Anim. Behav., 1968, 16, 1 0 8-113

THE Ek H ECTS OF ARTIFICIAL SELECTION FOR SLOW MATING IN

DROSOPHILA SIMULANS. I. THE BEHAVIOURAL CHANGES

BY AUBREY MANNINGDepartment of Zoology, University of Edinburgh

A previous study (Manning, 1967) examinedsome aspects of the control of sexual behaviourin female Drosophila, and two processes couldbe identified. The first determines whether afemale is `accessible' to the courtship of males .Young females are unreceptive for some 36 hrafter eclosion, but then, quite suddenly, theybecome receptive and will accept a male after afew minutes of courtship . Evidence was pre-sented which suggests that this rapid behaviouralchange occurs when the concentration of circu-lating juvenile hormone rises with the reactiva-tion of the corpus allatum after eclosion .

The second process controlling female sexualbehaviour was called `courtship summation' . Itinvolves the summation of the heterogeneousstimuli provided by a courting male which, oncea critical level of stimulation has been reached,induces the female to allow him to mount .

It was suggested that these two processes aredistinct, and we may summarize the evidence forthis conclusion as follows .

1 . Once females have become receptive, 95per cent of them accept males within 15 min ofcourtship and more than 70 per cent within 5min. No amount of courtship will make an un-receptive female accept a male normally . How-ever with receptive females it is possible tomodify the operation of courtship summationby slowing down the rate at which males candeliver courtship stimuli . (This is most easilydone by clipping their wings .) Such an operationgreatly increases the number of females whichaccept normally but only do so after long (20 to30 min) periods of courtship .2. The change from the unreceptive to the

receptive state is an all-or-nothing process,i .e. females are either completely unresponsiveto courtship or they accept within the normaltime range for receptive females . They do notbecome gradually receptive by requiring less andless courtship before accepting. This suggeststhat the courtship summation mechanisms arenot involved in the change . Inducing precociousreceptivity with implanted endocrine glands does

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not affect courtship summation, which operatesnormally and is not accelerated in treatedfemales.

The dual nature of the control of femalesexual behaviour was likened to the operationof the safety catch and trigger spring of a pistol .The former operates in an all-or-none fashion todetermine whether the latter is susceptible tothe pressure of the trigger finger.

This paper describes the behaviour of fliesfrom a line of Drosophila simulans selected forslow mating . The changes produced, when com-pared with earlier results from other lines alsoselected for slow mating, support the conclusionthat the two processes-the development ofreceptivity and courtship summation-are quitedistinct .

The behavioural results of this selection ex-periment with D. simulans are most striking andsome preliminary physiological analysis of geneaction has been possible . A subsequent paperwill report more fully on genetic analysis of theselected line.

MethodsThe base population of D. simulans was de-

rived from a cross between the `Jerusalem' stockkept at University College, London, and a localstock. The flies were reared on a standard agarcorn-meal molasses medium at 25±1'C andon a 12-hr-light 12-hr-dark cycle . All behaviouraltests were made within 3 hr of the flies' `dawn',which is the period of maximum activity .

Two types of behavioural measure were used :(a) `Single-pair matings' in small observation

cells, in which the duration of courtship requiredby a female before accepting could be measured .

(b) `Mass matings' in which fifty pairs ofvirgin flies were introduced into a half-pintmilk bottle and pairs were removed as they mated .This provided a continuous measure of theproportion of the flies which had mated sincethe start of the test, and was the method usedfor selection (see Manning, 1961, for furtherdetails) .

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MANNING: SELECTION FOR SLOW MATING IN DROSOPHILA

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ResultsThe Course of Selection and the Nature of theBehavioural Changes ProducedThe selection scheme was the same as that

used previously to select for slow mating in D.melanogaster (Manning, 1961) . The last tenpairs which remained unmated from a massmating on day 3 (day 0 = the day of eclosion)were used for breeding in each generation .Figure 1 shows the course of selection overtwenty-five generations expressed as deviationfrom unselected controls . The units are de-rived from the `mating speed' of each massmating, which is defined as the time taken fortwenty-five pairs (50 per cent of the flies) to mate .Where mating was very slow this figure wasobtained by extrapolation of a graph of pairsmating against elapsed time. As can be seen fromFig. I the slow selected line (SA) did not overlapcontrols after generation 4 and, in particular, asustained response to selection followed gener-ation 7. The actual values of mating speed hereare not important. Most of the figures for SAhad to be based on extrapolation from only a fewpairs. An average mating speed for unselectedcontrols is 2 .5 min and, in practice, any matingspeed above 30 min is best regarded as `veryslow'. When so few SA flies mated it becameunpractical to keep to the original criterion andwait for the last ten pairs which remained un-mated. Ten pairs were selected at random fromthe ones remaining after 30 min . In spite of this

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relaxation of selection pressure, the mating speedof SA continued to be far slower than that ofcontrols.

In previous selection experiment, (Manning,1961) two slow-mating lines of D. melanogastershowed marked changes in the behaviour ofboth sexes. Not only was their sexual behaviouraffected, but both males and females had greatlyincreased reactiveness to disturbing stimuli ofall types. The behavioural changes in the SA linewere quite unlike these .

