J. Exp. Biol. (1964), 41, 119-133 Iig

With 3 text-figures

Printed in Great Britain

THE LOCATION OF THE PHOTOPERIODIC RECEPTORSIN THE APHID MEGOURA VICIAE BUCKTON

BY A. D. LEES

Agricultural Research Council Unitof Insect Physiology, Cambridge

{Received 31 July 1963)

Although the photoperiodic reactions of insects and mites have been studied quiteextensively (Lees, 1955; de Wilde, 1962), the site of the photoreceptors is still unknown.The light pathway does not seem to involve the eyes. Thus Tanaka (1950) observedthat the photoperiodic control of diapause in the silkworm Antheraea pernyi was notaffected by cauterizing the lateral ocelli of the 4th-instar larva—a treatment thatresulted in the complete disappearance of these organs in the following instar.De Wilde, Duintjer & Mook (1959) have shown that adult Colorado beetles (Leptino-tarsa decemlineata) respond normally to day-length after the compound eyes have beencovered with black paint or destroyed with a cautery.

Less progress has been made in identifying the regions of light sensitivity. Geispits(1957) was able to fit an opaque hood over the head of a Dendrolimus larva which wasotherwise- continuously illuminated. With this covering in place for 12 hr. daily allthe insects subsequently entered diapause. But none did so when only the body wasshielded from light, showing that the photoreceptors were located on the head.Since the wavelength sensitivity of the visual and photoperiodic response showed somecorrespondence Geispits was led to the further conclusion that the eyes were in factthe photoperiodic receptors. But in view of the preceding evidence this conclusionseems questionable.

The earlier report of Belov (1951: cited in Danilyevsky, 1961) that the photo-periodic response in Antheraea pernyi is disturbed when certain 'retort-shaped'(chaetoid) bristles on the body are damaged is not confirmed by the recent work ofShakhbazov (1961). This investigator has observed that photoperiodic sensitivity ofthe 4th and 5th larval stages extends over into the pupa. And attention is drawnto the presence in the pupa of a clear area of transparent cuticle overlying the brain.Shakhbazov is inclined to regard this ' window' as the main light pathway, since, whenit is painted over, the effect is equivalent to an exposure to darkness.

Although 'negative* techniques, involving the elimination or silhouetting of dif-ferent body regions, may sometimes prove useful in locating the organ of perception,complementary methods of photostimulation are also needed. Yet it is by no meanseasy to provide an accurately located beam of light when the insect is actively feeding andmoving. Moreover, it is often impossible to surmount this difficulty by temporarilyimmobilizing the insect, since the effects of starvation sometimes mask the photo-periodic response (as in Leptinotarsa). Fortunately, the vetch aphid Megoura hasproved more amenable in this respect and satisfactory methods of illumination can bedevised. Some preliminary results have been published elsewhere (Lees, i960).

120 A. D . LEES

PRINCIPLE OF METHOD

In testing for photoperiodic sensitivity it is necessary to expose the selected regionand the rest of the body surface to different and opposed photoperiods. The Megouraclone used in the present experiments is known to have a critical daily photoperiodof approximately 14 hr. 55 min. at iS°C. (Lees, 1959, 1963). Parent insects rearedfrom the 1st instar on photoperiods that are within 15 min. of the critical lengthusually give birth to alternating sequences of egg-laying oviparae and parthenogeneticvirginoparae. But with shorter photoperiods of 14 or 12 hr. only oviparous daughtersare produced, and with a long 16 hr. photoperiod, only virginoparae (Lees, 1963).It is also known that the maternal photoperiodic mechanism controlling the fate ofthe embryos developing in the abdomen of the mother can readily be switched overby reversing the photoperiod during the larval period or early adult life of themother.

It should therefore be possible to demonstrate the approximate site of the photo-periodic receptors by exposing 'short-day' aphids to a supplementary period oflocalized illumination, provided the total duration of the two light phases (general andlocal) is made to exceed the critical photoperiod. If the area explored is light-insen-sitive the aphid will continue to produce oviparae throughout its reproductive life.On the other hand, if the light receptors are involved, we should expect to observe areversal in the progeny sequence, a series of oviparae being followed by a series ofvirginoparae.

METHODS OF ILLUMINATION

General illumination was provided by the fluorescent ' daylight' tubes with whichthe rearing cabinets were equipped. The light intensity was about 50 ft-candles atplant level. The sources used for localized illumination were lens-end torch bulbs eachof which was mounted separately in a brass light-proof housing (Fig. 1A-C). Shortlengths of metal capillary, prepared from hypodermic needles of different gauge(internal diameter 1-5, 0-5 and 0-27 mm.), and provided with a threaded base, couldbe screwed into bulb housing so that the pre-focused beam was directed down thelength of the capillary.

