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’.     

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.     

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.     

Physiological colour changes in Odonata eyes. A comparison between eye and epidermal chromatophore pigment migrations

Pigment migration in the eyes of Austrolestes annulosus and Ischnura heterosticta cause pronounced colour changes which superficially resemble those of Odonata epidermal chromatophores. In both species, the migratory pigment is confined to the distal pigment cells of dorsal ommatidia. When the pigment is concentrated around the base of the crystalline cones, a dense layer of Tyndall blue bodies produce bright ‘blue phase’ colours. Distal migration of the pigment disrupts the Tyndall effect and produces ‘dark phase’ (grey-brown) colours. As in chromatophores, eye pigments consist of a mixture of xanthommatin and dihydroxanthommatin together with an additional pigment, possibly ommin A, not found in chromatophores.As with chromatophores, eye pigments respond to change in temperature only, change in light intensity having no effect. The change from blue to dark phase (at 8°C) occurs at the same rate as in chromatophores, whereas the reverse change (at 20°C) is significantly slower. Equilibrium colours at constant temperature are variable but significantly different from those of chromatophores at 12°C and above. There is no diurnal variation in responsiveness as is found in chromatophores.Isolated dark phase eyes or undamaged pieces of eye are able to change to blue phase after temperature increase. Isolated blue phase eyes show little response to temperature decrease, isolated undamaged pieces show no response. A temperature difference between the eyes of the same intact insect may result in minor colour differences. Ablation of the optic tract or of tissue posterior to the optic tract prevents normal colour change from blue to dark phase. The above results indicate that eye pigment cells are structurally similar to Odonata chromatophores and are under similar environmental and physiological control.     

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.     

Circadian oxygen consumption rhythm of the flour beetle, Tribolium confusum

Oxygen consumption is circadian (about 24 hr) rhythmic in sterilized Tribolium confusum adults kept on a 12 hr light-12 hr dark regimen. High values for oxygen consumption occur shortly after ‘light-on’ and low ones at the beginning of the dark span. When the light-dark cycle is advanced by 6 hr, the oxygen consumption rhythm is phase-shifted within about 6 days; with a delay of the light-dark schedule (a return to the initial regimen) the oxygen consumption rhythm re-adjusts in 3 days. This directedness or polarity of the phase-shift adjustment constitutes a quantitative circadian system characteristic shared by organisms studied thus far.     

Events Surrounding the Early Development of Euglena Chloroplasts: VI. Action Spectra for the Formation of Chlorophyll, Lag Elimination in Chlorophyll Synthesis, and Appearance of TPN-dependent Triose Phosphate Dehydrogenase and Alkaline DNase Activities

Action spectra derived from dose-response curves measured for various processes associated with chloroplast development in Euglena gracilis var. bacillaris are presented. The action spectrum for chlorophyll synthesis during the first 36 hours of continuous illumination of dark-grown resting cells resembles the absorption spectrum of protochlorophyll(ide). The action spectrum for the preillumination phase of potentiation, during which preillumination followed by a dark period brings about lag elimination in chlorophyll synthesis when the cells are subsequently exposed to postilluminating light, shows a high peak in the blue region (at about 433 nm) with a small peak in the yellow-orange region (at about 597 nm); the postillumination phase yields an action spectrum very similar to that obtained for chlorophyll synthesis in continuous light in normal, unpotentiated cells, with peaks at 433 and 631 nm. Alkaline DNase and TPN-linked triose phosphate dehydrogenase, two plastid enzymes which are synthesized outside the chloroplast, yield action spectra which are consistent with protochlorophyll(ide) being the major light receptor. The action spectra which implicate pigments resembling protochlorophyll(ide) holochrome have blue to red peak ratios in the vicinity of 5:1 as does the absorption spectrum of the protochlorophyllide holochrome from beans; the action spectrum is not identical with the holochrome spectrum indicating that the Euglena holochrome may differ from the bean pigment in details of its absorption spectrum. The action spectrum for preillumination, shows a ratio of the blue peak to the red effectiveness of about 24:1. This suggests that preillumination is controlled by a photoreceptor different from the protochlorophyll(ide) holochrome.     

