3*5

Circadian organization of chitin in some insect skeletons

By A. C. NEVILLE

(From the Zoophysiological Laboratory B, Juliane Maries Vej, 36, Copenhagen, Denmark,and Department of Zoology, Parks Road, Oxford1)

With z plates

SummaryA circadian clock is shown to be involved in the control of macromolecular orientationof chitin by cells secreting and organizing insect endocuticle. Daily organization oflocust endocuticle into alternating lamellate and non-lamellate layers persists inconstant temperature (36° C) and constant darkness for at least a weeks; the free-running period is then about 23 h, so that after a number of days the circadian clockis 180° out of phase with the astronomical clock, with which it is normally phased.The rhythm is almost independent of temperature, with a Qlo of i'O4, as contrastedwith a Qlo of 2-0 for the actual rate of increase of endocuticular thickness. Locustepidermal cells differ in response to specific imposed environmental conditions accord-ing to their location in the integument. In some cells, constant low temperature un-couples chitin lamellogenesis from the circadian clock, provided that illumination(light or dark) is constant also: the result is continuously lamellate endocuticle. Inother cells constant light acts as an uncoupling factor, provided that temperature(high or low) is constant also: the result in this case is continuously non-lamellateendocuticle.

The circadian rhythm of chitin lamellogenesis persists in a cave cricket (Dolicho-poda linderi). A similar circadian lamellogenesis rhythm occurs in the endocuticleof nymphs and adults of the cockroach Periplaneta americana. A crossed-fibremultiple-ply endocuticle in the legs and wings of giant toe-biter water bugs (Belosto-matidae) also displays circadian organization, the chitin macromolecules in any onelayer lying in parallel fibres, at an angle of approximately 6o° to those in the nextlayer.

It is suggested that daily organization of the skeleton may be a general feature ofarthropods. Examples include the phenomena of timing of chitin lamellogenesis;chitin crossed-fibrillar organization; degree of fluorescence of the rubber-like proteinresilin; and mineralization of crayfish gastroliths.

IntroductionLOCUST solid endocuticle grows by daily increments, chronicling its progressby daily growth layers (Neville, 1963 a, b). The zonation is due to differencesin diurnal and nocturnal deposits, one pair of layers being deposited every24 h, and is the result of permanent changes in orientation of chitin crystal-lites occurring at the time of deposition. It is thus best detected in thepolarizing light microscope. In favourable conditions, adult desert locustsdeposit solid endocuticle day and night for 2 to 3 weeks after the final moult.When they are reared in laboratory cages with 12 h of day conditions (36° Cplus light, 75 to 150 foot candles from 10.00 to 22.00 hours) alternating with12 h of night conditions (260 C plus darkness from 22.00 to 10.00 hours),

1 Present address.

[Quart. J. micr. Sci., Vol. 106, pt. 4, pp. 315-25, 1965.]

316 Neville—Orcadian organization of insect skeletal chitin

locust adults produce a solid endocuticle in which each night layer has thechitin crystallites organized into several lamellae, whereas each day layer,although containing the same relative quantity of chitin, is not lamellate(Neville, 1965). In legs and wing veins, the chitin crystallites of the non-lamellate regions are oriented in fibrils along the axis of the respective organ.

Experiments with locust endocuticle (Neville, 1965), show that changingthe temperature and light conditions influences the type of cuticle being laiddown at the time. For instance, endocuticle grown during several days ofconstant-day conditions (36° C plus light) is in many regions of the skeletonnon-lamellate throughout. In other regions, which are always constant, thedaily pattern of organization into lamellate and non-lamellate layers is main-tained in spite of the imposed constant light and constant temperature condi-tions. Daily zonation is present in the endocuticles of material collected in thefield (Neville, 1963a) and even, as is reported below, in cave crickets growingin an almost constant habitat. This raises the question whether the dailylamellation rhythm is triggered by some exogenous rhythm other than lightor temperature, or whether it is driven endogenously by a circadian bio-logical clock. The results of experiments in which locusts were grown intemperature and light-controlled environments show that in solid endocuticlechitin lamellogenesis is under the timing control of a circadian rhythm,which is normally phased to daily environmental changes.

