equivalent than dark hr 0; the subsequent critical night length is therefore reduced. The extent of the photoreversal is related to light duration. The period of maximum light insensitivity (stage 2) is attained at the end of the fourth hour. From ca. dark hr 5 to just short of the critical night length light exerts an increasingly promotive action which favours the production of virginoparae. This dark process is not photoreversible. Stage 4, which begins at hr 9·5, marks the end of the timing sequence. Light will not then annul the non-promotive action of the previous long night.Light has three effects which are determined by its duration and position within the cycle. The two terminal effects, mentioned above, are associated with the interception of dark stages 1 and 3 by either short (1 hr) or longer photoperiods. Light also prepares or primes the dark period timer. Thus the critical length is increased, and timing accuracy lost, if the preceding photoperiod is less than ca. 6 hr. Light during stage 4 has a priming action but no terminal function. Repeated cycles are ‘read’ in various ways, depending on the cycle structure. For example, if light intercepts stage 3, a two-component cycle is interpreted as the overlapping sequence light/dark/light. One and the same photoperiod then acts terminally in respect of the preceding dark period and as a primer for the next dark period.There is also a mechanism for summing the promotive effects produced by repeated interruption of dark stage 3. With complex (four-component) cycles both halves of the same cycle may contribute. ‘Product accumulation’ falls below threshold if the frequency of presentation of a given promotive cycle is too low. This occurs if there are very long, relatively non-promotive dark components. Such cycles are accepted as ‘continuous darkness’.     

Insect photoperiodism: effects of temperature on the induction of insect diapause and diverse roles for the circadian system in the photoperiodic response

This review considers the effects of temperature on insect diapause induction and the photoperiodic response, and includes constant temperature, temperature cycles, pulses and steps in daily light–dark cycles, constant darkness and in constant light, all with reference to various circadian‐based “clock” models. Although it is a comparative survey, it concentrates on two species, the flesh fly Sarcophaga argyrostoma and its pupal parasite Nasonia vitripennis, which possess radically different photoperiodic mechanisms, although both are based upon the circadian system. Particular attention is given to the effects of daily thermoperiod in darkness and to low and high temperature pulses in conjunction with a daily light–dark cycle, treatments that suggest that S. argyrostoma “measures” night length with a “clock” of the external coincidence type. However, N. vitripennis responds to seasonal changes in photoperiod with an internal coincidence device involving both “dawn” and “dusk” oscillators. Other species may show properties of both external and internal coincidence. Although the precepts of external coincidence have been well formulated and supported experimentally, those for internal coincidence remain obscure.     

Reversal of pupal diapause in Sarcophaga argyrostoma by temperature shifts after puparium formation

Puparia from Sarcophaga argyrostoma larvae reared under short days were collected daily within 24 hr of their formation and divided into two groups: one which remained at the larval rearing temperature, and one which was transferred to a different temperature. Such temperature shifts after puparium formation can modify the subsequent incidence of pupal diapause. Temperature step-ups decrease the percentage of diapause; temperature step-downs increase it. The degree of this effect increases with the size of the temperature step. The effectiveness of a temperature step-up declines with increasing time after puparium formation.The percentage of diapause finally achieved in any group is a function of both the number of inductive (short-day) photoperiods experienced during larval life and the magnitude and direction of the subsequent temperature step. Temperature step-ups can permit the expression of photoperiodic information which would otherwise be masked. A model is presented to account for these findings.     

Photoperiodic sensitivity and diapause induction during ovarian,embryonic and larval development of the flesh fly,Sarcophaga argyrostoma

Sensitivity to the daily photoperiod, particularly with respect to pupal diapause induction, was studied during ovarian, embryonic, and larval development of the flesh flySarcophaga argyrostoma. Large flies were shown to have a greater number of primary follicles in their ovaries and to be capable of limited ovarian maturation in the absence of exogenous protein (autogeny). Such ovarian development occurred independently of photoperiod. However, long days experienced during embryogenesis caused more rapid development, and earlier larviposition, than short days. Short days during embryonic and subsequent larval development also induced pupal diapause, whereas long days led to continuous or non-diapause development of the pupae. Pupal diapause could not be induced by photoperiods during the vitellogenic phase of ovarian development. InSarcophaga argyrostoma, a maternal effect preventing pupal diapause among the progeny of files with a diapause history was not observed.     

