The control of sexual receptivity in female Drosophila

Anim. Behav., 1967, i5, 2~9-25O

THE CONTROL OF SEXUAL RECEPTIVITY IN FEMALE DROSOPHILA

By AUBREY MANNINGDepartment of Zoology, University of Edinburgh

A number of recent studies have revealed howneural and endocrine events interact to syn-chronize the sexual behaviour of insects withtheir reproductive physiology (Highnam, 1964a & b ; Barth, 1965) .

The endocrine glands most directly concernedare the corpora allata which secrete juvenilehormone. During an insect's larval or nymphalstages they show cycles of secretory activity, andcirculating juvenile hormone inhibits the assump-tion of adult characters at each pre-imaginalmoult (Wigglesworth, 1954) .

The nervous system can influence activity inthe corpora allata via modified axons which runto the glands from neurosecretory cells in thebrain and probably, in addition, by a conven-tional nerve supply . The corpora allata are in-activated during metamorphosis so that adultcharacters can develop. However, in manyinsects they must become activated again veryearly in adult life because the same juvenilehormone is required if the ovaries are to develop .

The re-activation of the corpora allata eitheroccurs 'autogenously', i .e. as an invariablesequel to metamorphosis, or may be triggeredby some event, such as feeding, which is norm-ally associated with suitable conditions forbreeding. There is remarkable variation betweendifferent insect groups, and even between closerelatives, in the details of control . Here we neednote only that the appropriate triggering stimuli-if any-are normally conveyed to the brainwhich then activates the corpora allata (High-nam, 1964a).

There are several ways in which a female'ssexual behaviour could be synchronized with itsovarian cycle. For example, receptivity might betriggered independently by the same eventswhich lead to activation of the corpora allata .Alternatively, receptivity might be initiated bythe hormonal changes which follow such activ-ation. A further possibility is that sexual re-ceptivity develops 'autogenously' and matingitself serves as the trigger which activates thecorpora allata. This latter appears to be thecase with the cockroach, Diploptera punctata,where females are receptive at metamorphosisbut do not develop eggs unless they mate

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(Engelmann, 1959)-a situation which has someresemblance to ìnduced ovulation' in mammals .Returning to examples where endocrine

changes precede those in sexual behaviour, thejuvenile hormone is known to operate in at leasttwo distinct ways . Direct hormonal control ofsexual receptivity has so far been demonstratedonly in the grasshopper Gomphocerus rufus.Here Loher (1962) has shown that allatectomizedfemales, although courted normally, do notaccept males . Implanting active glands leads tothe return of receptivity within a few days. Incockroaches, by contrast, the juvenile hormonedoes not affect the receptivity of females, but itdoes determine whether they mate or notthrough its control of pheromone secretion bythe epidermal cells. Without pheromone afemale cockroach is not attractive and malesignore her. Barth (1961) has shown that thesexual responsiveness of allatectomized femalesis unchanged, for if pheromone from intactfemales is rubbed onto their bodies they acceptthe males which now court them .

Immediately after mating most female cock-roaches stop secreting pheromone and eggproduction also ceases . Roth (1962) has shownthat it is neural feedback from the reproductivetract, distended by the development of the firstbatch of eggs, which causes the brain to inhibitcorpus allatum activity . Once the first eggcase has been laid-or the first brood of youngproduced in ovoviviparous species-the corpusallatum is disinhibited, more eggs develop,pheromone is secreted and the female once moreattracts the courtship of males. Barth (1965)points out that this type of link between phero-mone secretion and egg production is adaptivefor an insect which produces eggs in batches .A female is physically incapable of successfulmating whilst each batch is maturing .

The reproductive cycle of Drosophila providessome contrasts with that of cockroaches . Oncethe ovaries have developed, they remain in fullproduction and egg-laying can continue for aperiod of weeks (David, 1963) . This paperdescribes some behavioural observations on thecontrol of sexual receptivity in female Droso-phila and gives some preliminary account of itsphysiological basis .