The aphids were attached to the free ends of the capillaries either by suction or withthe aid of an adhesive. The first method was particularly convenient as the insectscould be returned to the plant without further handling by means of the simplearrangement shown in Fig. 1 A. Compressed air was blown through a filter pump,the side arm of which was provided with a 2-way, piston-operated tap, controlled by arelay and time clock (A). In the alternative position the vacuum was cut off and theline carried compressed air. A high-volume system of this kind permitted 10 or soilluminators to be attached in series and an adequate suction maintained even whenmany of the tubes were temporarily open. Reversal of the air flow served to prevent theinsect, with its slightly waxy cuticle, from adhering to its support after the vacuumwas cut off by clock A. Time clock and relay B operated a further tap which cut offthe supply of compressed air some minutes later.

The device used for collecting the aphids is shown in Fig. 1B. At the end of thesupplementary light period the insects were thrown against a sloping glass plate andwere deflected downwards towards the host plant (a germinating bean). The aphids

Location of photoperiodic receptors in an aphid 121usually discovered the plant within an hour or so but a 40 V. electrical barrier was in-cluded to prevent their possible escape.

As the suction developed across the smaller (0-27 mm.) diameter capillaries wasinsufficient to support the adult insect, an adhesive was required. Polyvinyl alcohol,which is water-soluble yet adheres adequately to the cuticle, was satisfactory for thispurpose. After placing a droplet of the pasty solution in position under the binocularmicroscope, the tip of the illuminator tube was pressed firmly against the insect andheld until the adhesive had set. After the period of supplementary illumination theadhesive was dissolved away by applying a droplet of water, and the aphid was returnedto the host plant. This operation was carried out with the aid of a red safelight.

With finer bore capillaries it was found impossible to keep the lumen pricked outand unobstructed. But fine plastic filaments, drawn from a viscous solution of poly-styrene in benzene and attached to the insect with polyvinyl alcohol glue, proved to

Time clockA

Fig. 1. A, plan of illuminators and suction device; B, arrangement used for collecting aphidsafter illumination; C, method of mounting light-conducting filaments in the illuminator.

122 A. D . LEES

be suitable for conducting light to the required part. Threads with a terminal diameteras small as 0-04-0-05 mm. were employed successfully. These were mounted, forsupport and protection, inside a small bore (0-27 mm.) metal tube. The wider,tapering end of the thread was in contact with the light bulb and the narrower free•end, which projected slightly beyond the metal capillary, was cemented in positionwith black paint (Fig. 1C). Although the light-conducting properties of the filamentare somewhat impaired by this procedure, which alters the refractive index of theexternal medium, the loss of light is immaterial because of the low threshold of thereceptor. Indeed it may be a positive advantage since high intensity illuminationwith its attendant light scattering prevents accurate localization of the beam (see p. 128).

Since the polystyrene threads are irregular in shape and differ in their light-conducting capacity, each selected filament was used to explore a number of differentareas, employing a succession of insects. To ensure that the light source remained asconstant as possible, the d.c. voltage was stabilized by including a continuouslytrickle-charged accumulator in the circuit; and the lamp bulbs (rated at 0-25 A. and2-2 V.) were under-run at i-8 V. to reduce ageing effects.

Other methods of illumination which avoid the troublesome procedure of repeatedlyattaching and detaching the insects from the illuminators were also tested. Althoughthese proved unsuccessful in this insect, they may be mentioned briefly.

One possibility is to attach particles of fluorescent substances to the body of theinsect which is then irradiated with ultra-violet. The principle of the method is basedon the assumption that the receptors will show a pronounced differential sensitivityto the exciting and emitted radiation or even that ultra-violet is photoperiodicallyinactive. Some experiments were made along these lines with anthracene crystalswhich fluoresce a brilliant blue. Unfortunately the range of wavelengths whichelicited the response proved to be unexpectedly wide and included not only the blueregion but much of the ultra-violet as well. The 3 65 m/i region (high-pressure Hg vapourlamp; Wratten 18A ultraviolet filter) was highly active; but, in addition, quite lowenergies of monochromatic ultraviolet of wavelength 254 m/i were also effective. Thesource in this instance was a low-pressure Hg vapour lamp used in conjunctionwith a liquid-gas filter of CoSO4-NiS04 and chlorine and an ultraviolet Woods glassfilter (Chance OX 7) (Bowen, 1946; Markham & Smith, 1949).

A radioactive luminous paint incorporated in a polyvinyl alcohol medium was alsotried. This material had a surface luminance of approximately 0-025 ft.-candles for alayer 0-5 mm. in thickness (10-15 times the luminance of a modern wrist-watch dial).But no response was obtained when a layer of this thickness was applied to the eyesand dorsum of the head. And it seems that either the intensity was inadequate or thatthe wavelength of the emitted light (predominantly yellow) was too long.