Developmental requirements of the univoltine species Chrysopa downesi: Photoperiodic stimuli and sensitive stages

Chrysopa downesi reproduces only in the spring and the resulting adults enter an aestival-autumnal-hibernal diapause which is primarily controlled by photoperiod. In the laboratory, constant photoperiods result in diapause induction and maintenance, whereas a series of short days followed by long days prevents or terminates diapause and promotes reproduction. The stages most sensitive to the diapause-averting stimulus are the free-living third instar, the third instar within the cocoon, and the pupa.C. downesi responds in different ways to three aspects of photoperiod: (a) an all-or-none response (diapause prevention or induction) to a sequence of two critical photoperiods, (b) an all-or-none response (diapause prevention or induction) to the difference between the long and short daylengths (a 4 hr difference is sufficient to avert diapause but a 2 hr difference is not), and (c) a quantitative response to the absolute duration of day (or night) length (after the short day requirement is fulfilled the rate of diapause termination is related to daylength).Differences and similarities in phenological adaptations and in photoperiodic responses of C. downesi, C. carnea, and C. harrisii reflect the degree of phylogenetic relationship between these closely related species.     

Manipulation of the length of the sensitive period,and the induction of pupal diapause in the flesh-fly,Sarcophaga argyrostoma

Larvae of the flesh-fly Sarcophaga argyrostoma were raised in short-day cycles (LD 12: 12) and at temperatures (18° and 20°C) in which short-day induction of pupal diapause was less than ‘saturated’. The cultures were then subjected to experimental treatments which modified the duration of larval development (the sensitive period). Overcrowding the larvae within a limited quantity of meat, premature extraction of the third instar larvae from their food, or exposure of the mature larvae to pure CO₂ for 24 h, were all found to accelerate puparium formation, thereby curtailing the sensitive period, and reducing the incidence of pupal diapause. Conversely, lengthening the sensitive period by allowing the mature larvae to wander in wet sawdust increased diapause incidence. The results are interpreted in terms of an interaction between the length of the sensitive period and the ‘required day number’, and also in the light of what is known about the endocrinological control of diapause and development in flesh flies.     

The flowering response of coleus in relation to photoperiod and the circadian rhythm of leaf movement

The flowering response of Coleus frederici and Coleus blumei x C. frederici is dependent on the photoperiod; both plants have a critical day length of about 12 hr. The inductive phase, defined as the period when light signals inhibit floral development, started 10 hr after the onset of darkness under 4 and 8-hr photoperiods, and 8 hr after the onset of darkness under a 12-hr photoperiod. However, a fixed temporal relationship between the inductive phase and the minimum leaf position was observed for Coleus frederici. The inductive phase always started 5 hr after the minimum leaf position. This evidence supports the theory that a circadian clock participates in the time measurement process of photoperiodic floral induction.     

Light-break experiments and photoperiodic time measurement in the spider mite Tetranychus urticae

To explain photoperiodic induction of diapause in the spider mite Tetranychus urticae (Acarina: Tetranychidae) a theoretical model was developed, consisting of two components, viz. a “clock” and a photoperiodic “counter” mechanism. The clock executes photoperiodic time measurement according to hourglass kinetics; the counter accumulates the photoperiodic information contained in a number of successive $\text{light}\text{dark}$ cycles by adding up the number of “long” and “short” nights experienced by the developmental stages of the mites sensitive to the photoperiod. The influence of the circadian system on photoperiodic induction is interpreted as an inhibitory effect exerted on the expression of the photoperiodic response; this effect is encountered only in certain photoperiodic regimes, where the circadian system and the photoperiod are out of “resonance” with each other. This “hourglass timer oscillator counter model”, devised to give a theoretical explanation of photoperiodic time measurement, the summation of photoperiodic information, and the influence of the circadian system on photoperiodic induction, proved to be consistent with experimental results obtained with T. urticae in both symmetrical and asymmetrical “skeleton” photoperiods, the latter based on diel as well as non-diel $\text{light}\text{dark}$ cycles.     