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ANIMAL BEHAVIOUR, 15, 2-3

MethodMaterialExperiments have been carried out with the

two sibling species D. melanogaster and D.simulans. There are quantitative differencesbetween them which will be noted when relevant,but the basic mechanisms involved are the samefor both species . The D. melanogaster were des-cendants of the `Pacific' stock kept at theInstitute of Animal Genetics in Edinburgh .The D. simulans were descendants of a crossbetween the `Jerusalem' stock kept at UniversityCollege, London and a local stock . The flieswere reared on a standard agar, corn-meal andmolasses medium at 25± 1 °C and, unless statedto the contrary, on a 12 :12 light/dark cycle .Virgin flies were kept in groups of twelve tofifteen in 3 x 1 in . vials with food, prior totesting. They were transferred to fresh food everysecond day.

Measuring ReceptivityThe sexual behaviour of most Drosophila

females is rather passive and here we shall mostlybe concerned with measuring whether they areprepared to accept males or whether they rejectthem . Drosophila males never attempt to mountfemales without performing some preliminarycourtship, although a few seconds may suffice .The courtship displays of D. melanogaster andD. simulans have been described elsewhere,(Manning, 1959) . Female receptivity was meas-ured using single pair matings in small Perspexcells 2 cm in diameter and 5 mm deep coveredby a glass cover slip. The time from the beginningof continuous courtship until the start ofcopulation-called the `courtship time'-wastaken as a measure of receptivity . Receptivefemales normally permit a male to mount themafter a few minutes of courtship . Males of bothspecies will usually court females of all ageswith equal persistence . Unless they did courtwell, males in the observation cells were re-placed ; females were recorded as ùnreceptive'if they had not accepted after 30 min courtship .(The justification for stopping tests at this timewill appear later .) Forced matings do occur,particularly in melanogaster, when the femaledoes not part her wings as the male mounts andshe can be seen struggling to push him off withher hind legs. However, unless she succeeded indoing so within 5 min, she was recorded asreceptive .

ResultsAge and Receptivity in Virgin Females

The majority of Drosophila adults emergefrom their pupae within an hour or two of`dawn'. If we call the day of eclosion `day 0',few females are receptive either on day 0 or day 1,but virtually all are receptive by the morning ofday 2. They remain receptive for several daysbut beyond day 6 in D. simulans or day 8 in D .melanogaster, an increasing proportion of fe-males prove to be unreceptive when testedthese observations are illustrated in Fig. 1 .

DAYS FROM ECLOSION

Fig. 1 . The receptivity of samples of virgin females atdifferent ages . Samples were of different sizes but nonewas less than 16. Age is represented on a logarithmicscale. Crosses represent D . melanogaster, circles D.simulans.

The change between day 1 and day 2 is mostdramatic and by testing at closer intervalsduring this critical period it was hoped to learnmore about its nature .

Drosophila courtship is best regarded as themeans whereby a male provides a female with astream of stimuli whose effects summate, finallyreaching a critical level where she accepts him .One of the most important stimuli given by themale comes from his wing vibration display,which drives a current of air over the female'santennae. The quantitative nature of the sum-mation process has been demonstrated by Ewing(1964) who showed that there is a linear re-lationship between a male's wing area and hiscourtship success .

We might ask therefore whether the develop-ment of receptivity in maturing females repre-sents a time when they require a graduallydecreasing amount of stimulation to reach thecritical level for accepting a male .

The light/dark cycle may influence the changein receptivity between day I and day 2 and inorder to make valid measurements at close in-tervals a culture of 'arhythmic' flies was estab-lished . These flies had been reared in continuous

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light at constant temperature for severalgenerations prior to testing and the normalrhythm of eclosion, mentioned above, hadbroken down . Samples of virgin females emerg-ing within a 5 to 6 hr period were measured forreceptivity at different intervals from eclosionusing mature (2-3 days old) males from the sameculture. The results are shown in Fig . 2 .

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MANNING : SEXUAL RECEPTIVITY IN DROSOPHILA

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Fig. 2. The receptivity of samples (minimum 16) of'arhythmic' D. simulans females over the transitionalperiod. Age plotted is the mean age of the sample, whoserange covered 6 to 7 hr.

As with normally reared females, receptivityis low at 24 hr of age but maximal at 48 hr, sothe lack of `timing' cues from the light/darkcycle has no effect. The changeover can first bedetected within samples of females whose meanage is 24 hr and samples are uniformly receptivesome 16 hr later .