The photoperiodic clock in the flesh-fly, Sarcophaga argyrostoma

Larval cultures of the flesh-fly, Sarcophaga argyrostoma, were raised in experimental light cycles with periods (T) of 21 to 72 hr, each cycle containing a photoperiod of 4 to 20 hr of white light. This ‘resonance’ technique revealed periodic maxima (～24 hr apart) of pupal diapause, thereby demonstrating an endogenous circadian component in the photoperiodic clock. The positions of these maxima of pupal diapause suggested that the oscillation, like that controlling the pupal eclosion rhythm in Drosophila pseudoobscura, is ‘damped out’ by photoperiods longer than about 11 to 12 hr, but restarts at dusk whereupon it runs with circadian periodicity in a protracted dark period. With photoperiods shorter than 12 hr, however, the two diapause maxima were less than 24 hr apart, suggesting that an additional component, possibly a ‘dawn hour-glass’, was modifying the position of the first peak.Both photoperiod and the period of the driving light cycle (T) were shown to affect the length of larval development (the sensitive period) and the number of calendar days needed to raise the incidence of pupal diapause to 50 per cent (the required day number, RDN). Peaks of diapause induction were shown to be the result of an interaction between a long sensitive period (slow development) and a low RDN, whereas troughs in diapause induction were the result of an interaction between a short sensitive period (fast development) and a higher RDN.Larvae of S. argyrostoma are unable to distinguish (in a photoperiodic sense) between 12 and 18 hr of red light (600 nm).     

Action Spectra and Adaptation Properties of Carp Photoreceptors

The mass photoreceptor response of the isolated carp retina was studied after immersing the tissue in aspartate-Ringer solution. Two electro-retinogram components were isolated by differential depth recording: a fast cornea-negative wave, arising in the receptor layer, and a slow, cornea-negative wave arising at some level proximal to the photoreceptors. Only the fast component was investigated further. In complete dark adaptation, its action spectrum peaked near 540 nm and indicated input from both porphyropsin-containing rods (λ_max ≈ 525 nm) and cones with longer wavelength sensitivity. Under photopic conditions a broad action spectrum, λ_max ≈ 580 nm was seen. In the presence of chromatic backgrounds, the photopic curve could be fractionated into three components whose action spectra agreed reasonably well with the spectral characteristics of blue, green, and red cone pigments of the goldfish. In parallel studies, the carp rod pigment was studied in situ by transmission densitometry. The reduction in optical density after a full bleach averaged 0.28 at its λ_max 525 nm. In the isolated retina no regeneration of rod pigment occurred within 2 h after bleaching. The bleaching power of background fields used in adaptation experiments was determined directly. Both rods and cones generated increment threshold functions with slopes of +1 on log-log coordinates over a 3–4 log range of background intensities. Background fields which bleached less than 0.5% rod pigment nevertheless diminished photoreceptor sensitivity. The degree and rate of recovery of receptor sensitivity after exposure to a background field was a function of the total flux (I x t) of the field. Rod saturation, i.e. the abolition of rod voltages, occurred after ≈12% of rod pigment was bleached. In light-adapted retinas bathed in normal Ringer solution, a small test flash elicited a larger response in the presence of an annular background field than when it fell upon a dark retina. The enhancement was not observed in aspartate-treated retinas.     

Common interruptions in the repeating tripeptide sequence of non-fibrillar collagens: sequence analysis and structural studies on triple-helix peptide models

Interruptions in the repeating (Gly-X1-X2)_n amino acid sequence pattern are found in the triple-helix domains of all non-fibrillar collagens, and perturbations to the triple-helix at such sites are likely to play a role in collagen higher-order structure and function. This study defines the sequence features and structural consequences of the most common interruption, where one residue is missing from the tripeptide pattern, Gly-X1-X2-Gly-AA₁-Gly-X1-X2, designated G1G interruptions. Residues found within G1G interruptions are predominantly hydrophobic (70%), followed by a significant amount of charged residues (16%), and the Gly-X1-X2 triplets flanking the interruption are atypical. Studies on peptide models indicate the degree of destabilization is much greater when Pro is in the interruption, GP, than when hydrophobic residues (GF, GY) are present, and a rigid Gly-Pro-Hyp tripeptide adjacent to the interruption leads to greater destabilization than a flexible Gly-Ala-Ala sequence. Modeling based on NMR data indicates the Phe residue within a GF interruption is located on the outside of the triple helix. The G1G interruptions resemble a previously studied collagen interruption GPOGAAVMGPO, designated G4G-type, in that both are destabilizing, but allow continuation of rod-like triple helices and maintenance of the single residue stagger throughout the imperfection, with a loss of axial register of the superhelix on both sides. Both kinds of interruptions result in a highly localized perturbation in hydrogen bonding and dihedral angles, but the hydrophobic residue of a G4G interruption packs near the central axis of the superhelix, while the hydrophobic residue of a G1G interruption is located on the triple-helix surface. The different structural consequences of G1G and G4G interruptions in the repeating tripeptide sequence pattern suggest a physical basis for their differential susceptibility to matrix metalloproteinases in type X collagen.     