Diapause‐inducing signals prolong nymphal development in the two‐spotted spider mite Tetranychus urticae

Female two‐spotted spider mite Tetranychus urticae are grown under different photoperiods and the photoperiodic regulation of diapause is examined. The photoperiodic response curve for diapause induction was of the long day–short day type, with critical day lengths (CDLs) of 2 and 12.5 h; diapause was induced between these CDLs. The preimaginal period is significantly longer in diapausing females than in non‐diapausing females; moreover, a significant positive correlation is detected between diapause incidence and deutonymphal period. Diapause incidence is high when long‐night photoperiods are applied against a background of continuous darkness in the stages including the deutonymph; this stage appears to be the most sensitive to photoperiod. These observations suggest that diapause‐inducing conditions inhibit nymphal development, particularly in the deutonymphal stage when photoperiodic time measurement for determination of reproduction or diapause is carried out.     

Aspects of the induction of diapause in a laboratory strain of the mite Tetranychus urticae

Some diapause characteristics were studied in a strain of the spider mite. Tetranychus urticae. which had been reared on bean plants in the laboratory for over 15 yr. The diapause induction response curve was of the long-day type, showing a sharply defined critical daylength of 13 hr 50 min. In constant darkness no diapause induction occurred, but with a photoperiod of 1L:23D diapause incidence was already complete. A thermoperiod with a 5°C amplitude induced diapause in combination with a short-day photoperiod only when the low phase of the thermoperiod coincided with the scotophase. The same thermoperiod did not induce any diapause in constant darkness. The photoperiodic reaction of the laboratory strain used in these experiments appeared to remain constant over a very long period of time and to be independent of the diapause history of previous generations of mites.Although photoperiodic sensitivity was demonstrated during the whole postembryonic development, sensitivity was maximal at the end of the protonymphal instar and declined rapidly during the deutonymphal instar. Only 2 inductive cycles of 10L:14D were required to induce up to 62% diapause if the mites were kept in continuous darkness during the remainder of their development. Long days or continuous light could reverse the inductive effect of a sequence of short-day cycles previously applied to the mites.Light breaks of 1 hr duration applied at different times during the dark period of a 10L:14D photoperiod generated a sharp bimodal response curve with two discrete points of sensitivity to the light breaks at 10 hr after ‘dusk’ and 10 hr before ‘dawn’, thus showing a remarkable similarity with the results obtained in light break experiments with some species of insects.     

Photoperiodic time measurement in the spider mite Tetranychus urticae: A novel concept

To explain photoperiodic induction of diapause in the spider mite Tetranychus urticae a new theoretical model was developed which took into account both the hourglass and rhythmic elements shown to be present in the photoperiodic reaction of these mites. It is emphasized that photoperiodic induction is the result of time measurement as well as the summation and integration of a number of successive photoperiodic cycles: the model, therefore, consists of separate ‘clock’ and ‘counter’ mechanisms. In current views involvement of the circadian system in photoperiodism is interpreted in terms of the hypothesis that the photoperiodic clock itself is based on one or more circadian oscillators. Here a different approach has been chosen as regards the role of the circadian system in photoperiodism: the possibility, previously put forward by other authors, that some aspect of the photoperiodic induction mechanism other than the clock is controlled by the circadian system was investigated by assuming a circadian influence on the photoperiodic counter mechanism. The derivation of this ‘hourglass timer oscillator counter’ model of photoperiodic induction in T. urticae is described and its operation demonstrated on the basis of a number of diel and nondiel photoperiods, with and without light interruptions.     

Effect of photoperiod and temperature on diapause in a Florida strain of the tropical warehouse moth Ephestia cautella

A photoperiodically-controlled diapause of the long-day, short-day type was identified in a brown-winged, yellow-eyed strain of Ephestia cautella (Walker). The proportion of larvae diapausing in very long photoperiods was less than in short photoperiods. The mean critical photoperiod, here defined as that photoperiod giving half the maximum percentage of insects that diapause in response to photoperiod at a given temperature, was between 12 and 13 hr for the long-day reaction at both 20 and 25°C. The principal sensitive phase occurred near the time of the last larval moult. The mean duration of diapause was 2–3 months at 20°C and slightly longer at 25°C. The optimum temperature for diapause development was near 15°C, all larvae pupating within 24 days after a 45-day exposure at this temperature. Diapause could be terminated whenever larvae diapausing at 20°C were exposed to as few as five long (15 hr) photoperiods at 25°C. Long photoperiods at 20°C, or short photoperiods (9 hr) at 25°C were less effective in terminating diapause.     