Figure 3 is a frequency histogram of thecourtship times of individual females measuredover this whole transition period from 24 to 40hr of age. It shows a clear bimodal distribution,with over 90 per cent of females either acceptinga male within 15 min or refusing him for morethan 30 min. This suggests that, for an individual,the change from the unreceptive state to thereceptive state is a rapid process. If femalesgradually increased their receptivity and thusdecreased the `quantity' of stimuli they requiredbefore accepting males, a much higher pro-portion of females would require courtshiptimes of intermediate length during this tran-sition period.

The change from the unreceptive to the re-ceptive state which occurs between day 1 andday 2 cannot be produced by courtship . Re-ceptive females accept males within 15 min butunreceptive ones are quite insensitive to court-ship stimuli . Continuing observations beyond30 min do not affect the distribution of theunreceptive group in Fig . 3 . At one time a period

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Fig . 3. The distribution of individual courtship timesover the transitional period between 24 and 40 hr of age .These data are taken from the samples used in Fig . 2 .They show clear bimodality with females either fully re-ceptive or unreceptive.

twice this length was used but, on day 1, femaleswere still unreceptive . Up to 7 hr of more or lesscontinuous courtship has been observed directedto young females without success .

At the other end of the scale, variations withinthe 0 to 15 min range of courtship will includecomponents due to differences in female `re-quirements', the intensity of the males' displaysand random variation from short breaks incourtship which inevitably occur when the maleloses contact in the observation cell . It cannotbe argued that the compression of courtshiptimes within a 15 min period is a characteristicof the neural mechanisms responsible for sum-mating courtship stimuli . Figure 4 shows thedistribution of courtship times for females onday 3 when courted by males whose wings hadbeen removed . Such males cannot provide wingvibration stimulation but continue to give thefemale certain visual and tactile stimuli . Oneassumes that the `courtship summation mechan-ism' is receiving stimuli at a lower density thannormal, but it obviously can operate under theseconditions and, although they are variable, manyfemales accept wingless males, but after a longerperiod .

The term 'switch-on' seems an appropriatedescription of the sudden, all-or-nothing changein receptivity which occurs in young virgin

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Fig . 4 . The distribution of courtship times when D .melanogaster females on day 3 are courted by winglessmales. The mode is shifted to 20 to 25 min of courtshipfrom the normal 0 to 5 min.

females . As Fig. 1 shows, virgins do not remainreceptive indefinitely. It might be asked whetherthere is also a rapid 'switch-off' process orwhether ageing females require an increasingamount of courtship before they will accept .Figure 5 shows frequency histograms for samplesof D. simulans females on day 16 and D. melano-gaster on day 25 when about 50 per cent ofthem are receptive in each case . Again there areclear bimodal distributions and females areeither fully receptive or unreceptive .

Other factors may be operating here whichwould tend to accentuate this bimodality . Un-receptive females of this age tend to repelmales by extruding their ovipositor, as comparedwith the `milder' repelling movements such aswing flicking and kicking used by immatureunreceptive females . Èxtrusion' is markedlyaversive to males and makes them turn away(Bastock & Manning, 1955) . Consequently itwas sometimes difficult to ensure that femaleswhich extruded received a genuine 30 min ofcourtship and on occasions males had to bechanged several times. The point may well beacademic, because the fact that a female doesextrude is probably a good sign that her re-ceptivity is 'switched-off' . Very few females wenton to accept males if they had first shown ex-trusion.

If ageing virgins which are unreceptive ontheir first test with a male are tested again onsubsequent days, usually some of them are re-ceptive. The older they are at the first test, theless likely it is that they will subsequently proveto be receptive again. It appears that 'switch-off' is not, at first, irrevocable, but a female


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Fig. 5. Courtship time distribution for samples of (a)D. melanogaster females on day 25, and (b) D. simulansfemales on day 16. Both show clear bimodality withfemales either receptive or unreceptive .

spends a period of several days during whichher receptivity fluctuates before it finally dis-appears .

