Circadian Rhythms and the Circadian Organizationof Living Systems

C OLIN S. PITTENDRIGH

Princeton University, Princeton, New Jersey

The writing of this paper has been influenced bystrongly held convictions. This does not concern thevalidity of the theoretical scheme it offers; it concernsthe need at this juncture in the study of “daily”rhythms for bold and explicit theory formation. Weare beset rather than blessed with an enormous numberof observations about a great diversity of organismsthat range from unicellulars through African violetsto man. Moreover, the fact that a majority of theseobservations is highly fascinating is itself a danger--the common danger threatening the biologist of mis-taking acquisition of more fascinating facts, and moreconcrete detail, for analytic progress. To make progressanalyzing circadian rhythms we must perceive whatthe problems are-or rather state what we take themto be-and proceed with accumulation of new informa-tion only as it tests, and alas probably eliminates,theory. The low life-expectancy of any detailed ex-planatory scheme in this field is no reason to eschewtheory formation altogether. On the contrary, I takeit there is an infinity of facts, relatively few of whichare necessary for an understanding of general principles;and that the function of theory is not only, or even prin-cipally, to state one’s best estimate of those principlesas to minimize the error in improving that estimatethrough discovery of new fact. The applied mathe-maticians and physicists from whom we are seekingmodels and analogs wish to know not all the facts butthe significant facts. And one can rebut the likelyobjection that which are significant is something knownonly in retrospect by insisting that present judgmenton the matter is necessary to proceed. Theory forma-tion, then, involves judgments-especially in a looselydefined field; judgment on what the problems are; andjudgments on which are the most pertinent of all theavailable observations. It is, in brief, a tool for what re-mains to be done. At any rate it is in this spirit thatthe present essay has been written; I have chosen foremphasis those facts I think are significant.

I have discussed everything in this paper so manytimes with my colleague, Victor Bruce, that I amuncertain where many ideas came from; several Iknow came from him. This, however, is not to implythat he condones all my interpretive ventures. I amindebted also to Ewald Pauming and Dorothy Minis;to my students Drs. Burchard, Roberts, and Menaker,

and Messrs . Swade, Plumlee, Weiss, Tobin, andGolden-they have all contributed to the experimentalresults used here. And finally I take pleasure in express-ing deep gratitude to Professors Aschoff and Bunningin whose laboratories I recently spent several profitablemonths while enjoying a Guggenheim Fellowship.

I owe a special debt to Mr. Swade for permission toreproduce and interpret some unpublished records ofhis on arctic mammals; it goes without saying thathe cannot be held to my views of their meaning. Hiswork and all the other experimental results from ourlaboratory reported here were made possible by fundsfrom several sources; from the Eugene Higgins Trust;the National Science Foundation; the Office of NavalResearch; and the Air Force Office of Scientific Re-search.

CIRCADIAN RHYTHMS; THE EMPIRICALGENERALIZATIONS

Table 1 is a summary of major empirical generaliza-tions about circadian rhythms. A general treatment ofthese rhythms might follow either of two leads affordedby the list. One would be the functional significanceand physiological implications of their temperature-compensated period. This has been the approachBruce and I have taken in several other discussions[1, 2, 3, 4, 5]; it is the approach which was stimulatedinitially by regarding daily rhythms as clocks [1]. An-other is afforded by those generalizations concerningthe ubiquity of circadian rhythms, their existence inboth single and multicellular systems, the fact they areself-sustaining, and, finally, that they are always innate.For what these statements challenge is the attitudethat “daily rhythms” are in some vague sense no morethan appropriate responses to daily change in theenvironment; that they are, as it were, secondaryadaptations superficial to the main physiologicalarchitecture of the organism.

What the generalizations listed, in fact, imply is theflat converse: that circadian rhythms are inherent inand pervade the living system to an extent that theyare fundamental features of its organization; and toan extent that if deranged they impair it. They suggest,indeed, that circadian rhythms present a major problemfor general physiology of the type Needham [6] hadin mind in stating that-“the organization of living

160 PITTENDRIGH

systems is the problem, not the axiomatic starting in a Drosophila culture was controlled by a singlepoint of biological research.” physiological oscillation.

This is the approach that has been adopted here. Indeveloping the evidence and implications of a pervasive THE 2-OSCILLATOR MODEL FOR THE DRO-

circadian organization a start is made through the SOPHILA ECLOSION RHYTHMS

experimental results which forced Bruce and me toabandon the view that the rhythm of adult emergences

It has been shown in earlier papers [4, 7 ] thattransients develop in the Drosophila rhythm when this

T ABLE I. EM P I R I C A L G E N E R A L I Z A T I O N S A B O U T C I R C A D I A N R H Y T H M S

The following abbreviations used in this table have also been adopted for brevity in later sections of the text. LD;light-dark cycle, which can be further specified as, e.g., LD 12:12 to denote a cycle of 12 hours light and 12 hoursdark. LL; constant light. DD; constant dark. CR; circadian rhythm. 7; period of an environmental or circadianrhythm, measured in hours. rra; period of a freerunning rhythm. ILL; period of a rhythm free running in constantlight and constant temperature. zDD period of a rhythm free running in constant dark and constant temperature.4; phase of a rhythm. A4; phase-shift.

References given to the generalizations listed are intended only as convenient guides to the extensive literaturebearing on each of them; they are, specifically, not intended as indications of the original or even principal authorityconcerned.

, I: CR’s are defined as those biological rhythms whose TFR IS AN APPROXIMATION TO THE PERIOD OF THEEARTH’S ROTATION [3, 4, 44, 46]

This remains the most powerful, though by no means, the only line of evidence justifying III, below.II: CR’s are UBIQUITOUS in living systems [4, 47]

This holds in the systematic sense of kinds of organisms, and the physiological sense of kinds of functions.The emphasis in the literature on rhythms of, say, locomotion and leaf movement reflects only ease of assay forthese “superficial” phenomena; rhythms of DNA synthesis, e.g., exist but are less easily followed routinely.

III: CR’s are ENDOGENOUS in the living system [3, 44, 47, etc.]This generalization is universally accepted; but one laboratory [48] retains some complex qualifications, and

would object to the generalization unless so qualified (See [48, 54] and Professor Brown’s question in the discus-sion following this paper.)

IV: CR’s are usually (if not always) SELF-SUSTAINING OSCILLATIONS [4, 47, 51]

/

V:

This is clearly the case in animals; some plant rhythms damp out but it is still not fully clear that this impliesreal damping of individual cell rhythms or merely their desynchronization which imposes an overt aperiodicityon the whole organism [51].

CR’s are INNATE [1, 4, 47, 49, 50]They are not learned from or impressed by the environment as so much of the older and even comparatively

recent literature has suggested. In those systems that are aperiodic if raised from egg or seed in constant con-ditions, periodicity is elicited by a single (non-periodic) stimulus that in Drosophila may be only a J&so sec.flash of light [1, 55].

VI: CR’s occur autonomously at both cell and whole-organism LEVELS OF ORGANIZATION [4, 47]They have not yet been sought sufficiently at levels lower than the cell for us to know whether they occur (au-

tonomously) even there.VII: TFR is characterized by a remarkably small variance in a freerunning sequence of cycles; the underlying system dis-plays remarkable PRECISION [4, 32, 52]

Observed standard errors of the period may be less than 2 minutes per day.VIII: TFR is not a fixed characteristic of the individual organism; it is open to spontaneous and induced shifts withina range of values [4, 32, 52, etc.]

The limits of this range may (but are not proved to) be characteristic of the individual.IX: Some SPECIES DIFFER clearly from others in the RANGE OF REALIZABLE TFR values [46]

There is a suggestion that in nocturnal species the range (measured as 3-o~ values) is biased below 24 hours; indiurnal species above 24 hours. In some species the range fully spans 24 hours (see Fig. 7, this paper).

X: TFR may show AFTER-EFFECTS of the regime immediately preceding the steady-state freerun being studiedEvidence for this new generalization is presented later in this paper.

XI: TFR is so slightly temperature-dependent that it is proper to emphasize its near-INDEPENDENCE OF T E M P E R A -T C R E [4, 53]

The known QlO’s range from -0.9 < ~1.2. This feature suggests the near-independence reflects a compensationachieved by a several-component system; and the temperature-compensation is taken by most workers to reflectfunctional significance of the system as a “clock” [4]; but see [48 and 54].

XII: TFR is LIGHT-INTENSITY DEPENDENT [44, 4 6 ]There is evidence of a fairly strong further generalization which I propose to call Aschoff's Rule. This c a n be

summarized by ~I.L > mo in nocturnal animals; ILL < TDD in diurnal animals.

CIRCADIAN ORGANIZATION

TABLE I.-Continued

161

XIII: CR’s are ENTRAINABLE by a RESTRICTED CLASS OF ENVIRONMENTAL PERIODICITIES [4, 9, 46]Light and temperature cycles are the dominant entraining agents (Aschoff uses the term zeitgeber [46]), and

in many species probably the only agents. There are many pertinent subsidiary generalizations concerning limitsof entrainment (narrower in more complex organisms), etc., which are fully treated in [9]. The present writerremains unconvinced by, but must note here, claims of Professor Brown [48] that unknown geophysical cyclesare “sensed” by organisms and, in fact, somehow explain the facts summarized by generalizations VII and XI.

XIV: The PHASE of a freerunning CR CAN BE SHIFTED BY SINGLE PERTURBATIONS in the light and/ortemperature regimes [3, 4, 32, 33, 53]

The character of the A+ response is a function not only of intensity and duration of the perturbing signal, but--especially-of the phase at which the CR was perturbed.

XV: TRANSIENTS always precede attainment of a new steady-state [3, 4, 7]This is true whether the former steady-state was disrupted by a single perturbation or by a A4 in the entrain-

ing cycle.XVI: CR’s have so far proved surprisingly INTRACTABLE TO CHEMICAL PERTURBATION [40]

But see [41] and later discussion in this paper.

is perturbed from its freerunning steady-state by singlesignals of light and temperature. Transients are de-fined as those intervals between peaks of eclosion thatare different from that recurrent interval which definesthe period of the steady-state. The striking differencesbetween light- and temperature-induced transientsoriginally prompted the 2-oscillator scheme Bruce andI developed for Drosophila; it has since been extendedto explain many other features of the system.

The essential feature of this model is its assumptionthat the physiological controls immediately underlying

/eclosion: 1) are themselves autonomously oscillatory(the B-oscillation in our 1959 paper); and 2) distinctfrom the light-sensitive (A-) oscillation that serves aspacemaker for the organism. The light-insensitiveB-oscillation is, in a sense, a peripheral system; it iscoupled to and phased by the A-oscillation and probablyrelies on this entrainment for the temperature com-pensation that characterizes the system as a wholeand derives from the pacemaker. The data on transientsand other effects generalized by the model imply theB-oscillation is temperature sensitive, probably in itscoupling to A- and certainly in the sense that it can bedirectly entrained (independently of its coupling toA-) by temperature cycles. (Figures 1 and 2.) Thedata imply, further, that there is some feedback ofB and A, but it is slight.

Whether light- or temperature-induced, the transientsreflect the motion of the B-oscillator. The light-inducedtransients lead to a new steady-state whose phase isboth clearly different from the previous one and fullydetermined by the phase of the previous light signal(Fig. 1): the light signal resets phase in the A-oscilla-tion, and the observed transient marks the motion ofB as it regains phase with the pacemaker (Fig. 2).When the temperature-induced transients subside,there is a trivial phase-shift (-2 hours) in the newsteady-state, and what shift there is bears no relationto the phase of the perturbation that induced thetransients (Fig. 1): the temperature step-down affected

the coupling of B to A, and the temperature dependentperiod of B is temporarily manifest but disappears asB regains its coupling to A which was nearly insensitiveto the temperature change (Fig. 2).

The alternative to the present model is that a singleoscillator, responding differentially to light and’ tem-perature, underlies the overt rhythm and hence thatits motion is reflected by the transients. Experimentssummarized by Fig. 3 discriminate between the al-ternatives. The test is made possible by a detailedknowledge of the system’s response to single 12-hourlight signals (Fig. 1); those beginning before sub-jective dawn reset phase via an advance; those fallingafter subjective dawn reset phase via a delay. Subjectivedawn is that point in a DD rhythm whose normal phasein an LD cycle coincides with the dark/light transition.The two alternative models predict radically differentpositions for subjective dawn while the system is in atemperature-induced transient. The figure shows thatthe prediction from the l-oscillator scheme fails; andthe prediction from the 2-oscillator scheme is re-markably precise. A signal coincident with the predictedsubjective dawn causes no reset; signals falling after itreset by delays; the one signal preceding it resets byan advance.