A Semidian Rhythm in the Flowering Response of Pharbitis nil to Far-Red Light: I. Phasing in Relation to the Light-Off Signal

Evidence is presented of an endogenous rhythm in flowering response to far-red (FR) irradiation, with a period of about 12 h (hence semidian rhythm), which persists through at least three cycles in constant conditions of continuous light at 27°C and has a marked influence on the flowering response in Pharbitis nil to a subsequent inductive dark period. The phase of the rhythm is not influenced by real time nor by the time from imbibition or from the beginning of the light period. Rather, it is fed forward from the beginning of the FR interruption to the beginning of the inductive dark period. The period of the rhythm is not affected by irradiance but is longer at cooler temperature. When there are two FR interruptions during the preceding light period, it is primarily the later one which determines the phase of the rhythm, although some interactions are evident. There appears to be an abrupt rephasing of the rhythm at the beginning of the inductive dark period. No overt rhythms which could be used as “clock hands” for the semidian rhythm were detected in photosynthesis, stomatal opening, or translocation.     

Perithecial Formation in Gelasinospora reticulispora: IV. Action Spectra for the Photoinduction

Action spectra for photoinduction of perithecia after different lengths of dark period were determined with apically growing mycelium of a sordariaceous fungus Gelasinospora reticulispora. When hyphae were exposed to monochromatic light in near ultraviolet and visible regions, reciprocity between intensity and exposure time was observed within the range of incident energy used. The resulting action spectrum determined after a dark period of 48 hours showed a peak at 460 nm with shoulders at 420 and 480 nm and another peak at 370 nm, indicating minima at 410, 430, and 470 nm. After 72 hours darkness the spectrum was very similar to the above, except that the major peak shifted to 450 nm and the near ultraviolet region was somewhat less effective. In both cases, wavelengths longer than 520 nm showed no effect.     

Action Spectrum for Resetting the Circadian Phototaxis Rhythm in the CW15 Strain of Chlamydomonas: I. Cells in Darkness

We have developed protocols for phase shifting the circadian rhythm of Chlamydomonas reinhardtii by light pulses. This paper describes the photobiology of phase-resetting the Chlamydomonas clock by brief (3 seconds to 15 minutes) light pulses administered during a 24 hour dark period. Its action spectrum exhibited two prominent peaks, at 520 and 660 nanometers. The fluence at 520 nanometers required to elicit a 4 hour phase shift was 0.2 millimole photon per square meter, but the pigment that is participating in resetting the clock under these conditions is unknown. The fluence needed at 660 nanomoles to induce a 4 hour phase shift was 0.1 millimole photon per square meter, which is comparable with that needed to induce the typical low fluence rate response of phytochrome in higher plants. However, the phase shift by red light (660 nanometers) was not diminished by subsequent administration of far-red light (730 nanometers), even if the red light pulse was as short as 0.1 second. This constitutes the first report of a regulatory action by red light in Chlamydomonas.     

Stages of diapause development in the pupa of Mamestra configurata based on the β-ecdysone sensitivity index

Four distinct stages of diapause development in pupae of Mamestra configurata held at 20°C can be recognized by means of the ‘β-ecdysone sensitivity index’. The latter refers to the ED50 of injected β-ecdysone required to break diapause in half of the treated pupae. Stage 1 begins with the newly-formed pupa, lasts about 3.5 days and is characterized by a rapidly falling ED50. Stage 2 lasts about four weeks during which the ED50 increases by almost 20-fold, from 0.27 μg/g at the beginning to 4.9 μg/g at the end. Stage 3 begins when the pupae are about 4 weeks old and lasts for about six weeks. Stage 3 is the stable diapausing state and is characterized by a virtually unchanging ED50. The onset of stage 4 occurs when the pupae are about 10 weeks old and is recognized by the beginning of a decline in the ED50. Stage 4 precedes the completion of diapause development and may signal the transition of the endocrine system to its active state.Pupal diapause deepens with time in M. configurata. The deepening process evidently occurs during stage 2 of diapause development. Pupae that were transferred to 5°C at the beginning of stage 2 failed to make the transition to stage 3 and were trapped in a very shallow diapause.     

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.     

Pigment Migration and Adaptation in the Eye of the Squid, Loligo pealei

The migration of the screening pigment was investigated in the retina of the intact squid. The action spectrum of pigment migration corresponds to the action spectrum of the visual pigment, rhodopsin, rather than to the absorption spectrum of the screening pigment. The total number of quanta required for a fixed criterion of pigment migration is the same, when the quanta are delivered over any period of time from 6 s to an hour or more. When less than 3–10% of the rhodopsin is isomerized, the screening pigment migrates out to the tips of the receptors with a time-course of 5–15 min, and back again over the same period of time. When rather more than 10% is isomerized, the outward migration takes 5–15 min, but the screening pigment does not migrate inwards, even after several hours in the dark. Indirect evidence suggests that the band of screening pigment, when it reaches the tips of the receptors, is approximately equivalent to a filter of 0.6 log units. The spectral sensitivity of the optic nerve response was measured, and was found to be broader than the absorption spectrum of squid rhodopsin in vitro; the broadness could be explained by self-screening, assuming a density of rhodopsin of 0.6 log units at 500 nm.     