The Development of the Reproductive SystemThe behavioural cycle outlined above can

be compared with some of the changes in thereproductive system which take place over thesame period .The rapid growth of the ovaries following

eclosion is illustrated in Fig . 6. Ovaries weredissected from females and photographed at aconstant magnification in a drop of DrosophilaRinger without a coverslip . The weight of thepositive, printed on double-weight paper at aconstant enlargement was taken as a measureof size. Thus the units in Fig. 6 are arbitrary,but growth changes are so marked that thismethod is adequate .The ovaries are very small at eclosion and

contain no mature eggs . On day 0 females feed

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Fig . 6 . The growth of the ovaries in virgin D . melano-gaster. The mean and two standard deviations of eachsample (between 5 and 15) are plotted . Age is repre-sented on a logarithmic scale. Open circles representfemales reared on normal food from eclosion, shadedcircles represent females 21 days old at the start ofmeasurements, which had been kept on a diet of 0 .2 Msucrose until that time and then given normal food . Inboth groups the maximum growth occurs between day Iand day 2 on normal food .

intensively and on dissection their crops proveto be swollen with yeast from the culture vials .By day 1 a few mature eggs are present but themost rapid growth occurs between day 1 andday 2, by which time the ovaries are practicallyat their maximum size. In virgins they declineslowly in size over a period of weeks, even whenthe flies are kept under optimal conditions .

The coincidence of maximal enlargement ofthe ovaries with the switch-on of receptivitymight suggest a causal connexion . Browne (1956)has suggested that it is ovarial developmentwhich determines receptivity in females of theblow-fly, Lucilia cuprina. He has shown thatunfed females with small ovaries do not mate .However, it is possible to exclude this explana-tion in Drosophila. Smith (1958) showed thatfemale D. subobscura which congenitally lackedovaries became receptive in the normal way .We do not have such direct evidence from D.melanogaster or D. simulans, but ovarial growthcan be effectively stopped by keeping femalesfrom eclosion on a diet of 0 .2M sucrose alone .Although they can muster enough protein forone or two eggs, the ovaries of such flies remainvery small, below the size for normal females onday 1 . Figure 7 demonstrates that protein-lessfemales nevertheless become switched-on atthe usual time .

MANNING: SEXUAL RECEPTIVITY IN DROSOPHILA

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It seems reasonable to transfer attention onestage back to some process which might be acommon causal factor for both switch-on andovarial growth . The activation of the corpusallatum is the obvious choice. Doane (1960)and King et al. (1966) have already describedthe overall growth and histological changes inthis gland following eclosion in D. melanogaster .It doubles in size during the first 12 hr of adultlife but thereafter shows little change for twoweeks or more . King et al. (1966) have evidencethat whilst the ovaries are growing the maturegland is releasing its hormone as fast as it isproduced and the fact that switch-on occursapproximately 24 hr after the corpus allatumattains full size is suggestive and worth investi-gating further.

It is extremely difficult to allatectomizeDrosophila but it is possible to inject extraglands from other donor flies. In this way onecan test whether switch-on is accelerated whenmature corpora allata are injected into flieswhose own glands have not yet grown . Thepreliminary results of this experiment havealready been reported with full details of thetechnique used (Manning, 1966) . Briefly, corporaallata together with the closely associatedcomplex of corpus cardiacum and hypocerebralganglion were injected together with a littleRinger into pupae on the day before eclosion .The donor flies were 6 to 7 days old virginfemales and control injected pupae received asmall piece of aorta from the same donors .Pupae were chosen rather than adult flies be-cause (a) they are far easier to inject and showlower mortality and (b) hormone from the in-jected gland presumably takes the same timeto operate as the recipient's own gland and todemonstrate a positive effect switch-on must beproduced by day 1 .

Figure 8 summarizes the results of this experi-ment ; 31 out of 32 flies receiving a mature corpusallatum complex as pupae were receptive on themorning of day 1 (Fig. 8a) compared with lessthan one third (2g) control injected flies (8b) .This difference is highly significant (X2 withYates' correction = 24 . 6 ; P<0 .001), whereasthere is no significant difference between injectedand non-injected controls (8c) . All the 16 control-injected females which were unreceptive on day 1proved to be receptive on day 2 in the normalway. On the other hand 12 allatum-injectedfemales which were receptive on day 1, werepreviously tested on day 0, a few hours afterthey emerged, but none were receptive then .

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MANNING : SEXUAL RECEPTIVITY IN DROSOPHILA

Table L The Ovary-Size of Receptive and Unreceptive Females on Day 1

Using the Wilcoxon test, comparing (a) with (c) P<0 .001 and comparing(b) with (c) P<0 . O1 ; (a) and (b) do not differ significantly.

The active part of the injected complex is likelyto be the corpus allatum itself, releasing juvenilehormone into the host flies' haemolymph .Apparently the hormone needs to be in circu-lation for at least 24 hr to produce switch-on.