No obvious differences in reactivity could bedetected, and the changes in sexual behaviourwere confined to females ; the males of SA usuallyshowed intense and persistent courtship withina few seconds of introduction to females . Figure 2shows graphs derived from mass matings when(a) SA males courted control females, (b) controlmales courted control females ; in both thesecases mating was rapid . Conversely either typeof male mated slowly when paired with SAfemales (c and d) . There are some indicationsthat the performance of SA males is superiorto that of unselected controls . It is quite possiblethat there has been selection for good courtshipamong SA males, since only the most persistentcan leave descendants if they are paired withSA females .The effects of selection on SA females are

obvious when observing mass matings . On day 3,control females are quite passive when courtedby males and accept them within a minute or

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Fig. 1 . The course of selection for slow mating over twenty-five gener-ations . In each generation the mating speed in controls is adjusted to avalue of 100 and the mating speed of SA expressed as a deviation fromthis, using a log scale .

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ANIMAL BEHAVIOUR, 16, 1

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Fig. 2. Graphs of mating speed when, (a) SA d d courtcontrol ?Y ; (b) control d d court control ?? ; (c) SA d dcourt SA?? and (d) control c16 court SAY?. Fliestaken from S18 during the course of selection .

two, many within a few seconds . By contrastSA females perform the most vigorous repellingmovements, extruding the ovipositor and twist-ing or lifting their abdomen beyond the reach ofa courting male. Normally such movements areshown only by elderly virgins which have be-come unreceptive, or by fertilized females whosereceptivity is inhibited by the presence of storedsperm in their seminal receptacles (Manning,1967). Extrusion of this type has a marked in-hibiting effect on courting males, which usuallyturn away (Bastock & Manning, 1955) . Conse-quently during a mass mating SA males oftenswitch their attention from female to female.Nevertheless most of them eventually persist andcontinue to court .The receptiveness of SA females was ex-

amined in more detail using single-pair matingsin observation cells. `Courtship time' was meas-ured from the start of courtship until the femalewas mounted, and males were replaced unlessthey courted persistently. Fortunately males willcourt females of all ages equally well and it wasusually possible to ensure that SA females re-ceived more or less continuous courtship through-out a 30-min observation period. Females whichhad not mated within 30 min were called 'un-receptive'. Such females are inaccessible to malecourtship and there is no evidence that continu-

ing observations beyond this time wouldappreciably alter the number of females re-recorded as unreceptive (see Manning, 1967, forfurther discussion on this point) .

Figure 3 shows frequency histograms forcourtship times of unselected controls and SAfemales over the first 3 days of adult life, duringwhich period normal females change from theunreceptive to the receptive state. Controlfemales showed the typical pattern of receptivityby day 2, but less than one third of SA femaleswere receptive either on day 2 or on day 3 . Thegreat majority did not become receptive and,although a few accepted males normally, agood proportion of those that did mate appearedto be `raped'. In such cases, although the femalemight be seen struggling to dislodge the malesthroughout copulation, she was recorded asreceptive unless she succeeded in doing so within5 min. SA females have been tested at variousages beyond 3 days, but the pattern does notchange and they never accept males in the normalway.

Corpus Allatum and Receptivity in the SA Line

If SA females fail to become receptive, thereis presumably some breakdown in the controlsequence between the activation of the corpusallatum complex and the action which a hormone-probably the juvenile hormone-has upon theneural or neuro-secretory mechanisms respons-ible for the change in behaviour . It has beenpossible to eliminate some of the potentialpoints at which breakdown could occur.

The corpora allata of SA females appear togrow and secrete normally after eclosion . This isreflected by the normal growth of their ovaries .A measure of size was obtained by weighingthe photographic image of untreated wholemounts of the ovaries, as described by Man-ning (1967). Table I shows that SA ovariesactually grow faster than those of normalfemales between day 0 and day 1 (the differenceon day 1 is significant, using the Wilcoxon test,P < 0 .01) but otherwise they show no changefrom the normal cycle .

This result offers strong circumstantial evid-ence that the failure of SA females to becomereceptive is not due to lack of juvenile hormone .This conclusion has been confirmed moredirectly by cross-implanting corpora allata(together with the fused corpus cardiacum andhypocerebral ganglion) between normal and SAfemales. Manning (1966, 1967) showed thatimplanting corpus allatum complexes from

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MANNING: SELECTION FOR SLOW MATING IN DROSOPHILA

mature females into pupae of normal flies justbefore eclosion, induced precocious receptivity .Almost 100 per cent of the host females werereceptive on day 1, whereas it is rare to find morethan 20 to 30 per cent `naturally precocious'females .

Table II shows the results of cross-implanta-tion between normal and SA females. The earlierwork revealed no difference between control-implanted pupae, which received a piece of thedonor flies' aorta, and controls which receivedno implant. Only the latter were used in thiscase. Corpus allatum complexes from SAfemales were capable of inducing precociousreceptivity in normal females, but the implant-

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Fig. 3 . Frequency histograms of courtship times for controls and SAfemales on days 1, 2 and 3 .