Night length measurements by the circadian clock controlling diapause induction in the mosquito Aedes atropalpus

Night interruption experiments were used to investigate the behavior of the clock controlling diapause induction in the mosquito, Aedes atropalpus. The data from these experiments indicated that the clock included a circadian oscillator which was phase set at dusk. Following this event the oscillator proceeded to drive a nightly rhythm of sensitivity to light. This rhythm included a photoinducible phase where light interruption inhibited diapause. The photoinducible phase was fixed, occurring 7 to 9 hr after dusk in all photoperiod regimens tested. The photoinducible phase was followed by a refractory phase, which continued until dawn. During the refractory period light did not inhibit diapause. These observations indicated that the circadian clock behaved like an interval timer which was set at dusk. The rhythm of sensitivity to light, an inherited time scale, limited the induction of diapause to seasonal periods when nights were longer than 9 hr. As a result, diapause was induced only when the daylength dropped below the critical photoperiod of L15:D9 (hours of light:hours of dark).A ‘T’ experimental design was used to confirm the importance of the length of the night in clock controlled induction of diapause in this mosquito.     

Comparison of the circadian eclosion rhythm between non-diapause and diapause pupae in the onion fly,Delia antiqua

The influence of pupal diapause on adult eclosion rhythm of Delia antiqua was investigated. When non-diapause and diapause pupae were exposed to various photoperiods at 15, 20 and 25 °C, both of them emerged as adults close to the light-on time, but the phase of eclosion varied with photoperiod and temperature. Moreover, there was a significant difference in the eclosion time between non-diapause and diapause pupae; the eclosion peak of diapause pupae was earlier than that of non-diapause pupae. When non-diapause and diapause pupae were transferred to constant darkness (DD) after having experienced LD 12:12 at 15, 20 and 25 °C, both showed circadian rhythmicity in eclosion. Although the free-running period (τ) decreased slightly as temperature increased in both non-diapause and diapause pupae, the latter tended to show shorter τ than the former. This observation suggests that the observed difference in eclosion time in LD cycles between non-diapause and diapause pupae is due to differences in τ.     

The action of skeleton photoperiods on flowering in Lemna perpusilla

The effects of 24 hr cycle skeleton photoperiodic schedulesinvolving two short light pulses on flowering in Lemna perpusillahave been studied. Simulation of complete photoperiods by correspondingskeletal ones is nearly perfect for all photoperiods up to 8hr and is unstable for periods of 9 to 13 hr. A jump in theresponse phase appears when skeleton photoperiods ranging from12 to 13hr are given. For all skeleton photoperiods longer than14 hr the phase is entrained so that it agrees with that givenby skeleton photoperiods of complemental lengths. That is, askeleton photoperiod of 18 hr is equivalent to that of 6 (=24–18) hr. Simulation is largely related to whether thesecond pulse is locked on to "dawn" or to "sunset" dependingon when it falls during the dark period following the firstpulse. The inductive action of skeleton photoperiods that gives unstableentrainment depends on the length of a preliminary dark periodgiven before the plant receives the first pulse, since in theseskeleton schedules the sensitive zone to the second pulse shiftswith the length of the preliminary darkness. Thus, we tentatively conclude that the circadian oscillationin L. perpusilla involves an entrainment mechanism and thatphotoperiodic induction is contingent on the coincidence oflight and a specific inductive phase in oscillation. (Received September 18, 1968; )     

Functional identification of an FMRFamide-related peptide gene on diapause induction of the migratory locust,Locusta migratoria L

FMRFamide-related peptides (FaRPs) are a type of neuropeptide, which participate in a variety of physiological processes in insects. Previous study showed that myosuppressin, being a member of FaRPs, initiated pupal diapause in Mamestra brassicae. We presumed that FaRPs genes might play a critical role in photoperiodic diapause induction of L. migratoria. To verify our hypothesis, flrf, a precursor gene of FaRP from L. migratoria, was initially cloned under long and short photoperiods that encoded by flrf gene identified from central nervous system (CNS). Phylogenetic analysis showed that the protein encoded by L. migratoria flrf gene, clustered together with Nilaparvata lugens (Hemiptera: Delphacidae) with 100% bootstrap support, was basically an FMRFamide precursor homologue. We noticed the availability of -RFamide peptides (GSERNFLRFa, DRNFIRFa) under short photoperiod only, which suggested their functions related to photoperiodic diapause induction. RNAi and quantitative real-time PCR (qRT-PCR) results further confirmed that the flrf gene promoted locust's diapause.