Either light or temperature, as a periodicity, canentrain the rhythm to a particular phase. In terms ofthe model, light achieves its ultimate entrainment ofthe B-oscillation and hence of the overt rhythm onlyvia entraining A to which B is coupled. Temperaturedirectly entrains B and in bringing the whole systemto equilibrium must do so via feedback of B on A. Wehave studied the simultaneous action of entrainingcycles of light and temperature including the effectof regimes in which the phase of these two agents issystematically altered from the normal. The normalphase relation (that of the field) is when dawn fallssomewhere near the low point of the temperative cycle;teleonomically [8] we should expect the two cycles toimpose a similar phase on the system when in that

162 PITTENDRIGH

SINGLE TEMPERNURE S16NN.S&/PHASE Of TEMPERATURE SIGNALS

26%-+-

16%a

oRl6lNAL FINAL

SINGLE LIGHT SlGtwspH#L-PWE

Of FLIES OFFLJcsl

PHASE OF LIGHT

DARK TO UOKTTRANSITION

CALCULATED

FIGURE 1. Light-and temperature-induced transients in the eclosion rhythm of Drosophila pseudoobscura. (Repro-duced from [7].)

Each horizontal row of points in A and B represents medians of Drosophila eclosion peaks in individual cultures,The cultures had previously been in a light-dark cycle with 1‘2 hours of light and 12 hours of dark, and the time scaleshown (which is the same for all three figures) represents hours elapsed since the last dawn of this 24-hour cycle.The seven cultures represented by A were in complete darkness for the time shown, and the temperature of eachculture was dropped from 26°C. (temperature at which the cultures were raised) to 16°C. at the time shown by theheavy diagonal line a-a, between hours 24 and 48. The diagonal line between hours 96 and 126 does not represent asecond series of signals; it is included only to mark the phases of the signals given 72 hours earlier. Vertical guidelines are given at 24-hour intervals after the last seen “dawn.”

Each of the thirteen cultures represented by B was exposed to a single 12-hour light signal beginning at the timeshown by the heavy diagonal line (between hours 24 and 48) and was subsequently left in complete darkness; as inA, the second diagonal (hours 96-120) is for comparison only and does not indicate a second series of signals. Theabsence of data after hour 120 in B is due to the fact that the experimental cultures involved had completed all eclosionby this time.

The stippled area in A and B is included to facilitate comparison of the ultimate phase-shifts caused by the tem-perature and light signals. The open and solid arrows on B and C direct attention to the existence of delays (long-period transients) and advances (short-period transients) in both the observed and calculated results.

1C gives a calculated behavior for the light-induced transients; it is derived from a mathematical approximationfor the 2-oscillator scheme given in [7].

In 1-A there are noteworthy features to cultures ii and iii. There is no peak between hours 48 and 72; the peak atat 72 hours was twice the average size clearly being a compound of two including the “missing” peak. The latter, thenwas forced into a phase-jump of the type analyzed in Fig. 6, and discussed in the text.

relation. Thus in the field the B-oscillation is derivingphase control from 2 sources; directly from the tem-perature and indirectly via A from the light cycle. Whena temperature cycle is imposed on an LD rhythm, n o

phase disturbance is in fact brought about when the lowpoint of the cycle is near dawn. But if the phase anglebetween the light and temperature cycles is changedwe observe a marked influence on the phase of the

CIRCADIAN ORGANIZATION

FIGURE 2. 2-oscillator interpretation of the Drosophila eclosion rhythm. Upper figure: The steady-state in DD andconstant temperature. Middle figure: A temperature-induced transient caused by temperature step-down from 26°C.to 16°C. at the point (TS) indicated. Lower figure: Response to light signal of 12-hour duration beginning at point(LS) indicated. Solid circles are medians of the eclosion peaks. The saw-tooth and square-wave form of A and B oscil-lations is pure convention. SD,, SD*, etc., are the subjective dawns in the cultures: They are determined by the lastseen dawn of the prior LD 12:12 regime. They are set to correspond with the downstroke of the A-oscillation’s saw-tooth form. The SD’s of the steady state are carried through the figure for comparison with the perturbed cultures.The eclosion peaks at the extreme right mark the steady-state phases of the control (C) temperature-perturbed (T)and light-perturbed (L) cultures.

m

IY

FIGURE 3. Tests to discriminate between 1- and 2-oscil-lator interpretations of temperature-induced transients.I: The 2-oscillator interpretation of the steady-state;cf. Fig. 2; A-oscillation is saw-toothed. Three successiveeclosion peaks shown.II: The 2-oscillator interpretation of the temperature-induced transient.III: Implications of a single-oscillator interpretation.IV: Experiments to decide where the subjective-dawnis during the temperature transient. Heavy lines on the“histories” of cultures 2, 3, 4, 5, 6, and 8 are 12-hourlight perturbations.&: Phase of control4 Reset: Phase of steady-state induced by the light-resets. See text.

rhythm that reflects this conflicting control. As the lowpoint of the temperature cycle is steadily moved to theright relative to a fixed light regime, the overt rhythmfollows it up to a point about 15-16 hours from dawnwhen a discrete phase jump ensues (Fig. 4). Of the360” of conceivable phase relative to the light cycle,

only 180” is realizable; there is a 180” zone of forbiddenphase relations. Identical effects have now been foundin Euglena by Bruce [9] and in the cockroach byRoberts [10].

Drs. Wever and Aschoff have pointed out to me thatthis result could be explained in terms of a singleoscillator driven simultaneously by two entrainingcycles; its phase would respond to phase conflict be-tween the drivers in the way we observe; there wouldbe only 180” of allowed phase. However, the 2-oscillatorscheme not only explains the 180” phase jump equallywell, but seems uniquely fitted to accommodate thefurther experiments shown in Fig. 5. The data have beengiven elsewhere [5] in their raw form. In the plot givenhere the median of each peak is plotted as a point,and successive days are plotted one below the other.There are four cultures involved. Each is driven bylight and temperature cycles and in each the degreeof phase conflict between the cycles differs. The steady-state phases of cultures I and II lie just to the left ofthe forbidden zone; those of III and IV just to theright. In cultures II, III, and IV there are minor peaksof activities each day lying on the other side of theforbidden phase zone from that on which the majorpeak lies.

In Fig. 5 (middle section) the light cycle is discon-tinued after day -2, and the transient approach ofthe 4 rhythms to new phase is obvious. They move tothe phase dictated by the persisting temperature cycle.This result would be expected on either a 1- or 2-oscillator interpretation. But not so the results in thelower section of the figure. Here both light and tem-perature cycles are discontinued on day -2. Thephase of the rhythms does not remain where it was,nor does it move to some compromise between that of

164 PITTENDRIGH

LOW POINT OFL TEMP. C Y C L E

16 -

\2t,. , , , ., , ,,(, ? . , ., .

4 8 12 16 20 wI

PHASE l JuM?’

DROSOPH\ IA

IH\GH PO\Nl O F

dTEMP CYCLE

I

12

16

20

144 8

II2 16 20 24

1 I

PWSE hAP-

COCKROACH

FIGURE 4. The phase of Drosophila and cockroachrhythms as a function of the phase angle between simul-taneously entraining light and temperature cycles..Temperature cycle sinusoidal. Abscissa : hours relativeto light cycle. Ordinate: phase angle between light andtemperature cycles measured in hours: In Drosophila,it is hours between dawn and low point of temperaturecycle; in cockroach it is hours between sunset and high-point of temperature cycle. Note in each case the phase-jump that develops at a critical phase angle betweenthe two entraining cycles.

the former light and temperature cycles. It moves tothe phase of the light cycle. This behavior suggeststhat during the steady-state imposed by conflictinglight and temperature same system strictly followedthe phase of the light cycle: its phase was registered inthe flies, for when all external control was removedthe system resumed the phase of the light cycle. This,of course, conforms with the earlier results on transientsafter single temperature signals: the feedback of B inA is slight and when displaced to an abnormal mutualphase A imposes its phase in B.

The phase relations of any coupled oscillator systeminvolve 180” of forbidden phase, for were the drivensystem to lie in that zone it would be transferringenergy into, not drawing it from, the driver. And thereare further observations in Drosophila that again makethis the preferred interpretation for the phase-jumpjust discussed. These further facts are summarized inFig. 6, redrawn from Fig. 4 in my 1954 paper [1] inwhich their significance was not seen. A 10” temperaturestep-down occurs in a freerunning DD rhythm athour-zero, which is subjective dawn as defined earlier;in the experiment shown it occurs a t SD-2. Two results

t t . ..ttI P lu iv

+ of +tw 4 C U L T U R E S

-I

-2

-3-4-5‘6mYS

tttt .1pm’p

LOW POINT OF EMP CYCLE I NTHE 4 C U L T U R E S

rrt NH

.

FIGURE 5. The eclosion rhythm in four Drosophila cul-tures (I, II, III, IV) driven simultaneously by light andtemperature cycles whose mutual phase angle is nearthe critical point. Cf. Fig. 4. Successive days plottedbelow each other. Points are medians of eclosion peaks.Upper figure: steady-state. In cultures II, III, and IVthe daily activity is bimodal; on some days minor peaks(ii, iii, iv) lie on the other side of the forbidden phase-zone from major peaks. Middle figure; light cycle dis-continued on day-2. Lower figure; both cycles discon-tinued on day-2. In this experiment culture III lay justto the left (instead of the right as usual) of the forbiddenzone. 4; phase.

ensue: one is the transient elongation of the nextinterval prior to the eclosion peak labelled E-3; E-3 isdelayed 12 hours. The second result is that a slightphase shift (-2 hours) shows up in the subsequentsteady-state. This means the subjective dawn (SD-3in the figure) was delayed only 2 hours unlike theeclosion peak, E-3. In the lower figure an identicaltemperature step-down occurs at hour-zero, but in aculture that continues to be entrained by an LD cycle.The light cycle checks t h e phase shift of subjectivedawn: the phase of the ultimate steady-state is normal.But the light fails to prevent the delay of E-3. Indeed

CIRCADIAN ORGANIZATION 165

rTEMPERATURE DROPS FROM 26. TO l6.C

E-l 1 E -2 /E-3 E - 4 E-5

FIGURE 6. Evidence of a forbidden and an allowed (AZ) zone of phase relations for the Drosophila eclosion rhythm.See text.

this delay is further increased by almost 12 hours(180”) as E-3 is forced over to the region of the nextdawn (SD-4). This is a very strong fact and immediatelyyields to the demands of the 2-oscillator scheme withits implicit zone of forbidden phase relations. InFig. 6 the vertical lines marked SD register the phaseof the A-oscillation. In the DD freerun the subjectivedawn (SD-3) slips two hours to the right as impliedby the phase of the subsequent steady-state; andconsequently the delay of E-3 still leaves this peak justwithin the allowed phase zone (AZ) relative to theA-oscillation. But the same delay places it to the rightof the allowed zone when A is pulled back -2 hoursby the light. A phase jump across the forbidden zoneis then imposed; E-3 is forced to its further delay.Similar effects are detectable in the details of Fig. 1A,but need not be spelled out here.

Finally it is noted that the postulated 2-oscillatorscheme with the implication of restricted phase relationsbetween the A and B components will also explain asingular previously unpublished feature of temperature-induced transients in the Drosophila system. Tem-perature step-downs produce, as noted, large transientdelays which are evidently observable only insofar asthe induced delay in the B rhythm still leaves it withinthe zone of allowed phase relations. Temperaturestep-ups, on the other hand, produce only a negligibletransient advance of the peak: advance is not realizableinsofar as it would bring the B rhythm into its forbiddenzone relative to A.

It may well prove that none of the observationsrecorded here absolutely and individually demand a2-oscillator scheme. But many, even individually,elude any obvious one-oscillator treatment; and,collectively, they render the 2-oscillator analysis ashighly probable as one can hope for until A and Bcan be identified concretely.

CIRCADIAN ORGANIZATION; A MULTI-OSCILLATOR SYSTEM

Our main concern is with a broader concept of therhythm problem which the 2-oscillator scheme for

Drosophila eclosion suggests. Harker [ 11) has also beenled recently to a 2-oscillator scheme for cockroachactivity rhythms, as were Brown and Webb [12] severalyears ago when studying the rhythm of color changein Uca. The broader implications of a a-oscillatormodel rest, therefore, on a much wider base than thedetails of the Drosophila case.

If we reject, as reason demands we must, the pos-sibility that in selecting eclosion in Drosophila, locomo-tion in the cockroach, and color-change in Ucu, wepicked by chance the only system in each organismmediated by a rhythmic component (B-oscillation)distinct from the light-sensitive pacemaker (h-oscilla-tion), we are forced to conclude there must be manydistinct oscillatory physiological systems in the in-dividual that are not themselves directly coupled tothe light-regime as entraining agent. We are forced, infact, to abandon the common current view that ourproblem is to isolate and analyze "the endogenousrhythm,” or “the internal clock,” and are faced withthe conclusion that the organism comprises a popula-tion of quasi-autonomous oscillatory systems. Theconsequences of such a view seem to me so fundamentalin any attempt to set our problems in perspective thatwe should pursue further both its meaning and theevidence for it before proceeding with its implications.