Table I. Ovary Growth of SA and Normal Virgin Females, from Eclosion to Day 6

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ation of normal complexes had no effect on SAfemales. Attempts to `flood' their system withthree complexes per SA pupa have so far beenunsuccessful with high mortality.

Discussion

These experiments certainly suggest that SAfemales have a normal supply of juvenile hor-mone and that if, as seems probable, this is theactive factor from the corpus allatum complex,the genetic changes affect one or more links inthe chain of `target organs' upon which thehormone acts . We have hitherto assumed thatit is the development of receptivity which fails inSA females, yet it is equally possible to argue

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Day 1 14 530 .4 127 . 6 14 321 .5 114.9

Day 2 15 1149 . 3 301 . 7 15 1137 . 5 195 .2

Day 3 10 1241 . 2 214 . 5 8 1012 . 5 181 . 3

Day 6 4 1723 . 2 256 . 1 4 1437 .5 234 . 8

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Table II . The Effects on Receptivity when Corpus Allatuni Complexes areCross-implanted between Normal and SA Females

that they become receptive, but the court-ship summation mechanisms are so stronglyaffected that the female is never brought abovethe threshold for acceptance .

It is impossible to distinguish between thesealternatives at present . In fact, whichever istrue, the effects in the SA line seem to be ratherexceptional . In other cases where selection forslow mating has been tried (Manning, 1961) it iscourtship summation alone which appears tohave been affected. Thus females of two separatelines of D. melanogaster with slow mating be-came receptive normally, i .e. they were un-receptive on day 1 and receptive by day 2 or 3,but they required rather more courtship thannormal females before they accepted males .There are various ways in which genes couldaffect courtship summation-by changing senseorgans, for example-without affecting thesummation process itself. Nevertheless if theeffects are central, they clearly can occur withoutaffecting the central mechanisms responsiblefor receptivity .

As far as it goes, the analysis of gene actionin the SA line reveals a situation strictly com-parable to that in some strains of mammals .Different inbred lines of guinea pigs show markeddifferences in the frequency with which theyperform various sexual behaviour patterns . Theperformance of these patterns is known to begreatly affected by circulating hormones, but thegenes affecting sexual behaviour differencesbetween the lines do not affect the endocrinesystem . They act on target organs in the nervous

The difference between normal females which received an SA complex andunimplanted controls is highly significant (x2 with Yates's correction =12 . 78; P < 0 .001) .

system which respond to hormones (Grunt &Young, 1953 ; Goy & Young, 1957) . Similarly,although an endocrine link operates in thenormal control of sexual behaviour in femaleDrosophila, this is not the link affected in the SAline. As with the guinea-pigs a neural targetorgan is the most likely site of gene action .

Summary1 . A slow-mating line of D. simulans has been

produced by artificial selection, in which thebehaviour of males is not affected but in whichthere are marked effects upon females .

2. The great majority of the females of thisline (SA) fail to become receptive on the 2ndday from eclosion, as do normal flies .

3. The development of receptivity followsthe activation of the corpus allatum-corpuscardiacum complex after eclosion. SA femaleshave normal ovarian growth, and their corpusallatum complex when implanted into normalhosts is capable of inducing precocious receptiv-ity .4. It is concluded that the failure of SA to

become receptive is not due to endocrine dis-turbances, but to some change in the neuraltarget organs upon which the hormone acts .

AcknowledgmentsIt is a pleasure to acknowledge my indebted-

ness to Miss M. C. Hill for her excellent tech-nical assistance throughout this work. I am grate-ful to the Science Research Council for a grantfor special research .

Normal females N Receptive onday 1

Unreceptive onday 1 but re-ceptive on day

2 or 3

Implanted with SA corpusallatum-complex as pupae 15 11 4

Non-implanted 13 0 13

SA females

Implanted with normal corpusallatum-complex as pupae 13 0 0

Non-implanted 22 2 0

MANNING: SELECTION FOR SLOW MATING IN DROSOPHILA

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REFERENCESBastock, M. & Manning, A. (1955). The courtship of

Drosophila meksagaster. Behaviour, 8, 85-111 .Goy, R. W. & Young, W. C . (1957). Strain differences in

the behavioural responses of female guinea pigs toalpha-estradiol benzoate and progesterone. Be-haviour, 10, 340-354 .

Grunt, J . A. & Young, W. C. (1953). Consistency ofsexual behaviour patterns in individual maleguinea pigs following castration and androgentherapy. J. comp. physiol. Psycho!., 46, 138-144.

Manning, A. (1961). The effects of artificial selection formating speed in Drosophila melanogaster. Anim.Behav., 9, 82-92,

Manning, A . (1966). Corpus allatum and sexual receptiv-ity in female Drosophila melanogaster . Nature, 211,1321-1322.

Manning, A. (1967) . The control of sexual receptivity infemale Drosophila. Anim. Behav., 15, 239-250.

(Received 17 February 1967 ; revised 22 April 1967 ;Ms. number : 726)