An attempt was also made to demonstrate light sensitivity by covering different areaswith various opaque materials. A commercial 'blackboard' paint, incorporating alight paraffin solvent, proved to be sufficiently non-penetrative to use on the head.Aphids were reared in short days, the relevant parts being painted over immediatelyafter the final moult. On the same day the general photoperiod (intensity 50 ft.-candles) was extended to 16 hr. A control series of untreated aphids placed in per-manent darkness after the final moult continued to give birth to oviparae until theend of parturition. Fifty aphids with either the compound eyes, the ventral or the dor-

Location of photoperiodic receptors in an aphid 123

sal surfaces of the head, or the entire head capsule covered, still responded withoutexception to the long photoperiod by switching over to virginopara-production.However, it was not possible to obliterate certain movable parts such as the basalarticulation of the rostrum, without preventing the insect from feeding; and it may wellbe that these areas still admitted light.

Certain parts, notably the compound eyes, have been destroyed with a R.F. cautery.The results are described below.

MATERIAL AND PROCEDURE

Although the photoperiodic mechanism can be reversed at any time during thelarval development of the mother (Lees, 1959), it is more convenient to work withthe adult insect, both for reasons of size and in order to avoid the difficulty of defining aspecific area in a moulting and growing insect. But the previous work has shown thatalthough it is possible to reverse the effects of a previous short-day treatment byplacing the adult on long days, the 'switch-over point' comes very near the end of theprogeny sequence. This is illustrated in Table 1 where the progenies of a number ofaphids are averaged. When the short-day treatment began during the prenatal period(this, of course, implies that the grandparent was so treated), and was terminated onthe day of the final moult, a mean number of 79 oviparae per female was producedbefore the switch-over, and 8 individuals failed to show any reversal at all (Table 1 A).Since any kind of manipulation inevitably leads to some reduction in fecundity,this particular procedure is of little practical use.

Table 1. The effect of different light cycles and of starvationon the reversal of the photoperiodic response

(S, short (12 hr.) day; L, long (16 hr.) day; Ov., oviparae; V., virginoparae.)

Mean length of successive

Expt.

A

B

C

D

E

Treatment of parents

Light cycle

Embryo, SLarva, SAdult, L J

Embryo, LLarva, SAdult, L

Embryo, LLarva, SAdult:

Days 1-8, LDays 8-15, S

As in C

As in C

Nutrition

Normal

Normal

Normal

Starved 8 hr. dailyfor 8 days

Starved 16 hr. dailyfor 4 days

No. ofparentstested

18

17

2O

17

2O

No.respon-

ding

IO

17

2 0

17

9

progeny sequences

(i) Or.

79

45

45

42

59

(2) V. (3) C

5 —

44 —

27 5

19 6

6 6

However, it has been found that the switch-over from oviparae to virginoparae isgreatly advanced if the parent is exposed to a double reversal of photoperiod, thefirst being effected at birth, and the second, as previously, immediately after the final

124 A. D . LEES

moult (Lees, 1963). The precise regime is as follows: the grandparent is first exposedto long (16 hr.) days at I5°C. for 4 days before the birth of the parent; at birth the latteris placed on short (12 hr.) days but is returned to the long-day regime immediatelyafter the final moult. Seventeen parents placed on this schedule produced an initialaverage sequence of 45 oviparae followed by a final sequence of 44 virginoparae(Table 1B). The possible reasons for the different effects of single and double reversals-are discussed elsewhere (Lees, 1963); here it will suffice to note that the photoperiodicmechanism begins to function some days (at least two) before birth; and it is suggestedthat the activation of the mechanism (including its presumed endocrine component)by long-day stimulation is accelerated by a previous partial activation.

In order to reduce the handling to a minimum it is also desirable to know the mini-mum number of long-day cycles that will effectively reverse the photoperiodicresponse. This information is given in Table 2. Whereas 2 cycles never resulted inreversal, 4 cycles were effective in about one-third of the insects tested. However, thereaction was not invariably positive until eight cycles were given. The averagedprogeny sequence of 20 aphids exposed to 8 long-day cycles immediately after thefinal moult are shown in Table 1C. The length of the initial sequence of oviparae,namely 45, did not differ from the sequence obtained when the whole of the adult lifewas spent in long days. Table 2 also illustrates a further point of some incidentalinterest, namely that the length of the initial sequence of oviparae appears to be in-dependent of the number of long-day cycles, provided, of course, a positive responseis forthcoming.

