Ent. e rp. & appL 8 (1965): 49--64. North-Holland P,hlishing Co., Amsterdan2 WING POLYMORPHISM IN APHIDS II. INTERACTION BETWEEN APHIDS BY BRUCE JOHNSON Waite Agricultural Research Institute, University of Adelaide, Australia A study has been made of the effect of "crowding" on both prenatal and postnatal control of wing development in aphids. In experiments on prenatal form control it was shown that brief controlled encounters of only a minute's duration between two adult aphids could cause them to switch from producing apterous to alate progeny. Aphids which had been exposed to contact with other aphids continued to produce alate progeny for several days. Evidence from a number of experiments suggests that the principle stimulus involved is tactile. In experiments on postnatal form control, it was shown that more alates developed among larvae which were reared together than among larvae reared in isolation. The appearance of winged forms of aphids has long been thought to be associated with crowding. Until recently it was thought that the factor causing alate production may have been a nutritional deficiency resulting from damage to the host plant caused by many aphids feeding on it. From a series of experiments in which adults and larvae of Brevicoryne brassicae and Myzus persicae were confined at different densities in small cages on their host plant, BONNEMAISON (1951) produced evidence which suggested that direct stimulation by other aphids could be a significant factor in promoting wing production. LEES (1961) found that when several young adults of Megoura viciae were kept together in a tube off their host plant for several hours they produced winged progeny whereas aphids kept isolated in tubes for the same time produced wingless progeny, thereby conclusively demonstrating that interaction between aphids was a significant factor influencing prenatal control of wing production in that species. This paper is one of a series concerned with the control of wing polymorphism in aphids. In the first paper (JOHNSON& BIRKS, 1960) the developmental process underlying production of the different morphs was described; this paper deals with the effects of interaction between aphids. In a further paper the effects of host, temperature and photoperiod on wing polymorphism will be discussed. MATERIALS AND METHODS The aphids used in this study were apterous alienicolae from a clone of Aphis craccivora Koch. The host plants were broadbean, (Vicia faba L.) which were grown in a glass house. Adult aphids were transferred either with a pair of fine pointed
W I N G P O L Y M O R P H I S M I N A P H I D S II .
I N T E R A C T I O N B E T W E E N A P H I D S
BY
BRUCE JOHNSON
Waite Agricultural Research Institute, University of Adelaide, Australia
A study has been made of the effect of "crowding" on both prenatal and postnatal control of wing development in aphids. In experiments on prenatal form control it was shown that brief controlled encounters of only a minute's duration between two adult aphids could cause them to switch from producing apterous to alate progeny. Aphids which had been exposed to contact with other aphids continued to produce alate progeny for several days. Evidence from a number of experiments suggests that the principle stimulus involved is tactile. In experiments on postnatal form control, it was shown that more alates developed among larvae which were reared together than among larvae reared in isolation.
The appearance of winged forms of aphids has long been thought to be associated with crowding. Until recently it was thought that the factor causing alate production may have been a nutritional deficiency resulting from damage to the host plant caused by many aphids feeding on it. From a series of experiments in which adults and larvae of Brevicoryne brassicae and Myzus persicae were confined at different densities in small cages on their host plant, BONNEMAISON (1951) produced evidence which suggested that direct stimulation by other aphids could be a significant factor in promoting wing production. LEES (1961) found that when several young adults of Megoura viciae were kept together in a tube off their host plant for several hours they produced winged progeny whereas aphids kept isolated in tubes for the same time produced wingless progeny, thereby conclusively demonstrating that interaction between aphids was a significant factor influencing prenatal control of wing production in that species.
This paper is one of a series concerned with the control of wing polymorphism in aphids. In the first paper (JOHNSON & BIRKS, 1960) the developmental process underlying production of the different morphs was described; this paper deals with the effects of interaction between aphids. In a further paper the effects of host, temperature and photoperiod on wing polymorphism will be discussed.
MATERIALS AND METHODS
The aphids used in this study were apterous alienicolae from a clone of Aphis craccivora Koch. The host plants were broadbean, (Vicia faba L.) which were grown in a glass house. Adult aphids were transferred either with a pair of fine pointed
50 BRUCE JOHNSON
tweezers or by applying to their dorsal surface a small diameter tube to which oral suction was applied. Larvae were transferred with a camel hair brush.