The question of exogenous versus endogenous rhythms has had specialprominence in the last few years in the general controversy involving theexistence or non-existence of circadian physiological clocks (summaries inHarker, 1964; Biinning, 1964). It has already been studied in an ever grow-ing variety of systems of living organisms. The present analysis does notseek merely to extend that list; rather it is intended to provide examples ofthe morphogenetical influence which a circadian clock may have uponchemical architecture at the ultrastructural level. In particular it will focusattention on the interactions of specific imposed environmental conditionswith the coupling which links the chitin orientation mechanism to the circa-dian clock in various cells.

Material and methodsThe work has been carried out mainly on the solid endocuticle of the hind

tibia of adult desert locusts, Schistocerca gregaria phasis gregaria Forskal(Orthoptera, Acrididae). Other insects used were adults of the cave cricket,Dolichopoda linden Duf. (Orthoptera, Raphidophoridae); the cockroach,Periplaneta americana (Dictyoptera, Blattidae); giant toe-biter water bugs,Hydrocyrius colutnbiae, Lethocerus cordofanus, and Limnogeton fieberi (Hemip-tera, Belostomatidae); and large beetles Oryctes and Heliocopris (Coleoptera,Scarabaeidae).

Material was frozen-sectioned either fresh or after fixation at 4° C for 48 hin neutral 4% formaldehyde, and examined by polarization microscopy inwater or glycerol.

Neville—Orcadian organization of insect skeletal chitin 317

Environmental changes in temperature and light influence the morpho-genesis of growing cuticle. Locusts were therefore reared in specially con-structed cages, consisting of refrigerators with ovens built inside them. Inthis way temperature could be changed rapidly in either direction. Aircirculation was provided by a fan, and illumination by a cold circular fluores-cent tube, so that temperature and illumination could be independentlyvaried, and preset to follow desired rhythms on electrical time switches.

All results were checked in at least 4 experimental animals and were con-stant in all cases for a specified treatment.

ResultsEvidence for a circadian clock influencing chitin orientation

The evidence for a circadian physiological clock influencing the timing ofchitin lamellogenesis in locusts is as follows. When locust adults are allowedto deposit endocuticle in constant warm night conditions (36° C plus dark-ness), throughout the course of several astronomical days the resulting depositsstill show a daily alternation of lamellate and non-lamellate layers. If lamel-logenesis is then stopped in certain regions of the cuticle (always constant)by 60 h in constant light (75 to 150 foot candles) at 36° C, a wide non-lamel-late zone appears in the sequence of deposits. If the locust is then replaced inconstant dark at 360 C the daily lamellation rhythm restarts (fig. 1). Therhythm is therefore independent of external clues involving light and tempera-ture, implying that chitin lamellogenesis is a circadian phenomenon. It canpersist in constant darkness at 3 6° C for at least 2 weeks.

As with all suspected circadian rhythms, the possibility remained that thedaily pattern could still be triggered by some unknown rhythmical eventoccurring at the same time on each astronomical day. True circadian rhythmsrun free in constant light (or dark) and constant temperature, but theyusually do so with a period which differs slightly from a precise 24 h; this iswhy the term 'circadian' (latin circa, about; dies, a day) was introduced(Halberg, 1959). If it could be shown that the free-running period of chitinlamellogenesis in constant darkness at 360 C differed from 24 h, this wouldexclude the possibility of exogenous triggering by an external factor which isphase-fixed in the astronomical 24-hour period.

In locusts adult emergence marks the beginning of the deposition of adultendocuticle. Accordingly, freshly emerged adult locusts were placed inconstant darkness at 360 C at 12.00 hours on day O of the experiment. Legsexamined at 16.00 hours on day 4 (i.e. 4 astronomical nights later) showed notonly 4 completed lamellate layers but also the beginnings of a fifth one. Ithas already been shown that lamellate layers are normally grown in nightconditions when locusts are reared in an alternating day/night environment(Neville, 1965). This therefore indicated that the free-running period ofchitin lamellogenesis in locusts in constant darkness at 360 C is less than24 h, since the suspected circadian physiological clock controlling the timing

318 Neville—Orcadian organization of insect skeletal chitin

of lamellogenesis was already gaining on the astronomical clock. Further legsamples at 12.00 hours on day 7 showed the non-lamellate deposit correspond-ing to that astronomical light period to be almost complete. More hind tibiaesampled at 17.00 hours on day 10 after the animals had experienced 10astronomical nights showed 11 completed lamellate layers in the endocuticle,indicating a gain of approximately 1 h per 24 h on the astronomical clock. Theresult cannot be stated with greater accuracy since the absolute duration ofdeposition of a lamellate layer is not known. The locusts at this stage were180° out of phase with the astronomical clock, since they were organizinglamellate cuticle in the astronomical day hours and non-lamellate cuticle inthe night hours.