Table 2. The relationship between the duration of long-day stimulationand the reversal of the photoperiodic response

No. of long-day (16 hr.)

cycles

2

468

1 0

No. ofaphids

2 0

48Si372 0

0//oresponding

93 i84

1 0 0

1 0 0

Mean no. ofoviparae in initial

sequence (responding??only)

—4240

4547

Since in the treatment outlined above the insects were returned to a 12 hr. photo-period after the 8th long day, there was often evidence of a second switch-over (fromvirginopara- to ovipara-production) in the progeny sequence (Table 1C). This is of nosignificance in the present context.

As the aphids must be removed from the plants during the period of supplementaryillumination, it is necessary to determine whether the photoperiodic response isinfluenced by temporary starvation. Accordingly, apterae were placed in empty speci-men tubes for a daily 8 hr. period for 8 days after the final moult. The ease of reversalof the photoperiodic mechanism in these insects was unimpaired, for a series of 17females transferred from short days to long days at the final moult produced an initialseries of 42 oviparae before switch-over (Table iD). However, the response wasdefinitely inhibited in a further group that was starved for 16 hr. daily for 4 days (nearthe limits of survival); only 9 out of 20 aphids responded, and in these the point of

Location of photoperiodic receptors in an aphid 125

reversal was postponed, occurring after an average of 59 oviparae had been born>(Table 1E). The fecundity was also reduced.

Since lengthy periods of starvation have undesirable consequences, an initial short-day photoperiod of 14 hr. was chosen (see Lees, 1963), and the period of supplemen-tary illumination reduced to 2 hr.

When the mode of attachment was by adhesives, the aphids were removed from theilluminators in deep red light. This was provided by a 15 W. tungsten filament lampand a Chance OR 2 filter with a steep cut-off between 630 and 590 m/i. Controlexperiments in which 20 aphids were exposed for the whole of the 2 hr. supplementarylight period to the red light showed that the wavelengths transmitted by this filterwere wholly ineffective in reversing the photoperiodic response.

The general procedure, based on these results, may be summarized as follows:apterae from the stock cultures (maintained in a long (16 hr.) photoperiod at i5°C.)were allowed to deposit larvae overnight on young bean plants (Viciafaba) growing in-short (12 hr.) days. These grandparental insects were then discarded. The developingparents continued to experience short-day conditions until the day of the final moult.The general photoperiod was then extended to 14 hr. (still a short day). At the end ofthe general light period the insects were attached to the illuminators for a supplementary2 hr. period of localized illumination, making a total daily light period of 16 hr. Theywere then returned to the plants. This procedure was repeated for 8 days, every effortbeing made to ensure that the illuminators were applied to the same site on eachoccasion. The experimental insects were finally returned to a 12 hr. photoperiod andtheir progeny were collected and classified. A positive response was indicated by aswitch from ovipara- to virginopara-production at about the 45th birth. Any indivi-dual that failed to produce at least 60 offspring was rejected. All experiments wereconducted at i5°C.

RESULTS

Fig. 2 illustrates the results of a series of experiments in which localized circularareas of the integument were illuminated for the duration of the supplementary lightperiod, as described above. When capillaries of the largest diameter were applied to thedorsal surface of the abdomen, the entire abdominal contents, consisting largely oftightly packed embryos, appeared brilliantly illuminated (Fig. 2 A). Yet none of theseembryos became a virginopara; all 18 parents tested continued to give birth to oviparaethroughout the period of parturition. In contrast, a positive photoperiodic responsewas invariably obtained when the head and thorax were illuminated from the dorsalaspect (Fig. 2A); all 15 parents later became virginopara-producers and had thereforeresponded to the supplementary light period. Since the ovarioles and their containedembryos do not extend into the head and thorax, this must mean that the control ofembryonic development is maternal and that the light receptor lies in the head region.

A more detailed exploration of the head and thorax was then undertaken, employingthe illuminators of smaller bore. Stimulation in the mid-line of the pro- and meso-thorax always produced negative results (Fig. 2 B). The sensitive area therefore appearedto lie on the head itself. This inference proved to be correct, for when the capillarywas located in the centre of the head (with the beam normal to the surface of thecuticle), a positive response was invariably obtained (Fig. 2C). On the other hand,

126 A. D. LEES

a much less consistent result was forthcoming when the lateral part of the head (and.one compound eye) was illuminated from the dorsal aspect, only 5 out of 13 apteraereacting positively (Fig. 2 C).

Compoundeye

Prothorax r°=\

1-0 mm.

Abdomen

Fig. 2. Results obtained with capillary illuminators. The denominator in each ' fraction'indicates the number of aphids (parents) tested, the numerator the number respondingpositively to supplementary illumination. Areas illuminated denoted by circles.

Two further sites on the head were tested. In Fig. 2D the capillary has beencemented over one compound eye so that the beam was directed inwards towards themid-line of the head. Five individuals responded out of 11. Stimulation of the frontalregion, as indicated in Fig. 2 E and F, yielded 2 positive responses out of 12.