All the experiments were conducted in constant temperature rooms set at 20 ~ . Batteries of eight 60 watt fluorescent light tubes were suspended 50 cm above the benches giving a light intensity of about 400 ft. candles at bench level. The lights were controlled by a time switch to give a 10 hour photoperiod, i.e. 10 hrs light and 14 hrs darkness per day. They produced an increase in temperature at bench level of 1--2 ~ while they were switched on, causing a small daily temperature fluctuation. All the experiments reported in this paper were conducted on aphids which had been maintained for several generations at 20 ~ and 10 hrs photoperiod. The effects of other conditions of temperature and photoperiod will be reported elsewhere.
The aphids were reared and maintained on leaf discs 1.5 cm in diameter which were punched out of young fully expanded leaves of broadbean plants. The discs were floated on Knops nutrient salt solution contained in small plastic vials with a diameter of 1.9 cm and these were kept in plastic trays containing 84 vials. The discs used in the experiments remained able to support aphids for over 2 weeks after being cut and two generations of aphids could be reared on them from birth to maturity. The aphids took about 6 days to develop from the 1st instar to adults. When too many aphids were crowded together on the leaf discs the general condition of the discs deteriorated rapidly and the aphids would not remain feeding on them.
Aphids feeding on leaf discs are undoubtedly exposed to an environment which differs in a number of respects from that which they normally experience when feeding on a plant. The primary difference is probably in the chemical nature of the sap they feed on. As a result of being severed from the plant, proteins are broken down into peptides and amino acids in the leaf discs, and other changes take place resulting in a gradual senescing and deterioration of the tissues. On leaf discs, aphids must remain on top of the disc whereas on whole plants they normally feed either on the undersurfaces of leaves or on stems and in both cases are suspended either vertically or upside down: they are rarely found in nature on the exposed upper surface of leaves. Leaf discs were floated upside down on the nutrient solution so that the aphids fed on the morphological lower surface of the leaf.
When young larvae were placed on discs they usually settled down to feed within an hour. Adult aphids were more inclined to step off the discs into the solution; they were therefore confined on them under small light-weight cages. The cages were made from slices of clear plastic tubing with an internal diameter of 3 mm and to one end of which a piece of nylon gauze was welded by heat. Under these cages the aphids soon settled down and when they were left overnight they were almost invariably found on the following day feeding and surrounded by young larvae which they had produced. The parent aphids were then removed and the larvae left uncovered on the leaf discs to develop. As long as there were not more than
WING POLYMORPHISM IN APHIDS 1I 51
12 to 15 larvae per disc the larvae remainded feeding on them until they became adult, at higher densities than this they sometimes walked off before reaching maturity. By transferring adult aphids daily to fresh leaf discs and leaving the larvae produced each day on the discs on which they were born, it was possible to rear each day's progeny separately for considerable numbers of adult aphids using a relatively small amount of bench space.
STUDIES ON P R E N A T A L F O R M CONTROL
Experimental procedure
When studying the factors influencing wing production, it is important to distinguish between prenatal and postnatal form control. In A. craccivora the form of developing aphids can be influenced either during late embryonic development or during the first two larval instars. The aphids begin development as presumptive alates and they continue along the alate course of development until and unless they are diverted from this to the apterous course of development (JOHNSON & BIRKS 1960). In the following experiments prenatal control was studied by exposing the parent aphids to different conditions and then placing them on leaf discs to reproduce. The parents were then removed and the larvae were left on the discs to develop to the fourth instar, when their form could be assessed. Aphids which produced fewer than 5 larvae were discarded. It has been found that when larvae are reared at a density of five or more per leaf disc at 20 ~ there is virtually no postnatal diversion (see p. 59) so that any larvae which developed into apterae when reared under these conditions must have undergone prenatal diversion and the proportions of the different forms therefore reflected prenatal form control.
Interaction between aphids favours alate production. To study this response it was necessary to ensure that apart from controlled encounters with other aphids, the aphids being studied were reared and maintained in total isolation. It was also necessary for the aphids to be maintained under general conditions which favoured aptera production for only then would it be possible to demonstrate an effect of an alate-favouring stimulus. Aphids which were reared in isolation from the first instar on leaf discs and were left to reproduce on their original discs produced a high percentage of apterous progeny. In the experiments which follow the aphids were reared in isolation and were then taken off their discs and starved in isolation in small cages for 2 hrs. This procedure was adopted to ensure that they became active before being exposed to the experimental treatments, as aphids frequently remained quiescent and unresponsive to any stimuli for considerable periods after being removed from their host. They were then exposed to controlled periods of contact with other aphids and immediately returned to fresh leaf discs where they were left
overnight to reproduce. The results of the experiments on prenatal control of form are expressed as the
percentage of parent aphids which produced 50% or more alate progeny on the leaf discs. Most aphids produced almost exclusively progeny of one form or the
52 BRUCE JOHNSON
other and relatively few of them produced mixed batches in which there were equal numbers of both forms. This is shown in Fig. 1. In over 80 % of aphids used in the experiments the predominant form accounted for more than three quarters of the progeny.