Halberg’s extensive writings (see [13], e.g.) revealthat mammals, like men or mice, can display any or allof the following rhythms: locomotion, body tem-perature, blood sugar, liver glycogen, eosinophil count,adrenal activity, phospholipid synthesis, RNA andDNA synthesis, cell-division, drug-specific sensitivities,etc. And this list is surely limited only by availableassay methods, and the time so far invested! It is,then, not a question of whether many rhythms coexistin an organism; that is a matter of simple fact. Theproblem is the much subtler one of delimiting re-sponsibility for all of them; of recognizing which areautonomous, and which are merely imposed or forcedon intrinsically non-rhythmic systems by the control-ling activity of a central oscillatory pacemaker.

It is possible that many rhythms do in fact belong in

166 PITTENDRIGH

the latter category. For instance, the known physio-logical links between adrenal activity and virtuallyevery other aspect of mammalian metabolism couldbe the basis for regarding whole complexes of rhythmsas only forced by the rhythmic activity of these im-portant endocrines; for regarding, in short, only theadrenal as a self-sustaining autonomous oscillator. Butour knowledge of all such physiological links in metazoainvolving humoral or nervous action is very non-specific as to causal detail, and they (the links) couldwell be nothing more than channels of coupling informa-tion serving for unilateral or mutual entrainment be-tween tissues that are independently oscillatory intheir own right.

The latter alternative is the one I believe the facts,collectively, imply. It is supported by some directevidence from tissue and organ culture. Enderle [14],many years ago, demonstrated a circadian rhythm ofgrowth in Daucus tissue culture; Bunning has claimed[15] the persistence of a circadian oscillation in excisedsegments of hamster intestine; and there is evidencefrom several workers [18] that cell-division manifestsa circadian periodicity in mammalian tissue culture.Finally, along these lines one notes that some degreeof autonomy in the oscillations of individual tissuesand organs is virtually demanded by the fact thatsingle cells-at least as protists-manifest fully self-sustaining circadian oscillations which are formallyidentical with those of multicellular systems [16, 17].Indeed, unless the protistan cell is radically differentin this respect the multicellular system is-literally-apopulation of autonomous oscillators. And its physio-logical organization must involve, as a major feature,communication channels whose principal function isnot to impose rhythmicity but merely to couple andhence appropriately phase oscillatory activities inherentin the individual subsystems.

The remainder of this section concerns observationson freerunning rhythms of activity and body tem-perature. None of them demands my inference ofautonomy in tissue and organ oscillations. But theyall reveal complexities in the rhythmicity of the wholeorganism that are most easily explained by it; and tothis extent they are part of my case for its validity.

We have observed in the past few years a widespreadphenomenon (roaches, lizards, mice, hamsters, finches)we call “after-effects.” These are detected in the periodof rhythms freerunning in DD. They have been foundto follow three kinds of immediate pretreatment.

The first is an aftereffect of entraining the animalto an atypical period. Figure 7 (lower section) givesdata for four male sibling hamsters that were firstentrained to a 23-hour day and later to a 25-hour day.After each entrainment they were released into DDwhere the steady-state DD was found to be larger afterthe 25-hour day than after the 23-hour day.

The second is an after-effect of constant light which,like entrainment, can modify 7. Roberts [10] has found

- +20 min.

- - 24 HOURS

- -12 min.

FIGURE 7. After-effects on the freerunning period ofhamster rhythms. Plotted points are medians. In theupper figure the vertically oriented histogram shows thedistribution of T values for this sample of eleven animals.In the lower figure TFR (25) is the value of 7 in the rhythmfree running after entrainment to a 25 hour day; TFR (23)same after 23 hour day. See text.

that cockroaches exposed to LL lengthen 7, and thislengthening persists at least in part when the animalgoes back to DD. Repeated exposures to LL gave anincreasing or accumulative after-effect on ODD in oneindividual. Figure 8 is another case in the mousePeromyscus. Here LL makes for an overt aperiodicity;in other individuals of this species it merely lengthens T[19]. The individual shown was returned to darknesson the indicated day and ran with an extraordinarilylong period for DD throughout several weeks. This isclearly an LL after-effect; the animal ultimately revertsto a typically short DD period.

The third category of after-effects has been foundfollowing transients. Figure 9 shows freerunningrhythms in a hamster and a finch. In each case the phaseof the freeruns is reset by single light signals of a 12-hour duration. In the finch the phase of the signal issuch as to cause a reset that is attained by advancing(short-“period”) transients; and when the transientssubside the steady-state has a much shorter periodthan the preceding one. Similar after-effects appear inthe hamster figure which includes resets involvingboth delaying and advancing transients; following theformer, 7 lengthens, following the latter it shortens.

CIRCADIAN ORGANIZATION 16 7

FIGURE 8. An after-effect of LL on the freerunning rhythm of the mouse, Peromyscus maniculatus. LL, constant light;DD, constant dark. See text. The data are presented as follows: each horizontal line in the left-hand of the figureis a day’s record from the running wheel; activity is indicated by the vertical pen marking on the line; these fuseto give a solid band during intense activity. Successive days are plotted one below the other. The entire record isreproduced on the right, displaced upwards one day, to facilitate visual following of long or short period rhythms thatrapidly scan across the 24-hour cycle.

0 6 I2 IS UHWRS

0

,,,,,,,,,,,,,,,,,,,,,,

i-1 1

FIGURE 9. After-effects of transients induced by 12-hourlight signals in a hamster and a finch. Plotted points areonsets of locomotory activity. In both animals the lightsignal shifts phase of the rhythm (as defined by the timeof activity onset). The new steady-state shows a period(7) that differs from that (r) of the prior steady-state;the sign of the difference in I reflects the character(delay, advance) of the transient(s) involved in thereset.

Finally, Fig. 7 (upper section) shows several observedrnn values for each of eleven hamsters. In eight casestwo values are plotted as larger circles, one open andone solid. In each case the open circle is the r value

observed in a freerun that immediately preceded anentrained steady-state and a second freerun; theT value for the latter is given by the solid circle. Attain-ment of the intervening entrained steady-state involvestransients by which the animal gains appropriate phasewith the light cycle. In 4 animals these transients weredelays, and in the subsequent freerun r was longer.The intervening entrainment was brief, a matter ofdays; but it is clear that in spite of the animal overtlyequilibrating with the light cycle some “inertial” effectof the transient persisted and imposed itself on thesecond freerun.

It is clear that if an individual is to be characterizedby its r-if, that is, there are genetic differences in thecontrol of r-this characterization must be expressedas a range of realizable r values. The system can bepushed within this range to any one of, presumably,many frequencies where it is stable, at least for awhile ; its state perpetuates itself.

The only strong conclusion to be drawn from theseremarkable properties is that the oscillatory systemunderlying the overt rhythm of an individual is in nosense simple. No familiar single oscillator behaves likethis. The system has no real eigenfrequenz determinedby a fixed set of parameters. Nor in turning to con-ceivable properties of a multi-oscillator circadian systemis there an obviously unique explanation; but a sug-gestion may be offered that leads us to other pertinentfacts. The observed frequency of the individual must

168 PITTENDRIGH

be a compromise of a spectrum of frequencies that wouldbe individually manifest if the constituent oscillationscould escape entrainment from the rest of the systemand freerun. The coupling mechanisms that bring aboutthe complex of mutual entrainments must involve dis-continuities, making possible a range of realizable sys-tem-frequencies.

There are now many cases where a freerunning systemgives evidence of comprising more than one componentwith different characteristic frequencies. The first isafforded by data from Menaker's [20] study of bodytemperature rhythms in hibernating bats. In thecase shown in his Fig. 1, a seven day record, the mainpattern of the rhythni is defined by a sharp rise andplateau of temperature once every 24 hours. But thereis a clear, lower amplitude peak in the pattern whichscans the main pattern at a distinctly lower frequence( -25 hours).

Three other examples come from Mr. Swade’sunpublished work on arctic mammals. Two of themconcern the effect of constant light on rhythms inmice. The first is the mouse whose LL aftereffect hasalready been noted (Fig. 8). Its ultimate reversion to atypically short DD frequency involves a remarkablephenomenon. The reversion is neither abrupt nor

JON

+OFF

gradual (both of which qualities have been seen inother cases). It is achieved by developing the moretypical frequency as a distinct component in the totalactivity pattern before the atypical frequency (LLinduced) has subsided. For about 10 days two distinctfrequencies coexist. The second mouse (Fig. 10) isreleased from LD into LL on the indicated day. Thefreerun initially shows the long period (low frequency)characteristic of LL action on nocturnal species. Thislong period continues until the activity onsets havenearly scanned a 24-hour cycle; it then abruptly changesto a shorter period (high frequency) which remainsstable. The remarkable feature is that if the phase ofthis ultimate short period freerun is extrapolated back,it coincides with the phase of the original LD steady-state. This surely implies that some component in theorganism had been running in the short period from theoutset of the LL regime; that the other oscillations,proximally controlling activity itself, had broken looseto freerun on their long period but are eventuallyrecaptured into entrainment by the rest of the system;and only recaptured, moreover, when they reached theirnormal phase relation with it.

This type of behavior is more fully manifest in Fig. 11for an arctic ground squirrel in LL. The light intensity

I , 2 4 H O U R S - ‘ IFIGURE 10. Rhythms in a red-backed Clethrionomys rutilus. Data presented as explained for Fig. 8. The first 35 daysinvolved an LD 12:12 regime which entrains the animal. Note the very gradual approach, via. transients, to theentrained steady-state. Lights go o n and off at hours indicated. Mouse released into LL on 36th day. Long period free-run follows, but switches fairly abruptly to a short period on the 31st day of the freerun. The phase of this latterfreerun extrapolates back to the phase of onsets in the prior steady-state. See text for interpretation.

CIRCADIAN ORGANIZATION 167

I , 24 HOURS ,-IFIGURE 8. An after-effect of LL on the freerunning rhythm of the mouse, Peromyscus maniculatus. LL, constant light ;DD, constant dark. See text. The data are presented as follows: each horizontal line in the left-hand of the figureis a day’s record from the running wheel; activity is indicated by the vertical pen marking on the line; these fuseto give a solid band during intense activity. Successive days are plotted one below the other. The entire record isreproduced on the right, displaced upwards one day, to facilitate visual following of long or short period rhythms thatrapidly scan across the 24-hour cycle.

6 I2 I0 UtwuRs

i

FIGURE 9. After-effects of transients induced by 12-hourlight signals in a hamster and a finch. Plotted points areonsets of locomotory activity. In b o t h animals the lightsignal shifts phase of the rhythm (as defined by the timeof activity onset). The new steady-state shows a period(r) that differs from that (r) of the prior steady-state;the sign of the difference in T reflects the character(delay, advance) of the transient(s) involved in thereset.

Finally, Fig. 7 (upper section) shows several observedrnD values for each of eleven hamsters. In eight casestwo values are plotted as larger circles, one open andone solid. In each case the open circle is the r value

observed in a freerun that immediately preceded anentrained steady-state and a second freerun; theT value for the latter is given by the solid circle. Attain-ment of the intervening entrained steady-state involvestransients by which the animal gains appropriate phasewith the light cycle. In 4 animals these transients weredelays, and in the subsequent freerun r was longer.The intervening entrainment was brief, a matter ofdays; but it is clear that in spite of the animal overtlyequilibrating with the light cycle some “inertial” effectof the transient persisted and imposed itself on thesecond freerun.

It is clear that if an individual is to be characterizedby its r-if, that is, there are genetic differences in thecontrol of r-this characterization must be expressedas a range of realizable r values. The system can bepushed within this range to any one of, presumably,many frequencies where it is stable, at least for awhile; its state perpetuates itself.

The only strong conclusion to be drawn from theseremarkable properties is that the oscillatory systemunderlying the overt rhythm of an individual is in nosense simple. No familiar single oscillator behaves likethis. The system has no real eigenjrequenz determinedby a fixed set of parameters. Nor in turning to con-ceivable properties of a multi-oscillator circadian systemis there an obviously unique explanation; but a sug-gestion may be offered that leads us to other pertinentfacts. The observed frequency of the individual must

CIRCADIAN ORGANIZATION 169

of the LL regime is increased on the day indicated. happens the band becomes two-parted. The secondMany days later two features become clear: first the part is a separate component moving at a much lowerdensity of activity at the beginning of each circadian frequency (longer period) than the main band. Theband ultimately becomes diluted; second, as this freerun of this low frequency component stops when,

FIGURE 11. Freerunning rhythm of the arctic ground squirrel (Spermophilus undulatus) in constant light. Data pre-sented as explained in Fig. 8. Intensity of LL is increased on the day marked by the dashed line running across thefigure and exceeding its edge. Arrows mark times of replacing burned out lamp bulbs from the bank of lights. Seetext.

170 PITTENDRIGH

having scanned the cycle of the main component, itregains entrainment in the early part of the mainactivity band. This is evidently its normal phase whencoupled to the rest of the system; the dilution of ac-tivity in this region, coincident with the freerun, dis-appears when the freerun is ended by re-entrainment.