Production of males and gynoparae by apterous viviparae of Myzus persicae continuously exposed to different scotoperiods

At 18–19°C, apterous viviparae of a holocyclic strain of Myzus persicae raised from birth under constant scotoperiods of 9 hr 26 min–15 hr darkness per diem deposited apterous viviparae, alate viviparae, and males, in that sequence. Under the shortest scotoperiod (9 hr 26 min) in which males were recorded, only a few were deposited at the end of reproduction. With increasing duration of the scotoperiods, the aphids switched to the exclusive deposition of males progressively earlier in their reproductive lives. Thus more males and fewer females were produced; however the proportion of wingless females increased. Similar trends were recorded when the number of prenatal exposures to each scotoperiod was increased from 0 to 7.Alatae developed into gynoparae at scotoperiods of 10 hr 29 min or longer, and into virginoparae at 9 hr 40 min or shorter scotoperiods. Gynoparae and alate virginoparae, as well as alates that produced both oviparae and viviparae were found at 10 hr and 10 hr 15 min.When apterous viviparae were transferred to scotoperiods of 10 hr 29 min or 15 hr only when they attained adulthood, they also deposited males but only toward the end of their reproductive lives.     

The semidian rhythm in flowering response of Pharbitis nil in relation to dark period time measurement and to a circadian rhythm

When seedlings of Pharbitis nil Choisy, cv. Violet, are exposed to a single inductive dark period at 27°C, brief interruptions with red light (R) can be promotive after 2–3 h of darkness but increasingly inhibitory to flowering up to the 8–9th h of darkness. This rhythmic response to R interruptions can be advanced in phase by > 1 h when the preceding light period is interrupted with far-red (FR) 2 h before darkness (FR -2 h) or with FR – 15 h, whereas FR –8 h or FR–22 h retard the rhythm. These shifts in the R interruption rhythm are paralleled by equal shifts in the length of the dark period required for flowering. Brief FR interruptions of darkness displayed a similar rhythm which was also advanced by FR –2 h and retarded by FR –8 h. We conclude therefore that the semidian rhythm in the light, which we have previously described, continues through at least the first 12 h of darkness, is manifested in the R interruption rhythm, and determines the critical night length. A circadian rhythm with a marked effect on flowering was also identified, but several lines of evidence suggest that the circadian and semidian rhythms have independent additive effects on flowering and do not appear to show phase interaction.     

The Luminosity Curve of the Protanomalous Fovea

Threshold spectral sensitivities (in the dark, or against bright colored backgrounds) are identical in the red-green range for both protanopes (dichromats) and protanomalous trichromatic color defectives. The latter, however, must have an additional photolabile cone pigment in the red-green range, and its presence is revealed by heterochromatic brightness matching through the spectrum (i.e. luminosity curves). The absorption spectrum of the anomalous cone pigment can be inferred from the protanomalous and protanopic luminosity curve, given reasonable assumptions as to how the different cone mechanisms pool their responses. Depending upon these assumptions, the pigment inferred is either (a) dilute solution of the normal red pigment (assumed density 1.0 for the deuteranope) or (b) similar in its absorption spectrum to the normal green pigment but shifted slightly toward the long wave end of the spectrum. Experimental attempts to choose between these alternatives have so far proved equivocal though (b) seems more likely on the basis of indirect evidence.     

Untersuchungen zur photobiologie und endokrinologie der Farbmodifikationen bei der Kohlweisslingspuppe Pieris brassicae: Zeitverlauf der sensiblen und Kritischen Phasen

The photophysiological control mechanism of pupal melanization and bile pigment content in Pieris brassicae has been analysed. The timing of sensitivity to yellow and blue light and to darkness during the sensitive period of the early pharate pupa has been studied by introducing short light or darkness interruptions into light or darkness periods. The release of proposed hormonal factors which regulate the pigment metabolism in response to light has been determined by ligations.It has been shown that the animals are sensitive to all light conditions and to darkness only during two short phases. These phases are different in time for the regulation of melanization and of bile pigment content. They are identical for the effects of yellow and of blue light and of darkness in the control of bile pigment content. But for the regulation of melanization the phases of sensitivity to darkness differ from those to yellow and blue light, both of which are identical.Ligation of the animals between the first and second thoracic segments at 1 hr intervals shows that a melanization-inhibiting factor is released from the anterior part of the body in response to each sensitive phase for yellow light, and a melanization-stimulating factor in response to each sensitive phase for blue light. Darkness, which results in intermediate melanization of the pupae, probably acts by the release of the stimulating factor at first which is subsequently followed by the release of the inhibiting factor at each sensitive period. Thus the effect of darkness is the result of counteractive endocrine effects in the periphery.