I L I l 1 I I I l I
Z ~o
i v tad o _
15 >-
,o IJA
(2, l.d 5 ta.
O IH I I I I I I t I I I I
IO gO 3O 4O SO 60 7O 80 gO I00
PERCENT ALATE I-ARVAE/INDIVIDUAL APHID
Fig. I. F r e q u e n c y dis t r ibut ion o f the percentage alate p rogeny p roduced by 1096 individual
parent aphids when left overnight on fresh leaf discs.
For all experiments a control series of aphids was kept. These aphids were treated in exactly the same way as the experimental series except that they were isolated throughout the experiment. The results of the controls for the individual experiments did not differ significantly between experiments (P < 0.001) and these have been pooled together in Table Ia.
Aphids confined together in cages
Aphids which had been reared in isolation were confined off their host in 3 mm plastic cages with another aphid for various periods. As a result of being kept together in the cages the aphids produced many alate progeny when they were returned to individual fresh Deaf discs (Table I, b, c). In the early experiments the aphids were confined together for several hours but much shorter periods were later found to be effective. Aphids which had been reared in isolation were then subjected to a number of different treatments in an attempt to analyse further the nature of the stimuli involved in this response.
Apterae of Aphis craccivora were confined in cages with alates of two different species, Myzus persicae Sulz. and Brevicoryne brassicae L. When confined with
WING POLYMORPHISM IN APHIDS 1i 53
either of these species virgins of A . craccivora produced alate progeny indicating
that the stimulus involved was not species specific (Table I, d, e).
Aphids were also caged for one hour with five first instar larvae. When they
were later placed on leaf discs they produced some alatiform progeny although the
response was not nearly as marked as it was when aphids were confined with other
adult aphids (Table I, f).
TABLE I
The influence of various treatments on prenatal form control in aphids which were otherwi,~e reared and maintained in isolation
No. of Average No. % alate Treatment of larvae/
adults producers * adult
568 8.4 68 7.6 20 8.0
a. Isolated controls: pooled for all experiments 12 b. Two aphids caged together for 6 hours 94* c. Two aphids caged together for 15 minutes 95* d. Aphids caged with an alate adult of Myzus per6i-
cae for 1 hour 27 7.3 93* e. Aphids caged with an alate adult of Brevicoryne
brassicae for 1 hour 30 5.9 87* f. Aphids caged with 5 first instar larvae for 1 hour 35 8.2 50* g. Aphids caged together in darkness for I hour 24 6.2 100" h. Two aphids caged alongside one another for 1
hour but separated by glass screen 36 6.8 8 i. Aphids caged together for one hour after ampu-
tation of antennae 44 7.2 87* j. Aphids caged with body of dead aphid for 1 hour 24 8.6 17 k. Aphids caged with another aphid, which had its
appendages stuck down with wax, for 1 hour 24 7.5 12 I. Aphids isolated on rim of vial for 1 hour 50 7.8 10
m. Two aphids on rim of vial for 1 hour 52 7.5 89* n. Aphids crawled over 10 times by another aphid;
off host 88 8.1 50* o. Aphids which were made to crawl over other
aphids 10 times 16 7.4 ! 2
§ Aphids which produced 50% or more alate progeny when confined overnight on fresh leaf
discs. * Difference between treatment and control series significant at P < .001.
TABLE II
Influence on prenatal [orm control of repeatedly touching aphids with a hair for 2 minutes
No. of Average no. % alate Treatment of larvae/ P
Adults adult producers
Controls 49 8.4 2 .006
Brushed 45 7.6 22
54 BRUCE JOHNSON
Visual, Olfactory and Tactile Stimuli
Virgin aphids that had been kept isolated off their host for 2 hours in the light were taken into a dark room and in total darkness the cages in which they had been isolated were emptied into a 2.5 cm X 5.0 cm glass tube which was then covered with gauze. After the aphids had been together in the tube for 1 hour they were tipped out into a large tray. After a few minutes the light was switched on and the aphids, which by this time had dispersed on the tray, were collected and again isolated in individual cages. At no time were the aphids closer than 3 cm to one another in the light. All of the aphids treated in this way produced alate progeny (Table I, g) indicating that the interaction effect could be produced in the absence of visual stimuli.
In a further experiment aphids were confined in very close proximity to one another in the light for 2 hours but separated by a glass screen. This was done by placing a small piece of glass from a coverslip in the small plastic cages, so dividing them into two chambers. One aphid was introduced into the cage on each side of the glass screen. Aphids caged alongside each other in this way produced apterous progeny, suggesting that visual stimulation alone is inadequate as an alate-promoting stimulus (Table I, h).