If it is a rise in juvenile hormone concen-tration which leads to switch-on, we shouldpredict that those females (some 20 to 30 per cent)from normal populations which become re-ceptive on day 1 will be those whose corporaallata have begun secretion earliest . This shouldbe reflected by larger ovaries in those femaleswhich become `naturally' receptive on day 1 .Table I shows the results of some observationsmade to test this prediction . Three groups arecompared for ovary size on day 1 :

(a) Èxperimentally' precocious flies which hadbeen injected with mature corpora allata aspupae.

(b) `Naturally' precocious flies .(c) Unreceptive flies .The data was analysed using a variant of the

Wilcoxon test suggested by Quenouille (1959)which confirms that both receptive groups (a)and (b) have significantly larger ovaries than theunreceptive group (c) [between (a) and (c)P<0.001 ; between (b) and (c) P<0 .01]. Group(a) included a number of flies with extremelylarge ovaries, but variance was high and thereis no significant difference between these fliesand those of group (b) .

The evidence thus supports the hypothesisthat hormone from the growing corpus allatumleads directly to the switch-on of receptivityand one might ask whether switch-off in elderlyvirgins occurs when the concentration of juvenilehormone falls below the critical level as a resultof ageing . Figure 6 shows that although ovarysize does decline slowly with age-and so does

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the size of the corpus allatum-this decline isnot so rapid as that of receptivity (Fig . 1) .Flies kept without protein for 3 weeks and thengiven normal diet, have enough hormone toallow their ovaries to grow substantially al-though they do not reach the same size as inyounger flies (Fig . 6). This suggests that hormonetitre remains quite high for a long period ; butit is difficult to know whether this titre is aboveor below that required for the initial switch-onof receptivity . The question could be resolved byinjecting corpora allata from young femalesinto elderly virgins which had become un-receptive, but this is a difficult procedure withhigh mortality. However, in the next section,some circumstantial evidence will be presentedthat an increased hormone concentration in anelderly fly does indeed delay switch-off.The Effects of Mating on Receptivity

Hitherto we have considered receptivity andits control in virgin flies only . In the naturalsituation most females will mate soon afterswitch-on and mating produces a number ofrapid and marked changes both to behaviourand to reproductive physiology. David (1963)has shown that whereas virgin flies have a slowturnover in the ovaries and lay few but largeeggs, fertilized females show a rapid turnover,laying large numbers of slightly smaller eggs .Virgin females tend to hold on to eggs which areotherwise mature and mating results in anenormous increase in the rate of egg-laying .Within 4 hr of mating the rate in D. melanogasterrises from less than I egg per female per hr toabout 20. Doane (1960) found that the corporaallata of mated females increase in size andremain larger than those of virgins for a con-siderable period,

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(c) Normal females,unreceptive on day 1 19 511 44 .2

HOURS SINCE END OF -COPULATION

Fig. 9 . The return of receptivity in 'arhythmic' D. melanogaster femalesafter completed (a), or interrupted (b) copulation with sterile males . Samplesare not less than 16, Ten min copulation produces complete 'switch-off' for48 hr, but only 30 or 40 per cent of females are affected by 5 min ofcopulation .

As far as sexual receptivity is concerned,Drosophila females, like those of many otherinsects, will not mate twice in quick succession .A newly mated female immediately repels maleswith the characteristic extrusion movementmentioned above . She also twists or elevates herabdomen out of the reach of courting males .Previous work (Smith, 1956 ; Manning, 1962)

has shown that the switch-off of receptivityfollowing mating has two components . Firstlythere is the result of copulation itself : this effectis probably mediated mechanically and trans-mitted to the brain via the nervous supply fromthe genital tract, although there is the possibilitythat some constituents of the semen exert achemical influence. This `copulation effect' isshort-lived and wears off unless-as is normallythe case-the mating has been fertile . The secondfactor is mediated by the presence of sperm andreceptivity remains switched off so long as thereis an adequate amount of sperm in the female'sseminal receptacle and spermathecae . Furtherexperiments have helped to clarify some pointsconnected with the `copulation effect' and the`sperm effect' .