Other fully explicit cases of dissociation of constituentrhythms in the system as a whole are given in Lobban's[21] paper in this symposium.

THE PHYSIOLOGY OF CIRCADIAN ORGANIZA-TION; AND ITS BREAKDOWN

The autonomy and potential dissociability of distinctoscillatory components in the circadian system is thecrux of the viewpoint, and its implications, I amattempting to develop. The normal temporal organiza-tion of the system must, a priori, involve maintenanceof identical frequencies among the several components,and maintenance of appropriate mutual phasing.How are these prerequisites fulfilled? There are twoanswers. First, in the field the system is entrainedunilaterally by the external cycles of light and tem-perature. This environmental entrainment actuallyfulfills a dual function: (a) it phases the whole systemappropriately relative to the external cycle of environ-mental change; and (b) in so doing it imposes uniformityof frequency on the multiple endogenous oscillations.How far it directly maintains their correct phasing isnot clear. This must depend, even in the field, to someextent on their mutual coupling and hence, mutualentrainment. In the absence of external cycles of lightand temperature the integrity of the circadian organiza-tion must depend exclusively on this mutual entrain-ment of individual oscillations to maintain both identicalfrequencies and appropriate phasing. There is nodoubt this mutual entrainment of constituent oscilla-tions is commonly sufficiently strong to maintainadequate organization even in rigorously constantconditions of light and temperature. But it is nowequally clear: (1) that these constant conditions, whichthe physiologist so assiduously cultivates, are oftendetrimental; and (2) that the damage they engenderderives from a breakdown of the innate circadianorganization. This breakdown is in all probability afailure of mutual entrainment among constituent oscil-latory subsystems leading to their dissociation and aloss of normal phase relationships. I should be explicitthat the statement that damage commonly develops inaperiodic regime is fact; and that its origin is in break-down of the circadian system is an interpretation I haveto justify in the present section.

The basic assumption in my interpretations is thatloss of proper phasing among physiological subsystemsis detrimental; and powerful as the indirect evidenceis (see below) there is only one fully direct demonstra-tion of this proposition. It is given by the outcome ofanother remarkable experiment by Harker [22] who,

to be sure, nevertheless does not commit herself to thegeneral view given here. Previous well-known studiesof hers [23] had already demonstrated an autonomousdaily rhythm in the release of neurosecretion from thecockroach subesophageal ganglion which persists evenwhen this organ is explanted into the body cavity ofother roaches. Harker has now shown that when suchsupplementary ganglia are daily implanted into theabdomen of an intact host roach they cause no damageif implant and host are in phase as to their circadianoscillations. If, however, the implant is 12 hours out ofphase with the host, the latter develops transplantabletumors in the mid-gut wall which lies below the out-of-phase implant.

THE ACTION OF AN APERIODIC LIGHT REGIME

There is no direct evidence that constant darkness isdetrimental to anything but green plants. But there isincreasingly strong evidence that constant light (LL) isdeleterious at least in some species, and that its actionis not due to an excess of light as such but to its effect onthe freerunning circadian system.

Several workers, beginning with Arthur and Harvill[24], have noted tissue damage in tomatoes grown inconstant light and constant temperature; and similardamage occurs in other plants. Hillman’s [25] study ofthe problem reveals what is surely the key to thephenomenon: no such damage develops in LL if theplant is simultaneously exposed to a 24-hour tem-perature cycle. A striking parallel to these results isprovided by the response of the Drosophila eclosionrhythm to LL under a constant vs. cycling temperatureregime. In LL at constant temperature the period ofthe rhythm increases to begin with, but the systembecomes rapidly aperiodic. However, such LL inducedaperiodicity does not develop if a 24-hour temperaturecycle is imposed [26].

The importance of the Drosophila data is that theybear directly on the response of a known circadiansystem to LL in a periodic vs. an aperiodic temperatureregime ; and invite the following interpretations forboth the tomato and the fly: (1) the action of LL is insome sense to disorganize the circadian system; (2) thata temperature cycle can maintain the organized stateotherwise disrupted by LL; (3) that the LL damageobserved in the tomato (and a different striking LLconsequence in the fly discussed below) is a consequenceof the circadian system’s disorganization; and (4) thatthis in fact amounts to a loss of mutual entrainment,and hence of appropriate phasing, among constituentoscillatory subsystems. The damage results, in brief,from a dysphasia of oscillations in the system; and thefact that these, no longer properly coupled, can freerunon their own individual frequencies explains the LLinduced aperiodicity of the system as a whole. That LLindeed affects aperiodicity via such an uncoupling ofconstituent oscillations is both plausible in terms of its

CIRCADIAN ORGANIZATION 171

other certain action and demonstrated to occur in afew cases like those illustrated in Fig. 11. The othercertainly known action of LL is to change the overtfreerunning period of the organism as a whole. But thereis evidence that this change in the frequency of thewhole system is itself a complex matter. Figure 12shows the entry of a mouse into constant light. The“period” of the freerunning rhythm lengthens (typicalfor a nocturnal species) but aperiodicity ultimatelydevelops because the period of activity cutoffs in-creases less than the period of the onsets. It is as thoughactivity time were determined by the phase angle be-tween two oscillations whose frequencies were dif-ferentially dependent on light intensity. It will be notedthat, on re-entry to DD, this same mouse begins with avery brief activity duration each day, but this expandsgradually; again the onset and cut-off of activity havedifferent frequencies. This increase in activity timepersists until the animal becomes aperiodic. One isstrongly attracted to the view that having lost (beenforced out of) normal coupling and entrainment in LL,constituent oscillations fail to regain entrainment inDD. Thus it is by no means excluded that the light-sensitive system itself may comprise more than oneoscillation [26] and that in LL reciprocal effects of lighton their period lead to breakdown of even the pace-maker’s (the light-sensitive system’s) own integrity.

It is of course directly to the point that the best,most fully explicit, case one could present showing thefreerun of a distinct oscillatory component was in ananimal after increase of LL intensity; and that theother cases strongly implying a freerun of dissociatedcomponents were similarly in LL.

Green plants are not the only organisms in which LLhas been found to impose real ‘damage. Mr. Swade’sstudies of LL action on arctic mice in constant tem-perature has been hampered by the fact that a majorityof them have died within a week of entry in LL. AndHighkin, who earlier reported damage in peas develop-ing from a constant temperature regime, now reportshere [27] that LL imposes a similar stunting in theseplants. Perhaps the most striking feature of Highkin’sstudy is its indication that this damage is not im-mediately repaired on re-entry to a periodic regime;it persists, at least as a dauermodifikation, in somesucceeding generations.

We have recently encountered a situation in Dro-sophila melanogaster which at the present state ofanalysis looks similar. In a deliberate attempt to finda system in animals sensitive enough to uncoverdetrimental effects due to interference with the innatecircadian organization, we have begun several analysesof the effects of periodic v s . aperiodic conditions on theexpression of genes with variable penetrance. One ofthose selected is a recessive allele tug responsible formelanotic pseudotumors. Figure 13 summarizes re-sults so far available. The homozygous stock has been

maintained in LD 12:12 at 22°C. by mass transfersince we acquired it. Several other lines have beenestablished in DD, LL, etc. Figure 13 shows that inthe line transferred to constant light the penetranceof tug fell rapidly: it dropped from 99 per cent to 40per cent in the first generation; by the fifth generationit was down 20 per cent, and from the twelfth generationonwards to the 24th (at present) it has remained below5 per cent. Penetrance in the LD control has meanwhilefluctuated from 69 to 99 per cent; and the same is trueof a DD line. The drop in penetrance in LL is not re-versed, at least immediately, on return of the stock toLD; this has been done twice, and the second return toLD is now in its 9th generation. Clearly the LL condi-tions have wrought some inherited change. It is note-worthy that the LL line has been crossed to the LD linethree times, and on each occasion (Fig. 13) there hasbeen a difference between the reciprocal crosses; and thedifference is overwhelmingly significant in the thirdinstance (Gen 39 LD x Gen 22 LL). In each case thepenetrance of tug is lower in those Fi’s obtained whenthe LL parent was female. There is to this extent someevidence of cytoplasmic change, which we were led tolook for by the suspiciously rapid drop over the first LLgeneration. This is further supported by a single experi-ment of Bruce’s in which the saline supernate from cen-trifuged homogenates of LD flies was added to themedium of LL cultures. Such supernates increasedtumor incidence but not significantly if they were sub-jected to UV and clearly not if they were autoclaved.This work is progressing, with attention being given tothe information needed to disentangle simple effects ofLL selection on penetrance modifiers from the moreintriguing possibilities of induced cytoplasmic change.

It is clear that if the interpretations of LL actionoffered here are correct, the severity of its deleteriousaction will be inversely related to the strength of themutual couplings among constituent rhythms: in-dividuals whose overt periodicity survives exposure toLL are individuals whose circadian organization resistsdissociation, and we should anticipate them to be lesssensitive both to outright LL damage, and to othersubtler modifications like that of tug penetrance inDrosophila.

THE ACTION OF AN APERIODIC TEMPERATURE REGIME

One can trace physiological consequences to anaperiodicity of the temperature regime also. But herethe evidence is not so much of outright damage dueto constant temperature; it is that performance isimproved by a circadian periodicity in the temperatureregime. The initial discovery of this effect we oweapparently to Went [28] who demonstrated beneficialaction of a daily thermoperiod in tomatoes. Thermo-periodism is now well known in plants but has previ-ously, to my knowledge, never been reported in animals.We have recently detected the phenomenon in Drosoph-

172 PITTENDRIGH

LL-DD

L -424 HOURSFIGURE 12. Rhythms in a red-backed mouse Clethrionomys rutilus. Data presented as explained in Fig. 8. LD, lightcycle entraining rhythm for several weeks; lights on and off at points indicated. The LD behavior is interesting andwill be discussed by Mr. Swade elsewhere. LL, constant light freerun; DD, constant dark freerun. Note that evenwhen the DD activity becomes distributed throughout the whole day clear periodicities can be discerned in the com-ponent bands. See text.

CIRCADIAN ORGANIZATION 173

6, 0FI

LL BACK/@ TO L D

4 6 8 ’ IO 12 14 16 IS 20 22 24 26 28 3t.J 32

0.6 n - 8 5 6

z.4n =802n =885

t2

0 EXTRACT OF LD01 E X T . UV’d0 E X T . AUTOClAVED0 COUTROL

G E N E R A T I O N S

FIGURE 13. The incidence of tumorous phenotypes in a stock of Drosophila melanogaster homozygous for the recessiveallele tug. See text. The large circles plotted at generations 16, 20, and 30 are for FI progenies of LD and LL crosses.The open circles give the values for the cross in which the female was from the LL line; the half black circles forthe cross in which the female came from LD. At two points (generations 13 and ‘23) a subculture from LL was trans-ferred to LD and maintained there for several generations.

i l a . We have been examining the penetrance of severalsex-linked recessive lethals provided by Dr. E. E.Novitski. These have been balanced in stock againsta multiple inversion x-chromosome (y SC* A-49 sns2 scs)which is a strong semi-lethal in homozygotes. While wehave not enough information on the penetrance of thevarious recessive lethals, it is already clear that the via-bility of females homozygous for the balancing chromo-some is markedly sensitive to the aperiodicity vs. peri-odicity of the temperature regime. Table 2 summarizesresults from several crosses. In each case the homozy-gotes are more frequent (as % total females) in theperiodic temperature regime than in the aperiodic.

In the same way as LL damage is interpreted here asdue to an imposed breakdown of circadian organiza-tion, the benefits of thermoperiodism may be interpretedas due to more effective entrainment of the system thana light cycle achieves alone.

DELETERIOUS AND BENEFICIAL EFFECTS OF CHANGE

IN FREQUENCY

If maintenance, or failure, of appropriate mutualphasing among constituent oscillatory subsystems isthe basis of thermoperiodic benefits and LL damage,we could well have predicted two other types of ob-servation which workers in the Earhart Laboratory inPasadena have reported. These “predictions” wouldhave stemmed from the postulate, made earlier, thatone role of the light-sensitive pacemaker is to maintain(or contribute to maintenance of) appropriate phaserelations in the rest of the system, and to do so byvirtue of entraining them. But as with all entrainment,the pacemaker can only achieve this end if its fre-quency is close enough to that of the other constituents :there are limits to entrainment as Bruce [26] hasemphasized.

TABLE 2. THE VIABILITY OF A SEMI-LETHAL HOMOZYGOTE

IN DROSOPHILA MELANOGASTER IN R E L A T I O N

TO THE PERIODICITY OF THE

TEMPERATURE REGIME

Numbers given are yields of females (heterozygotes,and semi-lethal homozygotes) in each of 5 crosses:balancer/recessive lethal X balancer. The recessive lethalwas different in each cross; its viability was the realobject of study and will be discussed elsewhere whenmore extensive experiments are completed. The balancerwas y sc4 A-49 snxt sc8, which is semi-lethal in homozy-gous form. In each cross duplicate matings were set up(5 pairs to a vial) and placed in different regimes as fol-lows: (i) CT; constant temperature, 21OC.; LD12:12;(ii) PT; 24-hour periodicity in the temperature regime;LD12:12; the temperature cycle was roughly sinusoidalwith a 16°C. amplitude and a mean at 22OC. In eachcross the per cent yield of semi-lethal females is greaterin the thermoperiodic regime. ~2 is significant at the 1 percent level in the first 4 crosses; at the 2 per cent in thefifth cross.

