Ent. exp. & appl. 9 (1966): 213--222. North-Holland Publishing Co., Amsterdam

W I N G POLYMORPHISM IN APHIDS III. THE INFLUENCE

OF THE H O S T P L A N T

BY

B R U C E J O H N S O N * Waite Agricultural Research Institute, University of Adelaide, Australia

The condition of the host plant can have an important influence on the development of winged forms in Aphis craccivora Koch. Prenatal form control can be influenced both by the condition of the host on which the parent aphids have been reared and by the host on wb.ich they are currently feeding while reproducing. Postnatal form control can be influenced by the host an which larvae are reared during: their first two instars. Whether or not the host in- fluences wing development and the extent of its effect depend upon other factors such as stimulation of aphids by other individuals. Prolonged periods of starvation both of parent aphids and of young larvae were shown to favour apterous development.

It is a c o m m o n observat ion that alate aphids are produced more abundantly on mature, old and wilting plant tissues than on seedlings and growing shoots. Despite this, attempts in the past to study the effect of the condition of the host on the control of wing polymorphism have not yielded consistent results, and different authors have been led to draw opposing conclusions. BONNEMAISON (1951), who made a comprehensive review of the literature on wing polymorphism in aphids, lists a number of authors who have claimed that the condit ion of the host is an important factor, and others who have concluded that the host was of little importance or that its influence was dominated by other factors.

I t is known that wing development in aphids is able to be influenced by a number of different factors other than the host-plant. These factors include temper- ature, photoperiod, at tendance by ants, and interaction between aphids. In study- ing the effect of any single factor it is essential to take into account, and if pos- sible control, all factors which may have an influence: under certain conditions a part icular factor may play an important and decisive role whereas under other

conditions its effect may be overshadowed. In this paper it is shown that the host can influence both prenatal and postnatal

fo rm control in Aphis craccivora, but whether or not it plays a decisive role is

determined by other factors.

MATERIALS AND METHODS

The materials and methods employed in the present study were substantially the same as those used in previous studies in this series which should be consulted for

* Present address: Department of Zoology, University of Tasmania, Australia.

214 BRUCE JOHNSON

details. (JOHNSON d~ BIRKS 1960; JOHNSON 1965). The aphids were aliencolae from a clone of A. craccivora and the host plant was broad bean, Vicia laba L. (var. longpod). The aphids were kept on bean seedlings and cuttings and on 1.5 cm diameter leaf discs floated on Knops nutrient solution. The aphid cultures were maintained and all experiments were conducted in a constant-temperature room at 20 ° ----- 2 °. Banks of seven fluorescent light tubes controlled by a time switch were suspended 50 cm above the benches and were switched on for 10 hours each day. The effect on wing production of different conditions of temperature and photo- period will be reported in a further paper.

Three types of host plant tissue were used in the experiments. Seedlings: Broad bean seeds were planted in John Innes compost and allowed

to germinate and grow for a few days in a glasshouse. The plants were used when they were about 6 cm high and a single pair of leaflets had unfolded.

Fresh lea/discs: Discs measuring 1.5 cm in diameter were punched out of mature leaves taken from halfway up the stem of 3 0 - - 5 0 cm high broad bean plants which had been grown in the glasshouse. Aphids were put on the discs within a few hours of the discs being cut. In one experiment, which will be indicated in the text, discs were cut from the leaves of seedlings.

Old lea[discs : Lea[discs were cut and were then kept in the constant-temperature room on Knops solution for seven days before being used. There was some variation in the condition of old discs and ones which had deteriorated badly were rejected. Most of them remained healthy but were slightly more yellowish than fresh discs. Aphids which were reared on discs from the first instar were put initially on fresh discs. By the time they had become adults, the discs they were on were several days old and would therefore be classed as old discs.

In studies on prenatal form-control, parent aphids were transferred to new discs as virgin adults having been reared from the first instar on specified host tissues. They were confined on the discs overnight under small plastic cages to reproduce. On the following day the cages and parent aphids were removed and the larvae which had been born were left on the discs to develop to the fourth instar, when the numbers of alate and apterous forms were coumed. The results of these ex- periments are given both as the percentage of "alate-producing parents", that is, the percentage of parent aphids of which half or more of their progeny developed into alates, and as the percentage of larvae which developed into alates among the total larvae produced by all aphids in each treatment.

When adult aphids are reproducing they tend to produce progeny of the one form unless there is a change in conditions that favours the production of the opposite form. Thus, when single adult aphids were confined for 24 hours on lea[discs in the constant temperature room where their environment remained relatively stable, they tended to produce larvae which were mainly either alate or apterous according to their previous treatment, with relatively few aphids producing equal or almost equal numbers of both forms (JOHNSON 1965). When aphids are left to reproduce on lea[discs for longer than this their environment undergoes change. The condition

WING POLYMORPHISM IN APHIDS n I 215

of the leafdisc rapidly deteriorates due to the increasing number of aphids feeding on it and the larvae tend to move about so exposing the mother to tactile stimula- tion. Both of these factors can influence prenatal form-control.

It has previously been shown that, when a number of larvae are reared together on leafdiscs at 20 ° and 10 tmurs light, there is very little postnatal diversion from .the alate to the apterous course of development (JoHNso~ 1965). In the experiments on prenatal form control, each of the parent aphids produced several larvae, so the percentage of alate and apterous forms which developed can therefore be taken as a reflection of prenatal control.