The dorsum of the head was next examined in greater detail by means of light-conducting plastic filaments. Owing to irregularities in the shape of the threads, lightintensities do not necessarily bear any relationship to the area of the tip. In practiceit was found useful to grade the filaments roughly by eye, comparing them with afilament of standard intensity. In Fig. 3 A-D, which shows examples of the results,the order of intensity was: A > B > C > D.

With a relatively bright source, such as A, responses were secured from nearly allparts of the head, including the eyes. The only exception was the site in the mid-linenear the posterior margin of the head capsule. With weaker illumination differencesin sensitivity became more apparent. Two positions of thread B in the mid-longitudinalline were positive as well as one inside the inner margin of the right eye. But two others-proved to be negative. Thread C, with a larger tip area but conducting relativelylittle light, was effective at only one site—in the centre of the dorsum. Two mid-line

Location of photoperiodic receptors in an aphid 127

positions of thread D were positive, one in the centre of the dorsum the other some-what closer to the frontal margin. Stimulation of the eye was ineffective.

On this evidence it appears that the eyes are not necessarily involved, althoughphotoperiodic responses are seen if the eye region is illuminated with sufficient bril-liance. On the other hand, the centre of the dorsum and the region slightly anteriorto this evidently have the greatest photosensitivity, despite the fact that the lightamber-coloured cuticle in this region appears to be featureless in surface view(ocelli are absent in the apterous form of Megoura). As sections of the cuticle and

, Pars intercerebrallsof brain

Suboesophagealganglion

~ \ -Thoracic ganglia.

Anterior lob« ofsalivary gland

Oesophagus

Posterior lobe _of salivary gland

Crop '

Fig. 3. A-D, some results obtained with light-conducting filaments. Positive and negativeresponses indicated by plus and minus signs. E, dissection of the head and prothorax toshow the disposition of some of the major internal organs.

128 A. D. LEES

epidermis also failed to reveal any structures that might reasonably be regarded asphotoreceptors, attention was directed towards the underlying internal structures.

An inspection of Fig. 3 E, which shows some of the more prominent organs of thehead and prothorax, immediately suggests that the photoreceptors may lie in thecentral nervous system. If this is so, certain regions of the C.N.S. can probably beexcluded. For example, illumination of the prothorax would be expected to involvethe suboesophageal ganglion and fused thoracic ganglia. Yet this treatment alwaysfails (Fig. 2B). The only light-sensitive region, namely the head capsule, is largelyoccupied by the protocerebrum which lies no more than 0-03 mm. beneath the cuticleand is separated from it only by the epidermis and a tenuous sheet of fat body cells.Since epidermis and fat body of a similar character occur elsewhere in the body, thereis no reason to implicate these tissues in photoreception. If the photoreceptors areindeed situated in the protocerebrum their probable location is in the intercerebralisarea between the two hemispheres.

When dissected brains are illuminated with a microbeam it becomes obvious that aconsiderable amount of light scattering takes place within the tissue. This property,which presents one of the major difficulties in locating the receptors, may be respon-sible for some apparent anomalies. For example, positive responses from the eye regionwith a bright source (Fig. 2 C, D; Fig. 3 A, B) do not necessarily indicate that either theeye or the optic lobes contain the photoreceptors. Similarly, the failure of the beam tostrike the protocerebrum may well account for some response failures, even thoughthe area illuminated is close to the receptors. This probably occurs when the illumi-nators are applied to the posterior margin of the head in the mid-line (Fig. 3 A, D);and it may be partly responsible for the two negative results shown in Fig. 3 B.

If the threshold of the receptor is low, the integument can be considered as trans-parent. One would then expect that an unscreened receptor located within the brainwould be non-directional. Fig. 2 C, D and E do indeed show that photostimulationeither from the dorsal, lateral or frontal aspects can all be effective. The lessereffectiveness of the lateral, and particularly the frontal, routes is not necessarily due toenhanced cuticular sclerotization or to additional anatomical obstructions in the lightpathway. It is more likely to be caused by the aphid's ability to reach the illuminatorswith the legs. The struggling movements which ensue reduce the fecundity sub-sequently and may have a deleterious effect on the photoperiodic process.

Prenatal photosensitivity

Previous work has shown that the photoperiodic mechanism in the parent aphidbegins to function several days before birth (Lees, 1963). The question therefore arisesas to whether the embryos are receiving light directly through the abdominal body wallof the grandparent or whether some less obvious process is involved. The possibilitythat the photoperiodic centre in the brain of the grandparent in turn influences theactivation of the centre in the embryo was thought to be particularly worthy of in-vestigation.