(1) The copulation effect. The exact time courseof the copulation effect has been studied usinga stock of D. melanogaster kindly provided byC. Auerbach. This stock has a peculiar arrange-ment of the sex chromosomes and when out-crossed yields males which are sterile becausethey lack a Y chromosome . Such males producesperm and mate normally but their sperm are

immobile and do not migrate into the seminalreceptacle of the female .

Sterile males were mated to 'arhythmic'females reared in constant light and the latterwere tested for the return of receptivity aftervarious intervals . Copulation normally lastsfor about 20 min in D. melanogaster and in somefurther tests, couples were separated after 5 or10 min to investigate how this affected switch-off.

Figure 9 shows the main results, which maybe summarized as follows . After full copulationwith sterile males, all females are unreceptivefor at least 12 hr, and all are once more receptiveafter 48 hr. Samples were very variable 24 hrafter a sterile mating, ranging from 4 per centto 98 per cent receptive. Ten min, or roughly 50per cent of the normal length of copulation, wassufficient to produce full switch-off, but 5 mincopulation was only effective in some 30 per centof females .

The return of receptivity after a sterile mating,as with the initial switch-on of a virgin, is arelatively sudden process with females either un-receptive or fully receptive .

(2) The sperm effect . Inseminated females re-main unreceptive for many days and Manning(1962) showed that the return of receptivity,which usually occurs 8 to 10 days after mating,is associated with the exhaustion of sperm byegg-laying . If egg production is suppressed bykeeping females without protein, their spermremains unused and they stay unreceptive . The

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sperm effect could be a mechanical one, medi-ated via the nervous supply to the sperm storageorgans, or it could be chemical, via some sub-stance released into the haemolymph . Haskell(1960) has evidence that a chemical influence isexerted by the stored sperm in some grass-hoppers, which also become unreceptive aftermating.

Drosophila females take about 24 hr to becomereceptive after their seminal receptacles arevirtually empty of sperm. This suggests thewaning of a chemical influence rather than theremoval of neural inhibition, but the criticalexperiments are very difficult to perform with ananimal of this size . In particular it is impossibleto denervate the genital tract as Haskell coulddo with his grasshoppers.

If the sperm do release some chemical into thehaemolymph (or cause the adjacent femaletissues to do so) it should be possible to makevirgin females unreceptive by replacing theirhaemolymph with some drawn from fertilizedfemales. Attempts have been made to do thiswith no success . A considerable proportion ofthe haemolymph of virgins on day 2 was re-placed by that from fertilized females . Mortalitywas high but six insects survived the operationwell and all were still normally receptive on day3. Of course, even if a circulating chemical isresponsible for maintaining switch-off it is quitepossible that a single injection is inadequate toproduce the effect. It would be better to implanta sperm-filled seminal receptacle into a virgin,

MANNING: SEXUAL RECEPTIVITY IN DROSOPHILA

but this requires the use of a large needle whichcauses too much damage . Sperm injected directlyinto the haemolymph do not survive for longenough to have any effect . For the moment theexact action of sperm is unresolved but, whetherits effect is mediated chemically or not, switch-off is certainly not produced by inhibiting thecorpus allatum and reducing juvenile hormoneconcentration . As mentioned earlier, the gland'sactivity probably increases following matingand egg production rises steeply.

There remain two aspects of the behaviour offertilized females which have bearing on thecontrol of receptivity .

Firstly, the return of receptivity when spermare exhausted is an all-or-nothing process, justas it is in virgins or in females who have beenmated with sterile males .

Secondly, a group of females which have beenfertilized and then allowed to exhaust theirsupply of sperm by egg-laying, contains morereceptive flies than a group of virgins of thesame age. Table II sets out the results of threecomparisons of this type, starting with a com-mon population in each case . In one, a group ofvirgins was compared with females mated once,in the other two, virgins were compared withfemales which had gone through two cycles offertilization followed by recovery of receptivity .