Cross No.

I

II

III

IV

remperature1

_

Hetero-zygotes Homozygote

_

s

-_

Homozygotesas er cent

Tota FemalesP

PT 249 34 12.0CT 383 9 2.3

PT 387 67 14.8CT 438 37 7.8

PTCT

358409

3115

4113

3112

8.03.5

PTCT

351317

10.53.9

V / PT 383; CT 294

6.43.9

-

174 PITTENDRIGH

It follows that the competence of the light-sensitivesystem to entrain the rest of the system should beimpaired by either of two circumstances: (1) by anexternal light cycle that in turn entrains the r of thepacemaker too far from that of the non-light-sensitiveoscillations; and (2), holding the light cycle fixed, bychanging the level of a constant temperature regimeand thereby also changing the freerunning frequency ofthe temperature-dependent, non-light-sensitive oscilla-tions too far from that of the pacemaker.

Effects interpretable in both these ways have beenreported from the Earhart Laboratory. Highkin andHanson [29] reported damage to plants which, althoughthey experience the same total light as others, receiveit in periodicities whose 7 deviates too far from 24hours. Went first uncovered the second effect in Africanviolets (Saintpaulia) and others have since done soin other species [30]. The growth of the plant, measuredat a particular constant temperature, is a function of7 in the prevailing light cycle. This again is not con-founded with total light received: over a long periodlight constitutes 50% of each cycle no matter what the7. This is essentially the Highkin and Hanson result;what is added is that the optimum 7 of the light cycleis a function of the temperature. At lower temperaturesthe optimum r is longer; at higher temperatures it isshorter. In present terms this means that the actionof the light-sensitive pacemaker on the rest of the systemis stronger the closer its frequency approximates the(slightly temperature-dependent) frequency of therest of the system it must entrain.

OTHER PREDICTABLE EFFECTS

If the interpretation developed above is correct, weshould expect two other sources of damage to thecircadian system which to my knowledge have notbeen looked for.

First, systems driven by simultaneous light andtemperature cycles should be sensitive to the phaseangle between these entraining agents, for (on hy-pothesis) they drive distinct components of the system.Where the light and temperature cycles are nearly 180”out of phase (cf. Fig. 4) they should induce, in severeform, that dysphasia of constituent subsystems I haveassumed underlies the lesions induced by LL and byabnormal light cycles, etc.

Second, we may also expect that such dysphasia willdevelop in experimental organisms subjected to largeand abrupt phase-shifts of the environmental entrain-ing agent. We do this in resetting hamsters abruptly by5 or 6 hours; and it is imposed on man nowadays whoabruptly shifts the phase of his circadian system whenflying from say, New York to Paris, or vice versa.The meaning Bruce and I have given to overt transientsimplies the system is in temporary and partial dysphasiaso long as they last. In mammals they may last forweeks. It remains to be seen whether any stress or

even damage is actually imposed by this type of phase-shift which the system has never been called upon bynatural selection to accommodate.

THE PHYSIOLOGY OF THE LIGHT-SENSITIVE

PACEMAKER

On the face of it, entrainment seems a simple affair;an endogenous oscillation, coupled to it by appropriatesensory inputs, “follows the light cycle”; but, in fact,as Bruce has shown us, the mechanism of entrainmentis far from understood. Its discussion here has twopurposes: (1) to emphasize recent progress in the com-parative study of the action of single light signals andthe response curves so obtained; (2) to outline a func-tional interpretation of these response curves thatleads to a general qualitative theory of entrainment.

It is convenient to begin by listing the facts that anycomplete theory of entrainment by light must explain,(1) The theory must be compatible with the knownaction of light revealed by the response of freerunningrhythms to single signals applied throughout the cycle;(2) It must explain how the freerunning circadianperiod is brought into precise match with that of theearth’s rotation;(3) How in so doing an adaptively appropriate phase ismaintained relative to the changing pattern of day-length and, thus, to the whole external cycle of en-vironmental change. This problem of phase controlspecifically involves the obvious differences betweennocturnal and diurnal species : as spring advances thephase of activity-onsets in the former must be delayedeach day to follow sunset delays; while, in the latter,it must be advanced to follow dawn. In the autumnthe converse relation holds.(4) It must be compatible with the facts concerningthe freerunning periods of circadian rhythms. Theseinclude: (a) the weak generalization that TDD is com-monly less than 24 hours in nocturnal species and morethan 24 hours in diurnal species; (b) that in some species(e.g., hamster), however, the range of Ann amongindividuals is wide, falling on both sides of 24 hours;and (c) that TDD is in some sense labile, or plastic, asdiscussed earlier in this paper.(5) It must be compatible with the known action ofLL on rr$n which is summarized by what I suggest wecall Aschoff’s Rule; Aschoff [44, 45, 46] is responsible formuch of the pertinent data as well as for recognizingthe generality involved. The rule states that QL > TDD

in nocturnal species but 7LL < Ann in diurnal species.There are some exceptions to this rule (e.g., Drosophila),but it is far stronger than that in (4a) above, and cer-tainly strong enough to demand explanation.(6) The theory must also clearly accommodate thefact that there are limits to the value of r which entrain-ment can enforce; and(7) Finally, it must be compatible with the phenomenaof frequency demultiplication.

CIRCADIAN ORGANIZATION 175

THE COMPARATIVE STUDY OF RESPONSE CURVES FOR

SINGLE LIGHT SIGNALS

Several workers [3, 4, 5, 7, 31, 32, 33, 34] have re-cently focused attention on this subject. The assump-tion has been that the response to such non-periodicperturbations should be a simple phenomenon in termsof which entrainment by periodic signals might beclarified. The most encouraging feature of the results isthe recurrence of a particular pattern of responses inwidely different species from unicellulars to mammals.This pattern is summarized in Fig. 14 for several typesof signals applied to Drosophila pseudoobscura. Thedata for 12-hour and 4-hour signals have been publishedbefore [3, 4, 7]. They are replotted here in a way thatfacilitates comparison with the new data for very shortflashes (>&CM) sec.), and for other species. The responseto a signal applied at a particular phase in the free-running rhythm (abscissa) is plotted as the phase-shift induced (ordinate). This shift may be attained ineither of two ways: by long (“delaying,” or phase-lag)transients, or by short (“advancing,” or phase-lead)transients. This difference in response is incorporatedin the curve by the convention of plotting shifts attainedby delays as positive values and those attained byadvances as negative values.

In addition to those for Drosophila the figure in-cludes curves for the hamster and the flying squirrel;the former is taken from an unpublished study [31]by Dr. Burchard in Princeton, and the latter fromDeCoursey’s [32, 33, 34] extensive recent analysis ofGlaucomys. The figure shows that in all species the signand magnitude of the response is a function of thephase at which the endogenous oscillation was per-turbed; there is a clear switch-over from phase-delaysto phase-advances in the subjective night. The com-parison of the species cannot be pressed too far atpresent; it would be confounded by differences in signalintensity, duration, and criterion of response. Thus,DeCoursey’s curve for Glaucomys is based on lo-minuteflashes at 0.5 ft.c., and the response is measured by themagnitude of the first transient following stimulation,whereas the curves from our laboratory use ultimatephase-shift after transients have subsided and involveseveral signal durations none of which is 10 minutes.In spite of these current complications there remains astrong qualitative convergence: all 3 species show bothadvances and delays, and in all 3 the switch occurs inthe subjective night. Bruce and Pittendrigh [35] havereported the same pattern from Euglena; it is evidentin Hastings and Sweeney’s [36] analysis of Gonyaulax,and in Ehret’s [37] of Paramecium. Roberts [10] foundevidence of it in cockroaches, and other unpublishedwork in Princeton has detected it in a lizard and afinch. We have, then, a new and major similarity toadd to the list of these properties of circadian rhythmswhich encourages the view that they indeed presentus with general problems.

0 6 I2 IS 24

FIGURE 14. Curves for the response to light of Drosophila,Glaucomys, and Mesocricetus (hamster). The responseis measured in terms of the change in phase of the rhythminduced by a single light perturbation. See text for differ-ences between DeCoursey’s response criterion (on whichher Glaucomys curve [ 3 2 ] reproduced here is based) andthe criterion used for Drosophila and hamster. Hamsterdata from Burchard [31].

The ordinate scale (see text for convention on + and- values) given for the “short” signal curve of Drosophilaapplies to the other graphs above and below.

The abscissa is given as time in hours from an arbi-trary hour-zero as phase reference. In Drosophila thiscorresponds with the phase of the last dawn seen in theLD 12:12 regime from which the cultures are releasedinto DD before perturbation by the assayed signal. Inthe hamster and GZaucomys hour-zero is taken as a point12 hours before the onset of activity which is at sunsetand corresponds therefore, at least closely, with hour-12on the Drosophila scale.

The >&cc second flash applied to Drosophila was ofvery high intensity. The 12 and 4 hour signals in theDrosophila and hamster work were 75-100 ft. c. ; the 10minute signals applied by DeCoursey to Glaucomyswere of much lower intensity, 0.5 ft. c.

A THEORY OF ENTRAINMENT BY PHOTOPERIOD

The meaning of the response pattern lies in themechanism of entrainment. It is of course recognizedthat we must ultimately explain it in terms of thedetailed physiology of the stimulus-response system,

176 PITTENDRIGH

and the structure of the light-sensitive oscillator(s);at present this is not possible. We can, however, alreadyperceive its functional significance for entrainment,and attempt the outlines of a qualitative explanationfor many of the seven listed generalizations which anycomplete theory must accommodate.

The functional significance of the response curve inGlaucomys has already been noted by DeCoursey [32].Combined with the innate rnn which is typically lessthan 24 hours in this nocturnal animal, the curveguarantees entrainment with adaptive phase control

* (Fig. 15). If a flying squirrel is set at any atypical phaserelative to an LD cycle, it will automatically regainadaptive phase and stay there; if set tonormal phase, the combined action of

the7DD

right of its< 24 and

of light falling on the advance section of the responsecurve drives it back to the left.On regaining normalphase it remains there because an equilibrium is de-veloped between an advance effect due to the innatelyfast rnn and a delay effect due to the light impingingon the delay section of the response curve. Displace-ment to the left is followed by the converse ; it bringsthe delay part of the curve into the light and forces agradual shift to the right which is arrested only whenthe delay part of the curve is moved out of the light,or so far out that its action is balanced by ;

The essential feature of this interpretation is that theentrained steady-state represents an equilibrium be-tween the rnn and the action of light which can beeither an advancing or aa delaying effect. It follows thatin a typical diurnal species (like a bird or lizard) inwhich rnn < 24 hours, an equilibrium will also developif it has a response curve of the general type discussed

[ha HAS M I N O R R O L E IN EQUlLlE3RlUH] w

DIURNAL

FIGURE 15. The interpretation of entrainment in termsof response curves for light. That section of the responsecurve that is embraced by the light (a 12-hour photo-period is illustrated) is plotted as either solid black(delay response) or cross-hatch (advance response).See text.

here; and that at equilibrium the advance section of theresponse curve must lie within the light period to offsetthe daily delay due to rnn. When these typical rn,,values obtain, both diurnal and nocturnal forms shiftphase appropriately as photoperiod changes. Its ex-pansion in the spring forces the nocturnal species tothe right, following sunset, because the photoperiodnow embraces more of the delay curve; conversely itappropriately forces the diurnal species to the left(following dawn) because more of the advance curve iscovered.

DeCoursey also noted that the action of LL onGlaucomys could be understood in qualitative termsfrom the response curve for that species. TLL > TDD inthe flying squirrel, following Aschoff’s Rule; and onecannot avoid the conclusion that this increase in 7 isrelated to the delay section of the response curvegreatly exceeding the advance section both in rangeand especially in amplitude. This interpretation of theaction of LL on T can, moreover, be extended to whatwe should expect of a diurnal species. 7 in diurnal formsshortens in LL; this would imply that in them theadvance section of the response curve must exceed thedelay section. And this is precisely what is demandedfor good phase control; a diurnal species should havethe converse of the Glaucomys (Fig. 15) curve.

Drosophila pseudoobscura is, however, a diurnalspecies and does not possess the hypothetical responsecurve of a diurnal species inferred from this line ofinterpretation: its advance and delay curves are nearlyequal. Indeed the area under the delay curve slightlyexceeds that under the advance curve. This is, however,an encouraging exception because D. pseudoobscuraalso violates Aschoff’s Rule in that TLL slightly exceedsrnn; and to this extent it is an exception that “proves”the present interpretation of Aschoff’s Rule. The de-tailed form of the Drosophila curves, which DeCourseyfound in conflict with her analysis of Glaucomys, pointsup additional aspects of the whole problem.