Thus chitin lamellogenesis is controlled by a circadian physiological clock,which has a free-running period of approximately 23 h in constant darknessat 360 C.

Temperature independence of the clock

A characteristic of circadian rhythms is their relative independence oftemperature. Thus the free-running experiment was repeated with freshlyemerged adults reared for 14 days and nights in constant darkness at 260 C.Resulting sections of hind tibiae showed regions in which daily lamellationhad persisted; they contained 14 lamellate and 14 non-lamellate layers,indicating a free-running period indistinguishable from 24 h at 260 C.Using the reciprocal of the period as indicating the rate at a given tempera-ture, the temperature coefficient Q10 of the rhythm between 260 C and 36° Cis 24 h/23 h or approximately 1-04. The rhythm is therefore nearly inde-pendent of temperature.

An estimate of the effect of temperature upon quantity of endocuticle de-posited was made by measuring endocuticular thickness from daily growthlayers at a well-defined locus in sections of hind tibiae grown in constantdarkness at various temperatures (220 C, 260 C, 320 C, and 36° C). Theresults gave a Qlo for thickness increase of approximately 2-0 at temperaturesbetween 220 C and 360 C.

FIGS. 1 to 4. Transverse sections of locust hind tibiae photographed between crossed polaroidsand all oriented for maximum retardation by lamellae. Figs, i and 3 with a first order redquartz plate parallel to the lamellae making them appear blue (i.e. black instead of white inthe photographs).

FIG. 1. Locust adult grown after emergence in 6 days of continuous darkness at 360 C (a);then with circadian lamellogenesis stopped by 60 h in constant light (75 to 150 foot candles)at 360 C (b). Circadian lamellogenesis restarts on replacement in constant darkness at 360 Cfor a further 48 h (c).

FIG. 2. Non-lamellate endocuticle grown in continuous light (75 to 150 foot candles) at36° C.

FIG. 3. Persistence of circadian lamellogenesis in endocuticle of a locust reared in con-tinuous light (75 to 150 foot candles) with a temperature rhythm.

FIG. 4. Persistence of circadian lamellogenesis in endocuticle of a locust reared in constanttemperature (36° C) with a light/dark rhythm.

FIGS. 1-4

A. C. NEVILLE

Neville—Orcadian organization of insect skeletal chitin 319

Orcadian organization of skeletons in cave crickets

If the endocuticular lamellation rhythm is circadian, it might be presenteven in cave crickets living in a constant environment. Accordingly, 6specimens of Dolichopoda linden Duf. (Orthoptera, Raphidophoridae) werecollected from a cave in the Eastern Pyrenees. The habitat was one of con-stant darkness at 130 C. One hind leg was removed from each specimen andthe corresponding legs left to grow on the animals for a further 24 or 48 h.When these legs were subsequently sectioned, daily growth layers werefound to be present, and the more mature legs contained one or two extrapairs of lamellate and non-lamellate layers as expected. The circadianrhythm can therefore persist in cave crickets.

Circadian organization of skeletons in giant water bugs and cockroaches

Rearing experiments have confirmed the presence of circadian zonation inthe solid endocuticle of the legs of nymphs and adults of a cockroach, Peri-planeta americana (Dictyoptera) and adults of a giant toe-biter water bug,Hydrocyrius columbiae (Hemiptera, Belostomatidae).

In cockroaches, the daily growth layering is due to layers with no organizedlamellae alternating with layers each comprising several lamellae. The mor-phology revealed by the polarizing light microscope is therefore comparableto that of locusts and grasshoppers, in which one lamellate plus one non-lamellate layer represents 24 h of skeletal growth.