The action of a given photoperiodic regime cannot of course be detected unless itis matched by a corresponding change in the progeny sequence. Now the smallfemale embryos present in the mother at birth are still undetermined and can be

Location of photoperiodic receptors in an aphid 129

caused to develop into either oviparae or virginoparae by selecting the appropriatephotoperiod during the postnatal development of the insect (Lees, 1959). Neverthe-less, prenatal (and particularly long-day) treatments can be shown by suitable methodsto exert a significant effect on the course of embryonic determination (Lees, 1963).One such procedure has in fact been used throughout the present work for acceleratingthe response of the adult insect to a changed photoperiod. This method, which isdescribed on p. 124, can also be used for studying prenatal light sensitivity. We maymerely recall here that aphids developing from birth to the final moult in short days,and afterwards in long days, produce a much shorter initial sequence of oviparae if thegrandparents have also been exposed to long days. This may indicate that the photo-periodic mechanism is reversed more readily by long days if it has already beenpartially activated during the prenatal period.

The system of illumination was the same as described previously, the principaldifference being that the generation treated were grandparents, not parents. Thecapillary illuminators were attached by suction either to the head or the abdomen.The tubes of slightly smaller diameter (0-5 mm.) used for head illumination were stilladequate to illuminate the entire brain with some brilliance. The procedure was forthe newly moulted adult grandparents to be exposed for 4 days to a daily 12 hr. periodof general illumination followed by 4 hr. of localized illumination. Larvae (the futureparents) born on days 4 to 5 were immediately transferred to a 12 hr. photoperiodwhich was extended to 16 hr. after the final moult. Their progeny was then collectedserially and, after classification, the consecutive series of oviparae and virginoparaewere averaged. The results are shown in Table 3. Previous experiments in which thegrandparents were exposed over the whole body surface to photoperiods of 12 or16 hr. are included for comparison.

When the abdomen alone was illuminated (Table 3, Expt. A), the parents subse-quently produced an average initial sequence of 42 oviparae followed by 34 virginoparae.The length of the first sequence hardly differed from the 45 oviparae produced by thedaughters of grandparents whose entire body surface had been exposed to a 16 hr.photoperiod (series D). Evidently then the photoperiodic mechanism of embryosin the last 4 days of prenatal existence is fully accessible to light transmitted throughthe body wall of the grandparents. The group of aphids deriving from head-stimulatedgrandparents (series B) gave birth to an initial sequence of 66 oviparae. Although thisappears to be significantly fewer than the 79 oviparae produced by aphids in series C,one would hesitate to infer that photostimulation of the head of the grandparentdefinitely promotes the later reversal of the photoperiodic mechanism in the parent.On the other hand, the highly significant difference between the means in A and B(t = 8-5 for 40D.F. :P = < o-ooi) surely indicates that direct appreciation of light anddarkness by the embryos is by itself quite adequate to activate the photoperiodicmechanism.

Elimination of the compound eyes

The illumination experiments have suggested that the eyes are not concerned in theperception of photoperiod. In order to check this conclusion, both eyes were destroyedwith a R.F. cautery immediately after the final moult. This series of aphids was exposedto the same lighting schedule as previously, namely, prenatal period, 16 hr. long day;developmental period, 12 hr. short day; adult period, 16 hr. long day. If the light

g Exp. Biol. 41, 1

130 A. D. LEES

pathway involves the eyes, we should expect the bunded insects to experience darknessirrespective of the photoperiod. Now, as permanent darkness is known to exert a weakshort-day effect (Lees, 1963), normal aphids placed in darkness at the final moultshould continue to produce oviparae. This expectation was fulfilled in a series of22 normal insects which gave birth to an average of 72 oviparae each and no vir-ginoparae. If then the photoresponse fails after the operation, the aphids shouldcontinue to produce oviparae in long days; but if it persists they may be expected toswitch over to virginopara-production. We have already seen that in normal insectsthis takes place after about 45 offspring have been born (Table 1).

Table 3. The effect on the photoperiodic response of parent aphids of exposingthe grandparents to supplementary periods of localized illumination

Treatment of grandparent

Lightregime

during hrs.Expt. ia-i6

Total dailyphotoperiod, hr.

Head Abdomen

16

No. ofparentstested

Mean no. ofoviparae in initial Mean no. of

sequence of virginoparae indaughters final sequence

± s.B. of daughters

42 ±1-4 34

16 13 66 ±27 16

12 12 79 ±2-6

D 16 16 18 45 ±2- 44

In Table 4 the aphids have been grouped according to the length of the initial seriesof daughter oviparae. In normal insects this sequence always numbered between 30and 60. In spite of heavy post-operative mortality, the 20 surviving aphids in theoperated series showed a virtually unimpaired fecundity. In 10 of these no switch-overto virginopara-production occurred even after 80-90 oviparae had been born. But10 individuals still reacted to photoperiod; indeed the reaction was completely normalin 5 individuals, the first virginoparae being preceded by only 40-60 oviparae. In the

Location of photoperiodic receptors in an aphid 131

others, however, the response was decidedly delayed, in one example occurring onlyafter 82 oviparae had been born.