Thus in females which have been fertilized,the switch-off which occurs with increasing age isdelayed compared with that of virgins . This isthe more striking because in other respects,

Table II. The Receptivity of Virgins Compared with that of Females which Have Been Mated SomeTime Previously

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1. Group 1 . Virgin YY 25 30 11 19------

- - - ---- ------ ------ 6.41 <0 .02

Group 2 . ?? fertilizedon day 6 27 24 18 6

2. Group 1. Virgin Y? 25 50 10 40------ - - - - --- ------ ------ 8 . 61 <0 . 01

Group 2. YY fertilizedon day 3 and 27 50 25 25on day 15

3. Group 1. Virgin 2? 42 20 1 19------ - - - - --- ------ ------ 11 . 39 <0 .001

Group 2. ?? fertilizedon day 3 and 42 20 12 8again on day24

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fertilization and egg-laying are known to acceler-ate the ageing process (Kummer, 1960 ; Smith,1958) .These observations provide circumstantial

evidence for the hypothesis that switch-off occurswhen the juvenile hormone titre sinks below acritical level. The increased activity of thecorpora allata which accompanies egg laying,is presumably responsible for the prolongationof receptivity in females which have mated .

DiscussionThe results given above go some way towards

analysing how the sexual responsiveness andreproductive physiology of Drosophila areintegrated .

Female sexual behaviour is controlled by twoseparate processes. The first, which we havecalled 'switch-on', determines whether a femaleis àccessible' to the courtship of males . Thesecond process may be called `courtship sum-mation' ; the mechanism responsible summatesthe various stimuli coming from a courtingmale until a critical level is reached, when thefemale allows him to mount . The action of thesetwo processes may be compared to that of thesafety catch and trigger mechanism of a pistol .The first determines whether or not the second isaccessible to the pressure of a finger . Switch-onand courtship summation are quite independentand evidence will be presented elsewhere thatcertain genetic changes can affect one processwithout affecting the other .

The experiments described here stronglysuggest that switch-on is produced as a directresult of the release of juvenile hormone into thehaemolymph . As Highnam (1964) points out,the fact that receptivity depends on activationof the corpus allatum does not necessarily meanthat the juvenile hormone itself acts on neuralmechanism involved . It may facilitate the releaseof stored neurosecretory material which is theactive factor . Roth & Barth (1964) have sug-gested that sexual receptivity in some femalecockroaches,_ which is independent of thecorpora allata, is controlled by neurosecretion .

Hitherto we have implicitly assumed - that thecorpus allatum, once activated, releases hor-mone spontaneously and that switch-on andovarian growth follow. This is not the onlypossibility . King et al. (1966) have compared indetail the histological changes occurring in thecorpora allata of normal D. melanogasterfemales with those in females homozygous forthe mutant `female sterile' (fes) . In both types


of female the corpus allatum enlarges over thefirst 12 hr from eclosion, but thereafter as theirovaries grow, the glands of normal females showlittle overall change and appear to be releasingsecretion as fast as it is formed . In contrast, theovaries of fes females are incapable of anygrowth, their corpora allata continue to enlargeand the cells of the latter appear grossly swollen .King et al . found that normal, undevelopedovaries grow rapidly when implanted into fesfemales. At the same time the corpora allataof the host females shrink and appear to releasetheir stored secretion . King et al . suggest thatthe gland does not release its hormone spontan-eously, but only in response to the demands ofthe growing ovaries. The latter may draw solutesfrom the haemolymph and this depletion, inturn, draws hormone from the gland cells .

This possibility would not be predicted on thehypothesis proposed which ascribes switch-onto the release of juvenile hormone . If the ovariesmust `draw out' the hormone why do D.melanogaster females, deprived of protein, be-come receptive although their ovaries remainvery much undeveloped? Again, Smith (1958)found that D. subobscura females which congen-itally lack ovaries altogether nevertheless be-come receptive normally. Females reared withoutprotein show some slight growth of the ovariesand one might suggest that some hormone couldbe drawn out in this case . The fact that injectedcorpora allata can induce precocious receptivity,and some signs of precocious ovarial growth,indicates that some hormone is released spon-taneously, at least from glands which weresecreting when removed from their donors .We need more experiments with normal and

fes females to elucidate this problem . CertainlyLarsen & Bodenstein (1959) using Aedesmosquitos and Johansson (1954) using themilkweed bug, Oncopeltus, have evidence thatthe activation of the corpus allatum precedesovarial growth in these insects and depends onthe females having fed . This may also be true ofLucilia_ where Browne (1956) found receptivitywas linkedd to ovary development . If we assumethat feeding activates corpus allatum secretionand ovarial growth (whichever comes first)then behavioural receptivity may be caused byhormone secretion as is suggested for Droso-phila .