It is obvious from the Drosophila curve for briefflashes that any usual photoperiod (8-16 hours) willembrace both the advance and delay sections of thecurve. The equilibrium during entrainment musttherefore involve (in addition to any effect from Ann)effects from both sections (advance and delay) of theresponse curve. In such cases, where both sections ofthe curve always fall in the light, the role of rnn >< 24in the equilibrium may be trivial; the equilibriumprobably depends almost entirely on the interactionof the morning advance and the evening delay causedby the light. One may well expect that in those specieswhere the rnn values are loosely distributed o n bothsides of 24 hours entrainment and phase control willbe of this type, thus explaining why the generalization(4a) listed earlier is weak.

The explanation of Drosophila entrainment in termsof the net action of the photoperiod is a flat reversal

CIRCADIAN ORGANIZATION 177

of the earlier position of our laboratory [1, 2, 3, 4, 5],in which we supposed the single dark/light transitionat dawn was the effective entraining signal. Thiscontradiction has led us to re-examine the bases of ourearlier view; they have proved wrong. It was based ona poorly executed experiment summarized in Fig. 2 ofa previous paper [5]. The several cultures were allraised in LD 12:12 and switched to the various photo-periods studied only on the day before data collectionbegan. The erroneous conclusion that phase was nearlyinsensitive to photoperiod was founded on the factthat--as one only now realizes and can perceive in theold data-the cultures never got out of transients inthe five days of observation. Recent experiments (Fig.16) prompted by the present interpretation of the re-sponse curve have shown that the phase of the Drosoph-i l a eclosion rhythm is in fact strongly dependent onphotoperiod.

The fact that a long duration signal (like 4 or 12 hours)embraces parts of both the advance and delay sectionsof the flash curve in Drosophila is surely responsible for

FIGURE 16. The phase of the Drosophila eclosion rhythmas a function of photoperiod. The upper figure plots thedistribution (under various photoperiodic regimes) ofeclosion throughout the day. The dawn of each photo-period is synchronized as a reference point for discussionof phase. Each curve is normalized to the same area andbased on mean values at each hour of the day from a 5day run in steady-state. The numbers on each curve indi-cate the duration (in minutes or hours) of the entrainingphotoperiod.

The lower figure, left of the break, replots the samedata; the phase of the rhythm (on the 24-hour abscissaof the square) is characterized here by the arithmeticmedian of the distribution (solid circle). The ordinateof the square is the photoperiod of the entraining signal.

The lower figure, right of the break, plots (open circle)the steady-state phase of the same rhythms after theyhave been released into DD, and transients have sub-sided. The dashed line gives the phases of the formerentrained steady-state for comparison.

the quantitative differences among the three responsecurves for this species given in Fig. 14. But the com-plexity of the system, even superficially, is greatly in-creased by this dual action of a single signal, and untilwe know how advances and delays are effected, andhow they interact, we cannot predict, even qualitatively,what the net action of a long signal should be. Thisdual action also imposes obvious difficulties in the wayof using phase-shifts to measure an action spectrum.

Two possibilities immediately suggest themselves onhow light acts, and since one of these is immediatelydismissable, at least for Drosophila, it is worth notingthem: (1) light exerts some continuous action on oneof the fundamental parameters of the oscillation, in away analogous to a continuing change in the resistanceof an electronic oscillator throughout a substantialfraction of its cycle; (2) the light is an effective signalonly as it comes on and goes off. In this view the 2transitiona (dark/light and light/dark) act as more orless discrete external perturbations. The known re-sponse of several metazoan photoreceptor preparationsprovides a clear concrete model for this. The onset of along light signal elicits an “on” discharge which rapidlydecays; the preparation then remains silent, thoughilluminated, until light is discontinued when an “off”discharge is produced. _

The continuous action approach has some attractivefeatures if one assumes the net action of a long durationlight signal is approximated by summing the areasunder those sections of the response curve it embraces.This approach yields computed effects for 12 and 4 hoursignals that are plausible estimates of the observedresponses; one gets an excellent model of phase controlalong the lines discussed earlier; and summing areasunder the advance and delay sections for Drosophilaand Glaucomys “predicts” a net action of LL com-patible with Aschoff’s Rule. But the hypothesis mustbe rejected in view of several recent observations fromour laboratory which Bruce [9] has noted in his dis-cussion, and also because of an experiment summarizedin Fig. 17 that was performed to test the hypothesis.Nineteen separate cultures of Drosophila were raised inLD 12:12 and ultimately allowed to freerun in DD.Each culture, however, received a different light signalon the last day before release into DD. All 19 sawdawn at the same time (that which obtained throughouttheir previous LD regime). But in each culture thelight was discontinued at a different time. The finalphotoperiod was thus different in each case. Accordingto the continuous action hypothesis the several photo-periods should have exerted a different net action onthe rhythm which is estimated by summing the areasunder the advance and delay sections of the responsecurve covered by each photoperiod. While this approachis clearly at best a very rough estimate, it does lead toclear qualitative predictions. For example, a 21-hourphotoperiod should shift the phase of the DD free-

178 PITTENDRIGH

OISERVCD PHASES

EXPECTATION ONCONTINUOUS ACTION

wyoo-rbmsfs

FIGURE 17. Experimental evidence for rejection of the continuous-action hypothesis of light effects; and that thelight-dark transition (“sunset”) of any photoperiod greater than 12 hours is an absolute phase-giver for the subsequentsteady-state. All cultures studies were raised in LD 12:12 (see text).

The left hand square in the figure has a W-hour ordinate and a 24-hour abscissa. The former measures the finalphotoperiod the cultures experienced before release into DD. From the response curve for flashes, plotted on the sameabscissa that measures the photoperiod, one estimates what total phase change (delay or advance) the final photo-period is expected to impose on the ultimate freerunning steady-state. The qualitative expectation is given by thedashed line on the right hand square. Note the change in the phase curve expected at 19 hours as further increaseof photoperiod begins to add an advance effect.

.

Observed phases of the steady-state are plotted as solid circles; and clearly force rejection of the continuous actionhypothesis.

running rhythm to the right but not as far to the rightas an l&hour signal: the 21-hour photoperiod includesa substantial advance effect missing in the 18-hoursignal. The results of the experiment are extremelyclear: the hypothesis is wrong. Any increase of photo-period beyond 12 hours causes a phase-shift to theright, and the new phase is always n X ~ 24 + 15hours after the last light/dark transition. This transition(as other experiments have implied) is thus an absolutephase-giver provided it is not followed by a dark/lighttransition within the next 24 hours. When this happensthe transitions interact; and the steady-state phaseresulting from their interaction is a sensitive function ofthe phase angle between them. It is, in short, a functionof the photoperiod. We need to know precisely how thetransitions act and interact before we can rigorouslyrelate the response curve for long-duration signal tothat for flashes, and derive a general quantitative theoryof entrainment that explains the dependence of phaseon photoperiod.

THE GENERAL PROBLEMS"Why this absurd concern with clocks, my friend?”

Walter de la Mare. “The Winged Chariot.”

Wilhelm Hufeland, a German physician, wrote a bookin 1798 on the art of prolonging life. He advised among

other things that one should heed the evangelist JohnWesley who is evidently responsible for the old jingle“Early to bed, early to rise, makes you healthy-etc.”Hufeland had, one likes to feel, an insight into ourproblems: for apart from this tortuous implication thathe suspected irregular entrainment would stress thecircadian system, he has two sentences, quoted byThienemann [38], that are an excellent caption for muchrecent discussion : “Die 24-Stundige Periode . . . . Sieist gleichsam die Einheit unserer natiirlichen chrono-logie.” His assertion here has been well sustained bythe brilliant work of later German naturalists-of vonFrisch, of Kramer, and their students-to whom weowe the truly wonderful discovery that bees and birds(and now many other metazoa) can measure the timeof day with an inner clock whose basic motion is a24-hour oscillation. It is true that many biologistsbefore 1950--including Pfeffer, Bunning, Kalmus, andKleitman in particular-recognized there were firstrate problems in the phenomena of daily rhythms. Butthe literature of the last ten years fully attests the keyrole played by the discovery of time-compensated sun-orientation in reviving and reformulating the interestattaching to them. As Bruce and I noted elsewhere [ 4 ]the evolutionary biologist refuses to suppose “clocks”appeared suddenly, de novo, in arthropods and verte-

CIRCADIAN ORGANIZATION 179

brates. He looks for simpler precursors and, even inignorance of Hufeland's suggestion, turns to dailyrhythms as potential time-measuring oscillations. Thisexplicit reformulation of rhythms as clocks was mainlyresponsible for establishing, in the last ten years, thetemperature-compensation of their period as a realgeneralization : it was deliberately sought as a functionalprerequisite of a good clock.

To some of us who were attracted to circadianrhythms by this line of thought the phenomenon oftemperature-compensation seemed the outstandingproblem; it challenges the usual emphasis on thetemperature dependence of cellular processes. To someworkers it continues to pose the major riddle and hasevidently been partly responsible for encouragingBrown to seek its explanation in terms of control byunknown geophysical periodicities. It was also partlyresponsible for encouraging Bruce and me [2, 4 ] tohave, initially, a too-sanguine hope that biologicalclocks all had the same basic mechanism. One felt themechanism for temperature-compensation must havebeen so hard-won by early cells that having onceacquired it living systems retained it in all later evo-lutionary development. Both lines of thought aresurely wrong. Burckhardt’s [39] recent paper on temper-ature relations of a stretch receptor in the crayfishreminds one that many aspects of the living system mustbe temperature-compensated; it maintains its organi-zation as such over a wide temperature range. One hashad no right to take for granted the proposition thatneurally transmitted information is probably frequencymodulated; not, at any rate, if one worries over temper-ature-compensation. For unless the spontaneousfrequency of a sense organ is compensated for temper-ature like the stretch-receptor Burckhardt studied, itwill be useless for anything except temperature sensing.The point is not that temperature-compensation failsto pose a first-rate problem; it is that circadian rhythmsa priori cannot be and are empirically known not to beunique in this respect. The compensation may be adifficult trick but surely one the organization turns allthe time, doubtless in diverse ways, in achieving allsorts of ends.

Their temperature relations therefore merge withthose other properties of circadian rhythms, discussedearlier, that focus our search for the general problemsin an underlying circadian organization as the realobject of our study.

The comparative physiology of circadian rhythms inhamster, Drosophila, and Euglena obviously concernssystems which in their concrete details are radicallydifferent. And the striking formal similarities theypossess must owe their origin to convergent evolutionimposed by a common demand for an inner temporalorder that matches that of the external environment.The magnitude and regularity of the daily change inthe environment is another thing one too easily takesfor granted. It is, in fact, little wonder that it has

clearly been an ever-present and intense selective agentwhich few-if any-species have escaped; and itremains to be seen how far even arctic, cave, and deepsea forms fully lack circadian systems. There is somereason to suspect we shall find them even there-butthis is another hazardous evolutionary prediction. Thepoint I have in mind is that many of these species havecomparatively recent ancestors more fully exposed tothe effects of the earth’s rotation; and, further, thatthe circadian organization selection has wroughtprobably serves broader timing functions than onlythose of phasing to the external world. Organization ina living system involves time quite apart from theperiodicity of the environment, and it may be that thepresence of such oscillations in every system one studiesreflects the exploitation of the circadian system for ageneral temporal ordering of constituent subsystems.If so, it may not be easily abandoned even in caves orthe deeps where external periodicity has been leftbehind. And, if true, it also gives its study-specificallythat of dysphasia in the system-added meaning.Nevertheless, the fact that the selection for circadianorganization must have been so widespread, strong,and of so long a standing, reduces the prospect of acommon concrete mechanism to vanishing point.

This is surely true of what are called B-oscillations inthe earlier discussion; and in spite of our [7] recent hopethat the universal light-sensitive oscillation mightprovide a common concrete mechanism there is nowlittle reason even for this. When Bruce and I raisedthis hope it was based on our finding a strongly charac-terized response pattern in Drosophila to single light-signals; on finding it elsewhere; and finally on failingto see adaptive meaning in it. One then guessed he hadsomething that could not be dismissed as another con-vergence induced by natural selection. But that isclearly not the case if the qualitative theory of en-trainment outlined above is valid. The response curvefor light is adaptive, and moreover reflection showsthat the pattern of the curve (which is all that differentspecies share) has to be as it is: only a morning advanceand an evening delay will give a stable equilibrium nomatter what the shape of the curve. Thus a morningdelay response would continue to shift the phase ofthe system to the right until either the delay part ofthe curve were forced into the dark, or the succeedingpart of curve (eliciting an advance response) weredragged into the light; only then will the system ceaseto shift its phase.