When parturition was at an end, the operated aphids were fixed and sectioned sothat the extent of the lesions could be checked. It was found that in all individuals theeyes and their dioptric apparatus had been completely destroyed, as well as every traceof eye pigment. In addition, the lesions often extended deeply into the optic lobes andwere accompanied by a marked vacuolization of the neuropile.

In the present context the significant feature is that some eyeless insects respondednormally to photoperiod. When the response was delayed or abolished altogether thismay merely mean that the injury was sufficiently widespread to involve the truephotoreceptors.

Table 4. The effect on the photoperiodic mechanism ofdestroying the compound eyes with a cautery

(Further explanation in text.)

Treatment

Eyes 1cauterized J

None

No.respond-

ing

1 0

17

No.failing torespond

1 0

0

30-40

00

6

40-50

30

6

DISCUSSION

Oviparae

50-60

2O

5

per parent

60-70

330

70-80

1

30

80-90

1

40

Transference experiments with several phytophagous insects and mites have shownthat their photoperiodically controlled diapause responses are not influenced by thelight regime under which their food plants are grown; and it has been concluded thatthese arthropods possess their own organs of perception (e.g. Way & Hopkins, 1950;Lees, 1953). The present work, in which an attempt has been made to locate thesephotoreceptors, has confirmed this view.

Although photosensitivity in Megoura is confined to the head capsule, the lightpathway does not necessarily include the compound eyes. This again is in agreementwith previous observations on blinded insects. The area with the greatest apparentsusceptibility to localized illumination of low intensity is the dorsum, just anterior ofcentre and in the mid-line. Since this is not far distant from the site of the medianocellus in the alate morph, this region was carefully searched for rudiments of thisorgan. As no trace of the cuticular lens, sense cells, red pigment granules or theocellar nerve was found, the possible role of the ocellus as a photoperiodic receptor canno doubt be discounted. Since, in addition, other specialized cuticular or epidermalstructures are also lacking (with the exception of a few trichoid sensilla), it seemsreasonable to assume that the photoreceptors lie beneath the cuticle within the proto-cerebrum.

If this argument is valid two main possibilities come to mind. Kennedy (1958) hasshown that various ' ordinary' neurones with no distinctive morphological character-istics may yet be photosensitive. An example is provided by the photoreceptorneurones in the caudal ganglion of the crayfish which exhibit normal electrical activityand control certain behavioural responses. Clearly, if the aphid photoreceptors are of

9-2

132 A. D . LEES

this kind, the prospect of identifying them is not encouraging. The second, and perhapsmore probable alternative, is that the neurosecretory system is implicated in the photo-periodic response. There is little doubt that the photoperiodic process in aphidsinvolves an endocrine component since the determination of the embryos in theovarioles (which are not known to be innervated) is controlled by a maternal centreremote from them. Other evidence suggests that the determination process mayinvolve the release of a hormone under long-day conditions (Lees, 1963). If thishormone proves to be a neurosecretion from the central nervous system, the relevantneurosecretory cells may of course be activated by other neural elements; but it alsoseems to be entirely possible that these cells could serve both as effectors and as photo-periodic receptors.

Although histological evidence that the neurosecretory system has such a functionis still lacking, it is perhaps worth noting that in Megoura cells of this type are presentin just that part of the protocerebrum suspected of maximum light sensitivity. Onegroup, consisting of a single cell pair, has recently been described by Johnson (1962)in A. craccivora. A second, and larger, group lies slightly to the anterior, on the dorso-frontal surface of the brain close to the mid-line.

It is generally accepted that responses to day-length in insects, as in other organisms,are mediated by specific photopigments. In this context Hille Ris Lambers (i960)has drawn attention to the chemical similarity of the aphin pigments (responsible forthe visible colour of aphids) to the well-known photodynamic agent hypericin.Although this resemblance should be borne in mind, it is perhaps worth noting thatthe distribution of aphins does not correspond with the locus of photosensitivity.The bulk of the green pigment in Megoura occurs in the abdomen, particularly in thefat body and embryos. Yet this part of the parent's body is not sensitive to photo-periodic stimulation. In contrast, the brain of Megoura, when viewed in white lightunder the binocular microscope, appears entirely colourless. However, easy visibilityis perhaps hardly to be expected if the pigment is confined to a few cells, is blue-sensitive and is in low concentration.