The system revealed in Drosophila seemsadaptive for an insect which can produce acontinuous flow of eggs as long as conditionsremain favourable . Drosophila can store sperm

for at least 2 weeks and this means it will beadvantageous to mate quickly after eclosion,even if food is scarce and no eggs have matured .Fertile eggs can then be laid soon after foodbecomes available.

It is not immediately clear why a femaleshould become unreceptive after a single mating .Unlike cockroaches, there are no physicalbarriers to prevent further mating and a femalecould keep her sperm supply `topped up', asopportunity presented itself. However, a singlemating provides at least a week's supply andit may be that the persistent attention of maleswould interfere with egg-laying . Further, copu-lating pairs are exposed to the greatly increaseddanger of predation. Both these factors maygive selective advantage to females which repelmales after mating once.

There is another factor which may accountfor the evolution of this behaviour, and whichaffects both sexes equally . By crowding fliestogether, it is quite easy to get multiple coatingsbecause females cannot avoid males and areraped. In such cases the use of geneticallymarked males has shown that new sperm ispacked into the coiled seminal receptacle ontop of that received from a previous mating.This means that sperm are used for fertilizingeggs on a `last in-first out' basis. Clearly,males whose sperm tends to cause the switch-offof a female's receptivity will be at a considerableadvantage, because their sperm will be used tothe full . Both males and females whose sons havesperm with this effect will be favoured .Patterson & Stone (1952) describe in detail

the marked ìnsemination reaction' which isfound in many species of Drosophila (particu-larly in the sub-genus Drosophila) . In thesespecies the presence of semen causes the epithel-ium of the vagina to secrete copious fluid andwithin an hour or two of mating the vagina isgreatly distended and blocked . Patterson sug-gests this reaction may serve-rather as thevaginal plug in rodents-to prevent furthermating. He discusses the evolution of theinsemination reaction in terms similar to thoseoutlined above . However, the action itselfpersists for less than 24 hr and we do not knowwhether such females are also affected behaviour-ally in the same way as D. melanogaster andD. simulans .

Summary1 . This paper describes various experiments

on the integration of reproductive physiology

MANNING : SEXUAL RECEPTIVITY IN D'ROSOPHILA 249

and behaviour in female Drosophila melanogasterand D. simulans.

2. The sexual behaviour of females is ascribedto two distinct processes . One determines thestate of receptivity, i .e. whether a female isàccessible' to stimulation from the courtshipof a male, the other summates the variousstimuli from courtship and eventually leads tothe female accepting a male .

3. Females are unreceptive to males on theday of eclosion (day 0) about 25 per cent arereceptive on day 1, the rest by day 2. Moreprecise tests place the transition period between24 and 40 hr from eclosion . In any individualthe change is a sudden one ; females are eitherfully receptive or fully unreceptive . Accordinglythis change is called 'switch-on' .4. The corpus allatum and ovaries show a

growth cycle parallel to that of receptivity .Evidence is presented that increase in juvenilehormone titre is responsible for the 'switch-on'of receptivity.

5. Virgin females remain receptive for manydays, but an increasing proportion become un-receptive after the first week of adult life .'Switch-off' like 'switch-on' is a rapid, all-or-nothing process . Females also become un-receptive immediately after mating and thisinhibition of receptivity has two components,(a) an effect of copulation itself, probablymechanical, which wears off after 48 hr, (b) aneffect due to the presence of live sperm, whichwears off once a female has exhausted the spermby egg laying, after some 8 to 10 days.

6. Old females which have mated and usedup their sperm are more often receptive thanvirgins of the same age. It is suggested that thisis so because their corpora allata are moreactive and the juvenile hormone concentrationis kept up above the critical level for longer .

7. The situation revealed in Drosophila iscompared with that found in other insects,particularly with regard to the role of theendocrine system .

AcknowledgmentsIt is a pleasure to express my gratitude to Miss

M . C. Hill for her excellent technical assistancethroughout this work . Mr Robert Loregnardkindly allowed me to quote some of his un-published observations on egg laying in Droso-phila . Dr Winifred Doane and Mrs EvelynRobertson both helped with details of theinjection technique used. Dr Margaret Bastockand Dr Arthur Ewing read and criticized an

250


earlier draft of the manuscript. Finally, I mustthank the Science Research Council from whomI held a grant for special research .

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(Received 30 October 1966 ; revised 11 December 1966 ;Ms. number : 701)

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The control of sexual receptivity in female Drosophila