There is perhaps a lingering hope that all thesecaveats to seeking a common concrete system haveclear pertinence only to multicellular systems; and thatthe single cell will have, or be, the common flywheel inall clocks. This remains to be seen. At present all weknow is that, formally, protists resemble multicellularsin their circadian rhythms; and these formal simi-larities-an innate circadian 7, temperature-compen-sation, and response curve to light-are what we know

1--

180 PITTENDRIGH

,

natural selection would demand irrespective of howachieved. Nevertheless, in taking stock of problemsand directions it is sure that work on single cells andsimple tissue cultures will provide considerable insights.At present the chemical attack has yielded little in spiteof the extensive attempts of himself and others thatHastings [40] has summarized for us. After a similarlylong list of negative results Bruce and I [41] have takensome encouragement from finding that the system canbe chemically manipulated: Euglena can be phase-shifted by exposure to D20 and when adapted to heavysater, presumably replacing at least some of theirhydrogen by deuterium, the cells freerun on a muchlower frequency than they do in HZO. One still knowsso little of what this implies that it remains to be seenhow far it takes us. Ehret’s [42] indications of nucleo-tides being involved are more promising in leading towell-known and central features of the metabolicsystem. Even so, assuming that present indications oftheir being involved mature to clear proof, the nextstep is none too clear. One suspects the search for adiscrete clock which the cell “has” may prove fruitless;that the search was founded in the first place on tooloose a use of language. One is entitled to say only thatthe cell “is” a clock, for he has no assurance yet thatany lower level of organization (mitochondria, forexample, remain unstudied in this respect) can auton-omously sustain a circadian oscillation. The suggestion,of course, is that again the problem goes bac k toorganizational features. The individual cell’s rhythm iscompensated for temperature, and if the explanation ofthis feature does lie in the properties of an organization,as such, we shall hunt in vain in the cell for an isolabletimepiece. But to abandon-at least on this account--search for meaning in Hiifeland’s statement that a24-hour period is the unit of biological chronologywould be to despair of the biologist’s real problem.This, as Needham asserted, is the problem of organi-zation.

Many years ago Biinning published the suggestion[43] that the photoperiodic effect was mediated bywhat one then called the endogenous rhythm; and headded to this basic suggestion a subsidiary hypothesisas t o how it did so. This latter postulated the existenceof a distinct scotophil (dark-loving) section of therhythm. A photoperiodic effect, like flower induction,was triggered or not according as to whether light fell,or did not, in the scotophil. This suggestion was made,I believe, before the photoperiodic problem was beingexplicitly discussed as a “clock” problem. Today wetake the implicit time-measurement (of night- or day-length) as reason to speak of the “inner clock” in-volved. It is not pertinent here to pursue the fact thatBiinning’s hypothesis has met with very little favoramong fellow botanists; to wonder why zoologicalstudents have never even considered it at least in theirpublished work; nor to attempt its evaluation in the

light of the existing data. My point in raising the issueis to make a necessarily brief attempt at greaterexplicitness in suggesting that a circadian organizationas such (not a discrete physical entity) is the time-measuring thing underlying the diverse phenomena thathave been responsible for our using the word “clock”in the first place. Photoperiodism is simply the easiestto tackle for such an explication; the bee’s zeitgedachtnisand the “clock” involved in sun-orientation are evenremoter prospects for analysis.

The attempt is based on several features of Figs. 4and 16 in this paper. First, Fig. 16 reminds us, asAschoff [44] has shown in more detail, that the steady-state phase of the circadian system is a strong functionof the photoperiod. This was not clear for Drosophilapreviously; and it is fair to comment that in discussingBiinning’s hypothesis other students of rhythms havefailed to emphasize sufficiently this obviously centralpoint. Thus it is fact-not a matter for discussion-thatthe circadian system does measure photoperiod; theconclusion that its phase is a “measure” of photoperiodhas precisely the same logical status as the conclusionthat flower-initiation or diapause-interruption is a“measure” of photoperiod. It is only the vagary ofconvention that excludes the phase control of rhythmsfrom that motley of phenomena labeled as photo-periodism, sensu "strictu."

The only real issue in the debate over Biinning’shypothesis is whether the individual organism usesmore than one device to estimate the same environ-mental parameter it exploits for several different ends.In proposing it probably does not, one is only voicingagain the essential feature in Banning’s proposition of1936. But I would prefer to avoid the special hypothesisof a scotophil and reformulate the essential propositionas follows: photoperiodic effects (sens. strict.) areaspects of the mechanism of entrainment of circadiansystems; that the entrained equilibrium of the systemis a characteristically different state for each photo-period; that above or below a critical photoperiod sharpdiscontinuities in the response mechanism are responsi-ble for imposing different states on the circadiansystem. On one side of the critical photoperiod phaserelations among constituent oscillatory elements allowa particular reaction sequence to proceed; on the otherside of that period widely different phase relationskeep this metabolic pathway closed.

* Returning to Fig. 16: The striking dependence ofphase on photoperiod breaks down at the suggestivevalue of 16 hours. Beyond that photoperiod phasebegins to shift back closer to dawn. But the changedresponse involves more than the system’s “phase” inany simple sense of this word. The adequacy of en-trainment begins to fail, and already at 20 hours onesees the beginnings of that aperiodicity which iscomplete under constant light. After release into DDthe freenmning system also indicates that entrainment

CIRCADIAN ORGANIZATION 181

by light involves more than merely establishing phaseof an oscillation whose form or state is invariant underdiverse photoperiods: spectacular transients (their du-ration varying with photoperiod) precede the newsteady-state whose phase differs substantially (andcharacteristically for each previous photoperiod) fromthe prior steady-state. The latter in fact is somedynamic equilibrium attained by the multi-oscillatorsystem responding to opposed perturbations-advance,delay-at the dawn and sunset transitions.

There is now a special significance attaching to ourearlier conclusion that the entraining signals of thelight cycle are the transitions in morning and evening.Change in photoperiod is a change in the phase anglebetween these signals. We know that when two en-training signals are coupled to and drive an oscillatorysystem the latter’s phase is not only sensitive to thephase angle but its response will necessarily involve

. sharp discontinuities of the type exemplified in Fig. 4.Indeed this figure can serve as a model to illustrate thepossibilities involved. It plots the phase of fly androach rhythms as a function of the phase angle betweena light and temperature cycle; and the essential featureis a phase-jump of 180” when that phase angle exceedsa critical value. A chemical oscillation in the cellcoupled directly or indirectly to dawn and dusk asopposing drivers will respond in the same way; onpassing a critical photoperiod, the phase-jump imposedon this oscillation would constitute the closing of aswitch that opened up previously impossible metabolicpathways. For, clearly, a reaction sequence can notproceed if an essential constituent is displaced fromothers by a 12-hour gap; and it will proceed if this gapcan be closed. In such a model the clock is not anentity; the time-measurement (that of a fixed interval-timer) is executed by the responses inherent in thedynamics of the circadian system.

We may yet be confronted with a somewhat wrysituation: the student of rhythms protests he has nocommon mechanism (in the concrete sense) to give hisfield the unity he would like; the student of insectphotoperiodism asserts his system (involving eyes andendocrine glands) bears only a superficial, functionalresemblance to that of flower-initiation by photoperiod.A n d yet both may be wrong in the sense that there arecommon mechanisms-built of different concrete parts-in circadian systems and photoperiodic effects every-where. These general mechanisms inhere in the princi-ples whereby constituent oscillatory subsystems arecoupled and mutually entrain each other; how in sodoing temporal organization is maintained within theorganism; how the system as a whole is coupled to themultiple periodicities of the environment; and howcritical phase-angle conflicts in the action of the lattercan be exploited at least for interval-timing. In briefthe prospect of a common mechanism to incite us isslim only if we are too preoccupied with the concrete

and neglect our real business of elucidating organi-zational features of the living system.

REFERENCES

1. PITTENDRIGH, C. S. 1954. On temperature independ-ence in the clock-system controlling emergencetime in Drosophila. Proc. Nat. Acad. Sci., 40:1018-1029.

2. BRUCE, V. G., and C. S. PITTENDRIGH. 1957. En-dogenous rhythms in insects and microorganisms.Amer. Naturalist, 91: 179-195.

3. PITTENDRIGH , C. S. 1958. Perspective in the studyof biological clocks. pp. 239-268. Symposium onPerspectives in Marine Biology. Berkeley, Calif. :Univ. of Calif. Press.

4. PITTENDRIGH, C. S., and V. G. B RUCE. 1957. An oscil-lator model for biological clocks. pp. 75-109.Rhythmic and Synthetic Processes in Growth,ed. Rudnick. Princeton: Princeton Univ. Press.

5. --.. 1959. Daily rhythms as coupled oscillatorsystems and their relation to thermoperiodismand phot operiodism . pp. 475-505. Photoperiodisma n d Related Phenomena in Plants and Animals,ed. Withrow. Washington: A.A.A.S.

6. N e e d h a m , J. 1937. Integrative Levels; A Revaluationof the Idea of Progress. Oxford Univ. Press.

7. PITTENDRIGH , C. S., V. G. BR U C E, and P. KAUS.1958. On the significance of transients in dailyrhythms. Proc. Nat. Acad. Sci., 4 4 : 965973.

8. PITTENDRIGH , C. S. 1958. Adaptation, natural selec-tion and behavior. pp. 390-416. Behavior and Evo-lution, ed. A. Roe and G. G. Simpson. Yale Univ.Press.

9. BRUCE, V. G. 1960. Cold Spring Harbor Symp. onQuant. Biol. Vol. 25.

10. ROBERTS, S. K. 1959. Circadian Activity Rhythms inCockroaches. Ph.D. Thesis. Princeton University.

11. HARKER, J. E. 1960. Internal factors controlling thesuboesophageal ganglion neurosecretory cycle inPeriplaneta americana L. J. Exp. Biol., 37: 164-170.

12. BROWN, F. A., JR., and H. M. WEBB. 1949. Studiesof the daily rhythmicity of the fiddler crab, Uca.Modifications by light. Physiol. Zool., XII: 136-148.

13. H a l b e r g , F. 1959. Physiologic 24-hour periodicityin human beings and mice, the lighting regimenand daily routine. Pp. 803-878. Photoperiodismand Relaied Phenomena in Plants and Animals,ed. Withrow. Washington: A.A.A.S.

14. ENDERLE, W. 1951. Tagesperiodische Wachstums-und Turgorschwankungen in Gewebekulturen.Planta (Berl.), 39: 530-588.

15. B u n n i n g , E. 1958. Das weiterlaufen der "physio-logischen Uhr" im Saugerdarm ohne zentraleSteuerung. Naturwissenschaften, 45: 68 (d).

16. BRUCE, V. G., and C. S. PITTENDRIGH. 1956. Tem-perature independence in a unicellular “clock.”Proc. Nat. Acad. Sci., 42: 676-682.

17. HASTINGS, J. W. 1959. Unicellular clocks. Ann. Rev.of Microbiol., 13: 297-312.

18. HUPE, K., AND A. G r o p p . 1957. Uber den zeitlichenVerlauf der Mitoseaktivitilt in Gewebekulturen.Z. Zellforsch., 4 6 : 67-70.

19. R a w s o n , K. S. 1959. Experimental modification ofmammalian activity rhythms. Pp. 791-800. Photo-periodism and Related Phenomena in Plants andAnimals, ed. Withrow. Washington: A.A.A.S.

20. MENAKER, M. 1959. Endogenous rhythms of body

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temperature in hibernating bats. Nature, 184:1251-1252.

21. L o b b a n , M. 1960. Cold Spring Harbor Symp. onQuant. Biol. Vol. 25.

22. H a r k e r , J. E. 1958. Experimental production ofmidgut tumors in Periplaneta americana L. J.Exp. Biol., 35: 251.

23. --.. 1956. Factors controlling the diurnal rhythmof activity of Periplaneta americana L. J. Exp.Biol., 33: 224.

24. A r t h u r , J. M., AND E. K. HARVILL. 1937. Plantgrowth under continuous illumination fromsodium vapor lamps supplemented by mercuryarc lamps. Contribs. Boyce Thompson Inst., 8:433-443.

25. ‘HILLMAN, W. S. 1956. Injury of tomato plants by

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

continuous light and unfavorable photoperiodiccycles. Am. J. Bot., 43: 89-96.

BRUCE, V. G. 1960. Cold Spring Harbor Symp. onQuant. Biol. Vol. 25.

H i g h k i n , H. R. 1966. Cold Spring Harbor Symp. onQuant. Biol. Vol. 25.

WENT, F. W. 1944. Plant growth under controlledconditions. II. Thermoperiodicity in growth andfruiting of the tomato. Am. J. Bot., 31: 135-140.

H i g h k i n , H. R., and L. B. HANSON. 1954. Possibleinteraction between light-dark cycles and endog-enous daily rhythms on the growth of tomatoplants. Plant Physiol., 29: 301-301.

WENT, F. W. 1966. Cold Spring Harbor Symp. onQuant. Biol. Vol. 25.