The skeleton of the giant toe-biter water bugs (Belostomatidae) is, how-ever, organized differently, being specialized for greater mechanical strength.The endocuticle is again grown as several layers beginning after adultemergence. In any one layer the chitin crystallites are oriented precisely inrecognizable fibre tracts (balken) and all of the fibres in any one layer areoriented in parallel. The direction of the fibres changes by about 6o° fromlayer to layer, and each layer represents 24 h of growth. Crossed-fibre struc-tures of this sort have been found in the genera Hydrocyrius, Lethocerus, andLimnogeton, and represent a multi-ply strengthening device. Similar crossed-fibre structures are seen in some large beetles, e.g., Oryctes and Heliocopris,but it is not yet known whether these also represent daily growth layers.The crossed fibrillar cuticle in honey bee antennae (Richards, 1952) is notcoupled to a circadian mechanism. In that case the endocuticle is grown in asingle day and the 15 to 20 fibril-direction changes all occur in the course ofthe one day.

In the case of Periplaneta and Hydrocyrius, the circadian organization ofthe skeleton could not be uncoupled from the clock by either constant-day orconstant-night conditions. Locusts are thus convenient experimental animalssince chitin lamellogenesis can be uncoupled from the circadian clock in somecells by constant-day conditions, whereas it remains coupled to the free-running clock in constant-night conditions. Factors involved in this un-coupling have therefore been further investigated in locusts.

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Neville—Orcadian organization of insect skeletal chitin 321

Uncoupling morphogenesis from the clock

For ease of reference, the experimental results of the following section aresummarized in table 1.

In many regions of the locust skeleton cuticle grown in constant light(75 to 150 foot candles) at constant temperature (360 C) is non-lamellate(fig. 2; see also Neville, 1965). Was this inhibition of lamellogenesis due toconstant light or to constant temperature or to a combined action of both ?To answer this, locusts were reared in an environment in which one of thesefactors was rhythmically varied (12 h of light, 75 to 150 foot candles alternat-ing with 12 h of darkness, or 12 h at 36° C alternating with 12 h at 260 C),whilst the other factor was in each case maintained constant. Both in con-tinuous light with a temperature rhythm (fig. 3) and in constant temperaturewith a light/dark rhythm (fig. 4), lamellae were still formed according to adaily rhythm. Thus in locust solid endocuticle it is necessary to maintainboth temperature and light constant together in order to prevent the epidermalcells from organizing chitin lamellae.

When locusts are reared in conditions of 24 h of light (75 to 150 footcandles) at 360 C alternating with 24 h of darkness at 260 C, over most of thehind tibia the 48-hour rhythm is followed, since the prolonged days overridethe effect of half of the circadian nights (figs. 5 and 6). Thus lamellogenesisonly occurs on every second astronomical night, when the imposed nightcoincides with the circadian clock night (fig. 9, c). The cuticle at the proximalend of the hind tibia (fig. 9, A) is strengthened by thickening on the side awayfrom the spines (fig. 9, B). In this region, the epidermal cells continue toorganize lamellae according to the circadian clock (fig. 7), so that lamellaeare formed on every astronomical night. Tracing lamellae around a transversesection of the proximal end of a hind tibia from the spine-bearing side to thenon-spiny side showed that the extra lamellate layers interdigitated with theones which were organized according to the superimposed 48-hour rhythm(figs. 7 and 8). Thus conditions which uncouple the morphogeneticalmechanism from the circadian clock in some cells need not necessarily do soin other cells. It is therefore important to define the locus of the cells whoseresponse to any given set of conditions is being investigated.

A constant temperature of 22° C is just adequate to allow locust cuticlesecretion to proceed, although at a much reduced rate. When locusts arereared in constant cold day conditions (220 C or 260 C plus light, 75 to 150foot candles) the resulting hind tibial endocuticle differs from region toregion in its differentiation. The chitin orientation mechanism is uncoupledfrom the circadian clock in all regions, but in some regions this producesabsence of non-lamellate layers whereas in others it causes absence of lamel-late layers. Thus, the thickened proximal region of the hind tibial endocuticle(fig. 9, A, B) is then continuously lamellate throughout, whereas the endo-cuticle in the rest of the hind tibia is non-lamellate throughout.