If olfactory stimuli were involved it was thought that these may be received through sensoria on the antennae. Accordingly the antennae were amputated at the base of the third segment in a number of virgin apterae and the aphids were then confined together for 1 hour in a cage. They all produced alate progeny (Table I, i), indicating that the antennae were not essential for the transmission of the stimulus from one aphid to another.
Aphids were also confined for 1 hour in cages with the bodies of other freshly killed aphids and with other live aphids of which the legs and antennae had been stuck together with wax so that they were unable to move. Neither of these treat- ments caused alate production (Table I, j, k).
The results of the experiments described suggested that a tactile stimulus was involved when aphids were caged together and an attempt was therefore made to apply an artificial tactile stimulus. A number of aphids were touched repeatedly on the legs, antennae and body with a bristle from a camel hair brush. They were then placed on fresh leaf discs. The results are given in Table II. More of the brushed aphids produced alate progeny than the controls and the difference was significant. However the treatment was very much less effective than stimulation by other aphids. The reason for this is not yet known but it is possible that either the precise way in which the tactile stimulus was applied may be important, or tactile stimuli may need to be reinforced by visual or olfactory stimuli to secure a full response.
Repeated encounters on a "Catwalk"
Aphids placed on the rim of a petri dish continue to walk round it for some time as long as they have overhead illumination and there is a screen around the
WING POLYMORPHISM IN APHIDS I1 55
dish to eliminate extraneous visual stimuli (IBBoTsoN & KENNEDY, 1959). An attempt was made to use this method to arrange encounters between two aphids. The diameter of the petri dishes which were available was too great, as the aphids frequently travelled in the same direction and, if they were moving at roughly the same speed, they rarely met one another. The rim of a small plastic vial 2 cms in diameter was then used. The vial was placed in water so that only its rim projected above water level. Two aphids were then placed on the rim. On their first few en- counters the aphids generally recoiled and appeared to be repelled by one another. On later encounters they generally stopped and probed and then one aphid often crawled over the other and proceeded on its way. Sometimes after meeting, one of the aphids would turn around and move off in the same direction as the other. Because of the irregularity of their behaviour, records were not kept of the numbers of encounters between individual aphids and the aphids were simply left on the rims singly and in pairs for periods of one hour. When they were returned to leaf discs the aphids which had been isolated on the rims produced mainly apterous progeny whereas those kept on the rims with another aphid produced mainly alate progeny (Table I, 1, m).
Aphids placed on top of one another
In the above experiments the numbers of encounters between aphids could not be experimentally controlled. In order to obtain a closer control of the meetings between aphids, enforced encounters were arranged. This was done by picking an aphid up with a suction tube and releasing it with its fore legs on another aphid so that it proceeded to crawl over the bottom aphid. As a standard treatment aphids were subjected to being crawled over in rapid succession 10 times by another aphid while on the laboratory bench. The whole process lasted between 1 and 2 minutes. This experiment was repeated on a number of occasions. While they were being crawled over, the bottom aphids remained perfectly still with their antennae held close to their bodies. After the treatment the aphids were picked up and returned to fresh leaf discs. These brief encounters were sufficient to induce many of the aphids to produce alate progeny (Table I, n).
In a further experiment twelve aphids were crawled over ten times by another aphid while they were still on their original leaf discs. During the treatment five of the aphids did not withdraw their stylets, six withdrew them but did not walk away until the treatment was concluded and one withdrew its stylets as soon as it was disturbed and continued to crawl about on the leaf disc even when the other aphid was placed on top of it. This aphid went on to produce apterous progeny; all the others produced exclusively alate progeny (Table III). All but one of the control aphids produced one or more apterous progeny indicating that the treatment of being crawled over ten times in situ on the host tissues had been effective in inducing alate production. The leaf discs used in this particular experiment were in poor condition by the time the aphids moulted into adults and there was consequently a high "basic" alate production among the controls and the threshold of response to
56 BRUCE JOHNSON
stimulation by other aphids was very low with the result that they readily responded to the aphids which crawled over them by switching to alate production.
TABLE I I I
Form of the progeny of individual aphids which were crawled over 10 times by another aphid while still on their original leaf discs.