BURCHARD, J. E. 1958. Re-setting a Biological Clock.Ph.D. Thesis, Princeton University.

D e C o u r s e y , P. J. 1959. Daily Activity Rhythms inthe Flying Squirrel, Glaucomys volans. P h . D .Dissertation, Univ. of Wisconsin.

--. 1960. Daily light sensitivity rhythm in a rodent.Science, 131: 33-35.

--.. 1960. Cold Spring Harbor Symp. on Quant.Biol. Vol. 25.

BRUCE, V. G., and C. S. P ITTENDRIGH . 1958. Reset-ting the Euglena clock with a single light stimulus.Amer. Nat., XCII: 295-306.

HASTINGS, J. W., and B. M. SWEENEY. 1958. A per-sistent diurnal rhythm of luminescence in Gon-yaulax polyedra. Biol. Bull., 116: 440-458.

EHRET, C. F. 1960. Cold Spring Harbor Symp. onQuant. Biol. Vol. 25.

H u g e l a n d , W. 1798. Makrobiotik. quoted in Thiene-mann, A. F. 1956. Leben und Umwelt. Rowohlt,Hamburg.

BURCKHARDT, D. 1959. Die Erregungsvorgangesensibler Ganglienzellen in Abhangigkeit von derTemperatur. Biol. Centralbl., 78: 22-62.

HASTINGS, J. W. 1966. Cold Spring Harbor Symp. onQuant. Biol. Vol. 25.

BRUCE, V. G., and C. S. P ITTENDRIGH . 1960. Theeffects of heavy water on the circadian photo-tactic rhythm in Euglena. In ma.

EHRET, C. F. 1960. Cold Spring Harbor Symp. onQuant. Biol. Vol. 25.

BUNNING, E. 1936. Die endonome Tagesrhythmikals Grundlage der photoperiodischen Reaktion.Ber. dtsch. bot. Ges., 64: 590-607.

ASCHOFF, J. 1969. Cold Spring Harbor Symp. onQuant. Biol. Vol. 25.--. 1952. Frequenzanderung der Aktivitatsperiodik

bei Mausen im Dauerdunkel und Dauerlicht.Pfliiger’s Arch., 255: 197-263.

46. --. 1958. Tierische Periodik unter dem Einfluss

47

48

49

50.

51.

52.

53

54

55

von Zeitgebern. Z. Tierpsychol., 15: l-36.B u n n i n g , E. 1958. Die Physiologische Uhr. Berlin:

Springer.BROWN, F. A., JR. 1960. Cold Spring Harbor Symp.

on Quant. Biol. Vol. 25.ASCHOFF, J. 1954. Angeborene 24-Stunden-Periodik

beim Kucken. Fleuger's Arch., 260: 170-176.HOFFMANN, K. 1957. Angeborene Tagesperiodik bei

Eidechsen. Naturwiss., 44: 359-360.WASSERMAN, L. 1959. Die Auslosung endogen-tages-

periodischer Vorgange bei Pflanzen durch einmaligeReize. Planta, 53: 647-669.

R a w s o n , K. 1956. Homing Behavior and EndogenousActivity Rhythms, Ph.D. Thesis. Harvard Uni-versity.

S w e e n e y , B., and J. W. HASTINGS. 1960. Cold SpringHarbor Symp. on Quant. Biol. Vol. 25.

BROWN, F. A., JR. 1959. Living clocks. Science, 1 3 0 :1535-1544.

PITTENDRIGH, C. S., and V. G. B RUCE. Unpublishedexperiments.

DISCUSSIONBROWN: I would like to caution about confusion of factswith hypotheses. Despite frequent claims, there is nologically defensible proof that the clocks underlyingcircadian rhythms possess a timing system, a self-sustaining oscillation, which is independent of a con-tinuous inflow of periodic information from the geo-physical environment. Such proof has been precludedby the fact that one can never establish through nega-tive evidence alone that nothing on the outside providesessential timing signals. One is not justified in makingthe assumption that circadian oscillations can be aconsequence only of being driven by fully independentoscillations of the same frequency. The very danger ofthis unwarranted assumption becomes especially evi-dent as we learn that there are no means for differ-entiating between frequency and phase changes. Wehave been duly impressed during the past two dayswith the readiness with which phases may be shifted inorganisms, and with the fact that free-running periodsare functions of the energy levels of the two principalphasing factors, light and temperature. The lastsuggests an obvious means by which the organismmight modulate, or alter, the frequency of any periodicenergy inflow through a means closely akin to ordinaryresponse to a stimulus. The conventional, and hithertohighly successful, approach to physiological problemshas been to exhaust first all proximal possibilities ofcause and effect before retreating to a position ofgreater autonomy of observed phenomena.

To prove the existence of an intrinsic timing system,we must take a positive approach and ferret out theactual biological timing mechanism. We must show thatits operational properties will account for fully auton-omous timing and yield rhythms with all the describedproperties of circadian ones. No one can doubt that aninherited clock-system is present in organisms. But ininsisting upon a self-timed, or fully autonomous, living

CIRCADIAN ORGANIZATION 183

clock, there always lurks the possibility that we arepursuing a ghost.P i t t e n d r i g h : Dr. Brown has made a variety of propo-sitions, only a few of which should be commented onhere; to treat them all as fully as I would like wouldtake more space than is warranted. I would onlyemphasize his last two sentences: apparently we all-Dr. Brown now included-agree “that an inheritedclock-system is present in organisms.” The remainingareas of dispute concern the issues in his last sentence.The question of the ghost is simple-either it is anaspect. of living organization, or an unknown geo-physical variable. My taste in ghosts suggests thelatter but, as scientist, I must agree that Dr. Brownmay prove right; and as scientist he will doubtlessagree he may prove wrong. We both will have somefun in any case.SIROHI: I would like to emphasize the idea of dawnand dusk which has been brought up by Pittendrighin this paper. Considered from a photoperiodic angle,dawn and dusk may be regarded as two points de-termining the length of a light period; the photoperiodbegins with dawn and ends with dusk. It may bementioned here that Pittendrigh did not give dueconsideration to the length of the photoperiod or lightintensity during photoperiod in his earlier postulationfor a biological clock model (Gatlinburg Symposium1957). He, however, made an attempt to interpretresults of Went, Hillman, and Highkin in the light ofhis model. More consideration of photoperiodic flower-ing response data, which are the basis of Bunning'shypothesis, should have been included in such interpre-tations.

Pittendrigh has indicated some very interestingresults which indicate that entrainment to differentphotoperiods (dawn and dusk) have a carry-over effectwhich can be observed under constant conditions.Recent work in photoperiodism with varying cycles(see Hamner’s paper) has shown that the effectivenessof a particular photoperiod in the flowering response isdependent on the length of the total cycle. I am awareof the fact that eclosion rhythm in Drosophila andflowering rhythms in plants have different character-istics. Severtheless, photoperiodic response to oddcycles strongly suggests the fact that in order to furtherelucidate the entrainment phenomenon of rhythms,studies pertaining to these cycles are very important.

I agree with Klotter that a biological clock modelshould be able to accommodate maximum biologicalresponses involved in rhythmic phenomena. Photo-periodism and flowering response, therefore, should begiven a due consideration in any such postulation.PITTENDRIGH: The interpretation of the Went, Hillman,and Highkin results given by Bruce and me in our 1959paper [5] was indeed made while we still thought dawnalone was involved in entraining the A-oscillation inDrosophila. Close inspection of our interpretation

there will reveal that it is independent of the way thelight cycle entrains the A-oscillation. Data on cycleswhose r is not 24 hours form an important basis for allstudies-those of Bruce and myself included-at-tempting to analyze entrainment phenomena.HARKER: There are two questions I should like to ask.First, is there any evidence that Drosophila pupae oreggs are sensitive to changes in light intensity? It ispossible that if they are not some of the transient peaksof eclosion are produced by animals which did notreceive the light signal.

Secondly, is there any evidence that the “dawn”effect in your photoperiod experiments is actually con-cerned with a periodic process? I have found that inPeriplaneta the onset of light is followed by an inhi-bition of activity about five hours later, regardless ofthe length of the light period. This inhibitory effect isnot repeated unless there is another “dawn,” that is, itis not periodic. It may affect the activity rhythm ofthe animal in a number of ways depending on the stageof the activity cycle on which it acts: as a result oneappears to get a different type of result with *differentlight periods. I suppose the onset of darkness mightaffect some animals in the same way; it is even possiblethat this might be so in Drosophila rather than that itshould be affected by the onset of light.P i t t e n d r i g h : The pupae, which are all that are rele-vant, are known to be sensitive to changes in lightintensity: one can entrain the eclosion rhythm with asine wave oscillation of light intensity that involves noabsolute darkness. However, I do not see the relevanceof this to the interpretation of transients you suggesthere. The transients (Fig. 1B) you refer to were pro-duced by discrete light signals imposed on DD rhythm;the question of change of intensity is surely not in-volved; it was infinite. In any case one should re-emphasize that the analysis of transients in Drosophilais not obscured by the fact the system is a population.At any rate the transient pattern for a Drosophilaculture is precisely that which is so beautifully shownby many single-animal systems like the hamster, etc.

There is more than one line of evidence that dawninteracts with dusk in affecting a periodic process inDrosophila. The strongest is partly covered by Fig. 16;the rhythm enters into transients on being released inDD, and the duration of these as well as the ultimatephase-shift (both properties of the oscillatory system)varies with the phase angle between dawn and dusk.The differences demand the conclusion that dusk wasnot the only parameter of the light signal involved inentrainment. This is also demanded by the fact thatthe phases of the freerunning rhythms do not form astraight line unless the photoperiod is greater than 12hours. aD e C o u r s e y : Dr. Pittendrigh has clearly indicated theplasticity of the freerunning activity rhythm frequency,and the importance of considering the effect of previous

184 PITTENDRIGH

conditions in obtaining response curves of organisms tosingle perturbations of light. In the study of endogenousrhythms with Glaucomys a similar slight plasticity ofthe freerunning rhythms of individuals has also beennoted. However, in the determination of the responsecurve of Glaucomys the small deviation of pointschecked at widely separated periods of time suggestthat the response curve had not changed even thoughthe animals had been subjected to various light regimesin a two-year study period.

Ball, HAROLD J.: The Effect of Visible SpectrumIrradiation in Oncopeltus Fasciatus (Dallas) andBlattela germanica (L.)

In the class Insecta mode of action of light hasbeen investigated in various ways. However, littleinformation concerning the effect of action spectra isavailable. This is unfortunate because light is one ofthe most important stimuli which helps to maintaininsects in a morphological form most likely to succeedat any given time.

The methods, materials, and apparatus used in theseexperiments can be found elsewhere [1].Tests with Blattella germancia (L.)--

B. germancia (L.) nymphs 10-13 days old wereexposed daily to “far red” irradiation (750 mp) for aperiod of 10 minutes. During the remainder of eachday the insects were maintained in holding cages underordinary room light conditions. A control group wasmaintained similarly under room light. Weeklyweighings were made to determine rate of growth.Results: During the first three weeks of treatmentweight differences between the “far red” treatedinsects and the controls were slight. By the end of the6th week of exposure all the treated insects were dead.Statistical analysis of the mean weights at the end ofthe fifth week revealed a significant difference in weightat the 5% level with the control insects weighing more.

Results of a 5 min. per day exposure to “far red” werecomparable. In another experiment, roaches maintainedunder (DD) conditions were significantly (5% level)heavier at the end of five weeks than were the controlinsects.Tests with Oncopeltus Fasciatus (Dallas)--

In general the results obtained using 0. Fasciatuswere similar to those obtained in the roach experiments.However, red light (630 mp) also produced deleteriousresults at exposures of 5, 10, and 20 min. per day.Control insects were significantly heavier (1% level)than either the “far red” or red-treated insects. Thered and “far red” light treatments inhibited thenymphal growth rate, caused a significant reduction inthe per cent of nymphs reaching imaginal form, andproduced adults which weighed less at maturation.Conclusions: The results of these tests indicate that thelonger wave lengths of light, red, and “far red” areespecially inhibitory to the species tested. The mecha-nism responsible for such growth inhibition is a subjectfor speculation. It has previously been suggested [2]that tissue of the central nervous system is reached byappreciable radiation from the visible spectrum underdaylight conditions. It has further been pointed out[1, 3] that the median neurosecretory cells may be theoverall controlling tissue for the type of growth inhibi-tion reported here.

REFERENCES1. BALL, HAROLD J. 1958. The effect of visible spectrum

irradiation on growth and development in severalspecies of insects. Jour. Econ. Ent., 61: 573-578.

2. Marcovitch, S. 1924. Migration of Aphididae and theappearance of sexual forms as affected by the rela-tive length of daily light exposure. Jour. Ag. Res.,67: 513-522.

3. THOMSEN, E. 1952. Functional significance of theneurosecretory brain cells and the corpus cardia-cum in the female blowfly, Calliphora erythro-cephala Meig. Jour. Exp. Biol., 29: 137-172.