When locusts are reared in constant cold night conditions (260 C plus

322 Neville—Circadian organization of insect skeletal chitin

darkness), the endocuticle of the thickened side of the proximal region of thehind tibia is once again continuously lamellate throughout, but that of therest of the tibia shows circadial organization with lamellate and non-lamellatelayers in alternation. Thus in either constant light or darkness, constant lowtemperature appears to be the main factor leading to the uncoupling of thecells producing the thickened proximal region of the hind tibia from thecircadian clock. In the other cells of the hind tibia, constant temperature(360 C or 260 C) plus darkness does not uncouple the chitin orientationmechanism from the circadian clock. On the other hand, constant tempera-ture (220 C, 260 C, or 360 C) plus light (75 to 150 foot candles) does lead touncoupling in these cells. Thus light appears to be the main uncouplingfactor for these cells.

Two further epidermal cell types are found in the locust wing. In alternat-ing days and nights, the cells which produce the radius plus media wingvein organize lamellate and non-lamellate layers in alternation. They areuncoupled from the circadian clock by any combination of constant conditions(e.g. cold night, cold day, warm night, warm day), when they deposit con-tinuously non-lamellate endocuticle. The cells producing the wing cuticlesituated between the radius and media and the cubital wing veins are per-manently uncoupled from the clock, producing continuously lamellateendocuticle even in alternating days and nights.

DiscussionWith the almost universal acceptance of the reality of biological clocks,

more and more indicator processes are being found to be coupled to them(examples in Harker, 1964; Biinning, 1964). It is therefore not too surprisingto find that the daily growth layers in some insect skeletons are also coupledto a circadian clock, and that this coupling can be influenced by variations inenvironmental conditions. In the epidermal cells producing the thickenedside of the proximal region of locust hind tibial endocuticle, constant lowtemperature uncouples chitin lamellogenesis from the circadian clock, pro-vided that illumination (light or darkness) is constant also. When thus

FIGS. 5 to 8. Transverse sections from different regions of the two hind tibiae of the samelocust photographed between crossed polaroids and all oriented for maximum retardation bylamellae. Locust reared in a 48-hour rhythm with 24-hour days and nights in alternation.

FIG. 5. Thickened side of tibia between regions 2 to 10 in fig. 9 A, B. Cells follow super-imposed 48-hour rhythm.

FIG. 6. Spiny side of tibia between regions 2 to 10 in fig. 9 A, B.FIG. 7. Thickened side of tibia between regions o to 2 in fig. 9 A, B.FIG. 8. Spiny side of tibia between regions 2 to 10 in fig. 9 A, B. In the regions shown

in figs, s, 6, and 8, the cells follow the superimposed 48-hour rhythm. In fig. 7 the cells remaincoupled to the circadian clock, so that the lamellate layers (34 h apart) interdigitate with thelamellate layers in fig. 8 (48 h apart) as shown.Figs, s and 6 are from leg removed after last 24 h of day conditions. Figs. 7 and 8 are from

other leg left on for a further 24 h of night conditions, and ending in an extra lamellate layer.Exocuticle is strongly birefringent but with individual lamellae too closely spaced to be

resolved.

FIGS. 5-8

A. C. NEVILLE

Neville—Circadian organization of insect skeletal chitin 323

uncoupled, these cells produce continuously lamellate endocuticle. By con-trast, in the epidermal cells producing the rest of the locust hind tibial

thickened non-spine side

spine sideL cells secreting thickened cuticle of

non-spine-bearing side of tibia« • * cells secreting spine-bearing side

of tibia

circadian clock

cuticle of spine sideof tibia

cuticle of thick-ened side of tibia

FIG. 9. A. Diagram of a locust hind tibia to show the locality of the proximal region (divisionso to 2). B. An outline of a transverse section through the proximal region of the hind tibiashown in A. Exocuticle is black: stippled endocuticle is that in which continuous lamellationcan be produced by growth in low temperature at constant illumination. The non-stippledendocuticle is that in which absence of lamellation can be produced by growth in high light-intensity at constant temperature, c. Diagram showing different results produced by differentcells by growing locusts in a 24-hour day/24-hour night rhythm. Dark areas, circadian clocknights (i) or superimposed 24-hour nights (ii). White areas, circadian clock days (i) or super-imposed 24-hour days (ii). Hatched areas, lamellate zones in resulting endocuticle. Dotted

areas, non-lamellate deposits. Total length of time axis 96 h.

endocuticle, constant light uncouples chitin lamellogenesis from the circadianclock, provided that temperature (high or low) is constant also. When thusuncoupled, these cells produce continuously non-lamellate endocuticle. Thepattern which emerges is that the response to the interaction between environ-

324 Neville—Orcadian organization of insect skeletal chitin

mental factors and the circadian clock is finally determined in the individualepidermal cell.