Crawled over 10 times Controls Parent No. Alate Apterous Parent No. Alate Apterous
% alate progeny 91% 62% Percentage of parents which produced exclusively 92 % 8 % alate progeny
It is apparent from these experiments that the aphids which were being crawled over were receiving some stimulation from the top aphid. Was the top aphid also stimulated by the experience? In one experiment a number of aphids that had been made to crawl 10 times over other feeding aphids were put on leaf discs and the
form of their progeny was assessed. These aphids mostly continued to produce apterous progeny indicating that they were apparently unaffected by the treatment
despite their contact with the bottom aphids through their tarsae and antennae (Table I, o). This result could be explained in either of two ways. Either these aphids were not being stimulated by the bottom aphids or else they were being stimulated but the repeated handling counteracted any stimulation that they had received. This latter possibility was tested by handling aphids which had been crawled over, by picking them up and releasing them several times. This treatment did not cancel the effect of their previous treatment and cause them to produce apterae. It appears therefore that the aphids which were picked up and made to crawl over other aphids did not receive the same stimulus from contact with the bottom aphid as that aphid received from them. These experiments further support
WING POLYMORPHISM IN APHIDS II 57
the hypothesis that the main stimuli involved in the production of the interaction response are tactile rather than visual, auditory or olfactory.
Age o[ aphids when exposed to contact with other aphids
In the experiments described so far the aphids were exposed to contact with other aphids after the final moult and before they had begun to reproduce. Aphids of other ages were also tested.
Aphids were reared isolated on individual leaf discs until about half way through the fourth instar. They were then collected and 50 were confined together in a 2" by 1" glass tube for 4 hours. They were then re-distributed on fresh leaf discs and allowed to develop to maturity and reproduce for one day after which they were removed from the discs and their progeny reared. Their progeny were predominant- ly alates (Table IV). Crowding experienced during larval development can thus have an effect on the form of progeny produced by aphids when they become adult and begin reproducing. In this experiment the larvae were not born until 36 to 40 hours after the parents had been caged together as fourth instar larvae.
TABLE IV
EJJec't on prenatal [orm control of confining aphids together Jor 4 hours daring the fourth larval instar
Treatment n
Controls: isolated throughout development 150 Crowded 4 hours in 4th instar 128
Average % alate number of producers larvae/adult
7.4 14 6.8 88
Old adult aphids were also exposed to contact with other aphids. These aphids had been reared on individual leaf discs until they became adult and were then transferred daily to new leaf discs for 5 days. On these discs they produced mainly apterous progeny. They were then collected and crowded together in a tube for 4 hours. There was a marked increase in the number of alates produced following crowding indicating that aphids which have been reproducing for several days are as responsive to contact with other aphids as young virgin adults.
Persistence o[ "'interaction" effect
In the previous section it was shown that larvae that were exposed to contact with other aphids in the fourth instar produced alate progeny when they became adult. This suggested that when adult aphids are exposed to contact with other aphids the effects may persist for some time, and an experiment was therefore conducted, to determine for how long the effects of a brief period of crowding could persist.
Aphids which had been reared isolated on leaf discs were divided into two
58 BRUCE JOHNSON
batches. In one batch the aphids were removed from their leaf discs, starved in
isolation for 2 hours and then replaced on fresh discs. The other aphids were treated in the same way except that they were confined together for 10 minutes in a 3 cu.cm cage before being replaced on fresh discs. Both lots of aphids were then
left on the discs overnight and at daily intervals were transferred to fresh discs. Their progeny were reared on the discs on which they were born. The results are given in Fig. 2 and the progeny produced by four typical individual aphids are
given in Table V.
I I I I I 1 I I I I I I
o o CROWDED (n =3o)
: : ISOLATED ( "=30)
- r I 0 0
Z -
U
5O I Ld
_J
0 I I I I I I I I I I I i I ~ s 4 5 6 7 8 9 io ii i~
DAYS
Fig. 2. Persistant effect on prenatal form control of crowding aphids together in a tube for 10 minutes as virgin adults and then transferring them daily to fresh leaf discs.
TABLE V
Form of the progeny o/ two typical aphids which had been caged ]or 10 minutes with other aphids as virgin adults, and of two isolated controls, over 12 successive days when transferred
* The first figure is the number of larvae which developed into alates, the second the number which developed into apterae.
As a result of being exposed to 10 minutes' crowding as virgin adults the aphids produced almost exclusively alate progeny for several days. Most of them gradually
WING POLYMORPltlSM IN APHIDS 11 59
reverted to aptera production over the following days although some continued to produce alates for the rest of the experiment. The control aphids on the other hand produced almost exclusively apterous progeny throughout the experiment.