Another view is that of L'Helias (1961) who considers that the photoperiodicresponse in Sappaphis plantaginea is dependent on photolabile pteridine pigmentswhich are believed to vary in concentration with day-length, particularly in theendocrine complex of the head. The experimental evidence on which this conclusionis based will be awaited with interest.

SUMMARY

1. The site of the photoperiodic receptors controlling the production of sexual andparthenogenetic females in the aphid Megoura has been identified by exposing adultapterae to supplementary periods of localized illumination.

2. The microilluminators devised for this purpose could be attached to the insects.In one type, the light beam was contained in a metal capillary; in another, light wasconducted to the required site by means of a fine plastic filament.

3. Photosensitivity is confined to the head of the aphid, the central region of thedorsum being particularly important as a light pathway. The compound eyes are notinvolved, although stimulation of the eye by a relatively intense beam can elicit theresponse, probably because of light scattering.

Location of photoperiodic receptors in an aphid 133

4. The response is unimpaired if the eyes are covered with opaque paint, and oftenpersists after they have been cauterized.

5. The photoperiodic mechanism in the embryo is actuated by light transmittedthrough the body wall of the parent.

6. The apparent absence of distinctive morphological features in the cuticle andepidermis of the dorsum suggests that the photoperiodic receptors are located in theunderlying protocerebrum. Neurosecretory cells, possibly in the pars intercerebralis,may be implicated both as receptors and as humoral effectors.

REFERENCES

BOWKN, E. J. (1946). Chemical Aspects of Light, 2nd ed. Oxford: Clarendon Press.DANILYEVSKY, A. S. (1961). Photoperiodism and the Seasonal Development of Insects. (In Russian.)

Leningrad State University Publications.GEISPITS, K. F. (1957). On the mechanism of perception of light stimuli during the photoperiodic

reactions of lepidopterous larvae. (In Russian.) Zool. Zhur. 36, 548-60.HtT.iF. Ris LAMBEBS, D. (i960). Some notes on morph determination in aphids. Ent. Ber., Deel., ao,

110-13.

JOHNSON, B. (1962). Neurosecretion and transport of secretory material from the corpora cardiacaof aphids. Nature, Lond., 196, 1338-9.

KENNBDY, D. (1958). Electrical activity of a 'primitive' photoreceptor. Arm. N.Y. Acad. Sci. 74, 329-36.LEES, A. D. (1953). Environmental factors controlling the evocation and termination of diapause in the

fruit tree red spider mite, Metatetranychus ulmi Koch (Acarina: Tetranychidae). Ann. Appl. Biol. 40,449-86.

LEES, A. D. (1955). The Physiology of Diapause in Arthropods. Cambridge Monographs in ExperimentalBiology, No. 4. Cambridge University Press.

LEES, A. D. (1959). The role of photoperiod and temperature in the determination of parthenogeneticand sexual forms in the aphid Megoura viciae Buckton. I. The influence of these factors on apterousvirginoparae and their progeny. J. Ins. Physiol. 3, 92—117.

LEES, A. D. (i960). Some aspects of animal photoperiodism. Cold Spr. Harb. Symp. Quant. Biol.25,261-8.LEES, A. D. (1963). The role of photoperiod and temperature in the determination of parthenogenetic

and sexual forms in the aphid Megoura viciae Buckton. III. Further observations on the maternalswitching mechanism in apterous aphids. J. Ins. Physiol. 9, 153—64.

L'HELAIS, C. (1961). Le role des pterines intermediares photosensibles et thermosensibles dans lagenese des hormones du complexe endocrien de l'insecte. Ann. Biol. 37, 367-92.

MARKHAM, R. & SMITH, J. D. (1949). Chromatographic studies of nucleic acids. I. A technique for theidentification and estimation of purine and pyrimidine bases, nucleosides and related substances.Biochem. J. 45, 294-8.

SHAKHBAZOV, V. G. (1961). The reaction to the length of daylight and the light receptor of the pupa ofthe Chinese oak silkworm Antheraea pernyi G. (In Russian.) Dokl. Akad. Nauk SSSR, 140, no. 1,

TANAKA, Y. (1950). Studies on hibernation with special reference to photoperiodicity and breeding ofthe Chinese Tussar-silkworm. J. Seric. Sci. Japan, 19, 580-90.

WAY, M. J. & HOPKINS, B. A. (1950). The influence of photoperiod and temperature on the induction ofdiapause in Diataraxia oleracea L. (Lepidoptera). J. Exp. Biol. 37, 365—76.

DE WILDE, J. (1962). Photoperiodism in insects and mites. Ann. Rev. Ent. 7, 1-26.DE WILDE, J., DUINTJKR, C. S. & MOOK, L. (1959). Physiology of diapause in the adult Colorado beetle

(Leptinotarsa decemlineata Say). I. The photoperiod as a controlling factor. J. Ins- Physiol. 3, 75—85.