In plants several aspects of morphogenesis may be controlled by a circadianclock operating upon a photoperiodic system (Biinning, i960). However,Lees (i960) thinks that an interval timer may be adequate to explain photo-periodic mechanisms in arthropods. Several examples of the effect of photo-periodism on insect morphogenesis at the tissue level are well established(e.g. onset of diapause, Lees, 1956; diapause development, Beck and Alex-ander, 1964; polymorphic differentiation in aphids, Lees, 1959). Also,interference with the circadian system in cockroaches can lead to tumourformation (Harker, 1958). The results presented above show that the cellularcontrol of the orientation of macromolecules at the ultrastructural level, mayalso be linked to a circadian clock; this could have possible implications inresearch on control of orientation of cellulose macromolecules in plant cellwalls. Indeed Preston (1952) has already speculated on the possible correla-tion between the number of orientation changes in the crossed-fibrillar wallorganization in conifer tracheids with the number of days of growth. Anestablished coupling between a biological clock (period 18-5 h) and ultrastruc-ture occurs in the daily layering of starch grains in potato and tobacco, butnot in wheat (Buttrose, 1962).

In most cells of the locust hind tibia continuous light at constant tempera-ture uncouples chitin lamellogenesis from the clock, whereas the rhythm isstill expressed in continuous darkness at constant temperature for at leasttwo weeks. This is consistent with the fact that 'periodicity fade-out' by un-coupling from the physiological clock usually occurs more quickly in constantlight than in constant darkness both in animals and in plants (Biinning, 1964).In locust cuticle constant light intensity could only produce uncoupling ofchitin orientation from the clock provided that temperature was constant aswell. A similar interaction of constant light with constant temperature acts todamage the leaves of tomato plants (Hillman, 1956).

It is significant that the period of the rhythm remains almost independentof temperature whereas the amplitude of growth increases with temperature.The actual temperature coefficient value (Qlo) recorded for the orientationprocess (1-04) is similar to those of the circadian clocks of other organisms,including other insects (Sweeney and Hastings, i960). Since under con-trolled conditions in the laboratory in alternating days and nights the depositsremain in phase with the imposed conditions, the circadian clock must beresynchronized to normal day/night cycles.

The fact that cave crickets living in permanent darkness at constanttemperature show daily growth layers in the skeleton might at first sightsuggest that the rhythm has persisted for countless generations. Dolichopodais, however, a carnivore living fairly near to the cave mouth and makingnocturnal excursions out of the cave for food at night (Vandel, 1964). It maytherefore receive weak phase-setting signals from moonlight. The periodcould not be deduced in the present experiments but might be expected to

Neville—Circadian organization of insect skeletal chitin 325

differ appreciably from 24 h. Park, Roberts, and Harris (1941) found this tobe true of the period of activity in the cave crayfish Cambarus pellucidus.

Daily organization of the skeleton may possibly be a general feature ofarthropods. Thus chitin lamellogenesis in locusts and cockroaches, chitincrossed-fibrillar organization in Belostomatids, variation in fluorescence ofresilin (Neville, 1963c) and mineralization in a crayfish gastrolith (Scuda-more, 1947) are all known to be daily phenomena. Apart from the crossed-ply cuticles, the functions of the other kinds of daily growth layers remainobscure; perhaps they are merely a reflection of a rhythmical metabolism.

It is a pleasure to thank Professor T. Weis-Fogh for providing encouragement andfacilities in his laboratory. I am grateful to Professor Weis-Fogh and ProfessorJ. W. S. Pringle, F.R.S. for comments on the manuscript; to the Agricultural Re-search Council for financial assistance during the tenure of a Research Officership; toSt. John's College, Oxford, for the award of a Fereday Fellowship; and to the Anti-Locust Research Centre, London, for supplies of locusts.

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