STUDIES ON POSTNATAL FORM CONTROL
Experimental procedure
Larvae which have already been diverted from the alate course during their embryonic development can not be caused to develop into alates no matter how extreme the conditions to which they are subsequently exposed; larvae which have not already been diverted, and which therefore begin their larval development with an alate potential, can be diverted from the alate course in the first or second instar (JOHNSON & BIRKS, 1960). In studying postnatal form control it is therefore necessary to obtain larvae from parent aphids which have been exposed to alate-
�9 favouring conditions and in which a minimum of prenatal diversion has taken place. The larvae used in the following experiments were obtained from parent aphids which had been reared on leaf discs and then crowded together for several hours in a tube. The parents were then left overnight on detached bean leaves and the larvae deposited by them were collected the following morning in an aspirator and im-
mediately used in the experiments. It is also necessary when studying postnatal control to expose the larvae to
conditions which favour some degree of apterous development. If this is not done all or most of them will develop into alates and no effect of crowding will be able to be demonstrated. In the following experiments the larvae were reared on fresh leaf discs. These favoured a small but significant production of apterae. Seedlings favour apterous development more strongly but they could not be used because it was not possible on whole plants to ensure that the larvae remained in close proximity to one another (see also discussion).
Larvae crowded on host
Larvae were distributed on leaf discs at densities of one, five and ten per disc and left to develop to the 4th instar when their form was assessed. The results are
TABLE VI
Effect on postnatal form control of rearing larvae at different densities on leaf discs from tile first instar
No. of larvae/ Total No. of % Alates leaf disc larvae
1 311 64 5 442 88
10 566 92
60 BRUCE JOHNSON
given in Table VI. Although high percentages of alates were produced in all batches there were significantly more among the aphids reared at 5 and 10 per leaf disc than among those at 1 per disc (P < 0.001). Several experiments were conducted in which larvae were reared at one and two per leaf disc. There was a small but
consistently higher percentage of alates among the larvae reared at two per disc.
The pooled results of these experiments are shown in Table VII.
TABLE VII
Effect on postnatal form control of rearing larvae at densities of I and 2 per leaf disc
one/leaf disc
Alates 813 Apterae 323
Totals 1136
Pooled Z a = 15.84 (1 d.f.) Heterogeneity Z 2 = 5.30 (8 d.f.)
P < 0.001 P > 0.70
two/leaf disc
792 209
1001
Larvae crowded off their host
First instar larvae obtained in the same way as in the previous experiments were
confined together off their host plant. In this experiment the larvae were treated in three different ways. Some were transferred directly to fresh leaf discs, some were
starved in isolation for seven hours under small plastic lids and others were starved for seven hours crowded together in a single plastic lid with a capacity of 3 c.c. All the aphids were then reared at one per leaf disc. The results are given in Table VIII . Fewer larvae developed into alates among those which had been starved in isolation than among those which were not starved at all. This suggests that the period of starvation was having an apterising effect. But, because the larvae which were
crowded had a higher percentage of alates than the larvae which were starved in
isolation it is apparent that the effect of starvation can be counteracted by crowding. Thus, as with prenatal form control, interaction between aphids can also be im- portant postnatally and it is effective whether or not the aphids are on their host
plant when they encounter one another.
TABLE VIII
Effect on postnatal form control of caging first instar larvae together and in isolation for seven hours off their host, then rearing them isolated on leaf discs
Crowded or
Isolated
A. Isolated B. Isolated C. Crowded
Period of No. of larvae % alates starvation
Direct transfer 242 7 hours 286 7 hours 353
Difference between A and B; and B and C significant at P < 0.001.
52 22 60
WING POLYMORPIIISM IN APHIDS II 61
D I S C U S S I O N
Whether individuals of A. craccivora develop into alatae or apterae can be influenced both during prenatal and postnatal development. With postnatal form control, the larvae respond to stimuli received directly from the environment. With prenatal form control, on the other hand, the embryos are exposed to two different kinds of factors which may influence their course of development. Factors such as temperature, photoperiod and possibly nutrition, may influence the embryo directly. LEts (1964) has already shown that in Megoura v&iae photoperiod can act directly on developing embryos through the mother's cuticle. The course of development of embryos can also be influenced by the mother. This is apparent from the fact that very brief contacts between parent aphids can influence the form of progeny they produce, and also from the finding that when parent aphids are decapitated they immediately switch to producing apterous progeny (JOHNSON & BIRKS, 1960). This effect of decapitation is also interesting in that it indicates that the mother influences the form of its progeny just before or at the time the embryos are born.
The way in which the mother influences its embryos is not known but it is likely that some chemical agent is involved as there is no nervous connection between them. As the embryos pass down the oviduct their follicle epithelium is ruptured and it is possible that they may then be exposed to substances produced by the mother. If this is so, the chemical agent would be classifiable as a pheromone, a chemical produced by one animal and influencing behaviour or developmental processes in another animal.
In A. craccivora the aphids begin development as presumptive alates and they proceed to develop into alates unless or until they are diverted to the apterous course of development (JOHNSON & BIRKS, 1960). Several different factors, including temperature, photoperiod, host plant condition and contact with other aphids can all influence wing production. Thus, although the original ontogenetic course of development of the aphids is towards the alate form, whether or not they continue to follow this course, can be influenced not only by factors which favour apterous development and so promote diversion, but also by factors which favour alate development such as contacts with other aphids. Whether or not diversion occurs, appears to depend upon the integration of stimuli of several different kinds, some of which may favour alate development, others apterous development. With prenatal form control this integration may take place both in the mother influencing whether or not a stimulus is despatched to the embryo, and in the embryo itself.
The experiments described in this paper confirm the findings of BONNEMAISON (1951) and LEES (1961) that the "crowding" stimulus can be independent of the host in a sense that aphids can respond to direct stimulation by other individuals. It was shown that with aphids two individuals can constitute a "crowd". The experiments also indicate that very brief periods of stimulation of as short as 1 minute can produce a response, and that they can have an influence which persists for many days. The precise sensoria concerned in this response have not yet been identified. It appears likely that the principal stimuli are tactile. It is possible that
62 BRUCE JOHNSON
olfactory and visual stimuli may also be involved: the fact that it was not possible to demonstrate any effect of these does rule out the possibility that tactile stimulation may normally be reinforced by stimuli of other kinds.
Under the experimental conditions described in this paper very brief contacts were effective in influencing form control. When maintained under other conditions the aphids do not respond so readily. In a further paper it will be shown that the threshold of response of aphids to stimulation by other aphids can be influenced by temperature, photoperiod and the condition of the host plant.
The amount of stimulation by other individuals that aphids living in a colony on a plant would receive, would depend on the .degree of activity of the rest of the aphids in their vicinity. This, in turn, would be a direct reflection of the condition of the plant. On plants in poor condition and on wilting plants, aphids tend to withdraw their stylets and wander about more readily than they do on more favourab'.e plants, and they are also more restless in that they move their legs and antennae about more freely while feeding. Tactile stimulation may, in fact, commonly be brought about in this way rather than by aphids which have actually withdrawn their stylets and are crawling about. Alate production is thus more likely to occur on less favourable plants not only because of any direct influence the plant may have on form but also because of the influence of the plant on the behaviour of the aphids. Conversely, on very favourable plants not only will the nutritional status of the host directly favour apterous dev.elopment but the reduced locomotor activity due to favourable feeding stimuli will ensure that a minimum of tactile stimulation of the aphids by one another will take place. Any factors which result in reduction of locomotor activity in aphids will lead to reduced frequency of contacts between aphids and thus will contribute towards a reduction in alate production in the colony.
It is possible that the apterous-favouring effect of ant attendance of young larvae (JohNSON 1959) may also be due to reduced locomotor activity. Attendance by ants is known to reduce the tendency of aphids to move about and also to increase their rate of feeding (BANKS & NIXON 1958). Thus ants modify the behaviour of aphids in such a way that mutual stimulation of the aphids by one another is reduced, and food uptake increased. Both of these factors could be influential in preventing alate development. On the other hand it is still possible that where aphid form is influenced by ant attendance the aphids may also respond directly to the attentions of the ants.
Many contradictory results have been obtained in previous studies of the factors influencing wing polymorphism in aphids (see BONNEMAISON, 1951). It is suggested that some discrepancies may have been due to small differences in the rearing or handling of aphids. Because brief contacts between aphids can produce a significant persistent response, it is apparent that in studies on wing polymorphism the insects used must be isolated throughout their lives. In the past this has not been done and the aphids must have been exposed to uncontrolled contacts with other individuals.
WING POLYMORPHISM IN APHIDS 11 63
There are a number of instances of animals having their behaviour influenced by
stimuli which they have exper ienced for a brief per iod ear l ier in their lives. Im-
pr int ing can be regarded as a phenomenon of this kind. A n o t h e r instance is the
"handl ing" effect in newly born rats where brief handl ing of the animals has an
influence on their t emperamen t which persists throughout their lives. A m o n g
insects it has been found by ELLlS (1963) that when two locust hoppers which
have previously been isolated are kept together for as shor t a per iod as 30 minutes
in the first instar, their behaviour becomes modif ied in the direct ion of phase
gregar ia and in the 4th instar they still show gregaria pa t te rns of behaviour . The
persis tent nature of the response of aphids to contact with other aphids appears to
be a fur ther instance of this type of phenomenon.
ZUSAMMENFASSUNG
FLOGELPOLYMORPHISMUS BEI BLATTLA'USEN. 11. WECHSELW1RKUNG ZWISCHEN DEN APHIDEN
In grol3en Blatflauskolonien werden leichter Gefliigelte gebildet als in l~leinen. Dafiir gibt es zwei Ursachen. Bei hoher Dichte ver~indern die Aphiden die Physiologie der Pflanze der- gestalt, dab bei den L~iusen, die an ihr saugen, die Gefliigeltenbildung gef6rdert wird; und bei hoher Befallsdichte vermehrt sieh fiir die Aphiden auch die Wahrscheinlichkeit, stimulatorische Wirkungen aufeinander auszuiiben. In der vorliegenden Arbeit wird die Natur und Wirkungs- weise der Wechselwirkung zwischen Individuen von Aphis craccivora Koch untersucht.
Die Blattliiuse wurden bei 20 ~ und 10stiJndiger Photoperiode in v611iger Isolation auf kleinen Blattscheiben gehalten und weiter vermehrt, die - - aus Bl~ittern ihrer Wirtspflanze heraus- gestanzt - - auf einer Niihrl6sung schwammen. Unter diesen Bedingungen brachten sie iiber- wiegend ungefliigelte Nachkommen hervor. Zur Erzeugung gefliigelter Nachkommen konnten sie durch kurze Begegnungen mit anderen Aphiden angeregt werden. In einigen Versuchen wurden die Blattl~iuse fiir verschiedene Zeitabsehnitte in kleine Kiifige zusammengesperrt, in anderen wurden sie herausgefangen und veranlabt, in rascher Folge mehrere Male iibereinander hinwegzukriechen. Beide Behandlungsweisen bewirkten, dab die Aphiden vonder Produktion ungefliigelter zur Produktion gefliigelter Nachkommen umschalteten. Eine Anzahl von Ver- suchen bereehtigen zu der Annahme, dab weder visuelle noch olfaktorische Reize dabei mit- wirken, und es ist zu vermuten, dab in der Hauptsache Berfihrungsreize den Ausschlag geben. Der Versuch, einen entsprechenden kiinstlichen Reiz zu setzen, hatte nur zum Teil Erfolg.
Die Blattliiuse, welche einem Kontakt mit anderen Liiusen ausgesetzt gewesen waren, wurden t~iglich auf frische Blattscheiben weitergesetzt und ihre Nachkommenschaft auf den Blatt- scheiben aufgezogen, auf denen sie geboren wurde. Auf diese Weise l ieges sich zeigen, dal3 kurze Begegnungen zwischen Blattl~.usen mehrere Tage lang einen EinfluB auf die Form der Nachkommenschaft beibehalten k/Snnen, die sie danach produzieren.
Die Fliigelentwicklung von Aphis craccivora kann auch w~ihrend der postnatalen Entwicklung beeinfluBt werden und es lieB sich nachweisen, dab auch hierbei die Wechselwirkung zwischen den Larven einen wesentlichen Faktor darstellt. Isoliert auf Blattscheiben aufgezogene Larven entwickelten sich weniger h~iufig zu Gefliigelten als solche, die in Dichten yon zwei oder mehreren je Blattscheibe gehalten wurden.
64 BRUCE JOHNSON
REFERENCES
BANKS, C. J. & NIXON, H. L. (1958). Effects of the ant, Lasius niger L., on the feeding and excretion of the bean aphid, Aphis ]abae Scop. J. exp. Biol. 35 : 703--711.
BONNEMAISON, L. (1951). Contribution ~ l'6tude des facteurs provoquant i'apparition des formes ail6es et sexu6es chez les Aphidinae. Ann. (piphyt. 2 : 1--380.
ELLIS, P. (1963). Changes in the social aggregation of locust hoppers with changes in rearing conditions. Anita. Behav. 11: 152--160.
IBaOTSON, A. & KENNEDY, J. S. (1959). Interaction between walking and probing in Aphis [abae Scop. J. exp. Biol. 36 : 377--390.
JOHNSON, B. & BIRKS, P. (1960). Studies on wing polymorphism in aphids. I. The developmental process involved in the production of the different forms. Ent. exp. & appl. 3 : 327--339.
JOHNSON, B. (1959). Ants and form reversal in aphids. Nature, Lond. 184" 740. LEES, A. D. (1961). Clonal polymorphism in aphids. R. ent. Soc. Lond. Syrup. 1 : 68--79. - - (1964). The location of the photoperiodic receptors in the aphid Megoura viciae Buckton.