R E S U L T S

Prenatal Form Control First instar larvae were placed singly on fresh leafdises and on seedlings and

left to develop in isolation. After moulting into adults but before beginning to reproduce they were transferred individually to fresh leafdiscs on which they were left overnight. They were removed the next day and the larvae they had deposited were left to develop to the fourth instar when their form was assessed. The results are given in Table 1A. The aphids which had been reared on seedlings nearly all produced exclusively apterous progeny. Most of the aphids which had been reared on leafdiscs produced mainly apterous progeny and a few alates, while a small but significant number produced predominantly alate progeny. As all of the aphids in this experiment were kept on similar leafdiscs after becoming adults, any difference between the two groups must have been due to the conditions they experienced during their own larval development. It appears therefore that aphids which have been reared on seedlings produce alate progeny less readily than aphids reared on leaf discs.

In the experiment just discussed a high percentage of apterous progeny was produced by the aphids regardless of the host they had been reared on. By ex- posing the aphids to an additional alate-promoting factor it was possible to demon- strate more clearly the effect of the host on prenatal form control. In the following experiment aphids were exposed as virgin adults to a crowding stimulus (JOHNSON 1965). They were reared in isolation on either leafdiscs or seedlings as in the previous experiment. After moulting to adults, but before they had deposited any larvae at all, they were collected and then kept crowded together for one hour at a density of 5 per cage in small plastic cages with a capacity of 1 cc. They were then placed individually on fresh leafdiscs where they were left overnight to reproduce. The results of this treatment are given in Table IB. Both the aphids which had been reared on leafdiscs and the aphids reared on seedlings responded to the crowd- ing treatment by producing more Mate progeny when compared with the aphids in experiment A which were not crowded and which had produced predominantly apterous progeny. But, whereas nearly all of the aphids which had been reared on leafdiscs and were then crowded produced predominantly alate progeny, only 30%

216 BRUCE JOHNSON

of those reared on seedlings produced mainly alates. It appears therefore that the condition of the host on which aphids have previously been feeding can influence the extent to which they respond to contact with other aphids. That is, the host can apparently influence their threshold of response to other alate favouring factors.

In the experiments described so far all the aphid,s were transferred to fresh leaf- discs as virgin adults so that the differences attributable to the host were due to the host on which they had been feeding during their development and not to the one on which they were feeding while reproducing. This suggests that nutrition may be important in the control of form, either through the general level of nutrition of the aphids or through the availability of some more or less specific substance or substances in their diet. Were aphids also able to be influenced by the host on which they were feeding while they were reproducing?

Aphids were reared in isolation on leafdises and on seedlings as in the previous experiments. They were then transferred as virgin adults to old leafdiscs, and left overnight to reproduce. To minimise any possible influence of the leafdises on postnatal form control (see page 218), all larvae were transferred to fresh leafdiscs within 24 hours of being born. The results are given in Table IC. Many more alates were present among the progeny of the aphids which had been reared on discs than on seedlings, and the proportion of alates produced was much higher than among aphids reproducing on fresh leafdiscs (Table IA).

The increased alate production in this experiment compared with the previous experiment could have been brought about by either a direct or a n indirect in- fluence on prenatal form control of the host the aphids were on while reproducing. Apart from any direct influence the host may have had, involving a response of the parent to some property of the host tissue, the host may also have had an indirect influence through affecting the degree of "crowding" of the parent aphid by its own larvae. It has previously been shown (JOHNSON 1965) that exposure of adult aphids to contact with larvae can stimulate them to produce alate progeny. Aphids con- fined on leafdiscs with their own larvae can be stimulated by them to begin producing winged offspring if the conditions are such that the larvae are active and move about on the disc after being born. Fresh leafdiscs are more favourable for inducing settling in larvae than old leafdises; on old discs the larvae crawl about for longer before settling down to feed and in doing so they may stimulate the parent and so cause her to produce alate progeny. However, it is not possible to account for the increased alate production in this experiment solely in terms of a response of the parents to crowding by their own larvae, since some of the aphids produced exclusively alate progeny whereas this did not occur at all when uncrowded aphids reproduced on fresh leafdiscs (Table IA, C). It is apparent therefore that prenatal control of form can be influenced directly by the host that aphids are on while they are reproducing. It is apparent also that at least some of the aphids responded immediately on being transferred. This suggests that the influence of the host in these cases may have involved a response to taste rather than nutrition.

WING POLYMORPHISM IN APHIDS I I I 217

TABLE I

Effect on prenatal form control of the condition of the host on which aphids have been reared. The aphids were exposed to different treatments as virgin adults.

% Aphids % Aphids Treatment Host on which which % of aphids which No. produced produced Total Alate P*

Expt. as virgin aphids were of No. of (X 2 adults reared adults > 50% exclusi- Pro- alate vely alate Progeny geny test)

progeny progeny

Transferred Leafdiscs 123 11 0 1013 15 directly to ~ .001

A fresh leaf- discs. Seedlings 59 0 0 516 2

Crowded to- Leafdiscs 92 95 35 765 96 gerber in a cage for 1

B hour, then ~.001 transferred to fresh leafdiscs. Seedlings 54 30 6 398 25

Transferred Leafdiscs 81 46 10 619 46 directly to ( . 001

C old leaf- discs. Seedlings 41 5 0 376 8

* Based on numbers of adult aphids which produced more than 50% alate progeny.

Ef fec t of Starvation

A l t h o u g h aphids r e spond to o ld leafdiscs b y p roduc ing alate progeny, expos ing

them to severe s tarvat ion, b y keep ing t hem off their hos t for a per iod , can have

the oppos i te effect of causing t h e m to p roduce ap te rous progeny. This was shown

in the fol lowing exper iment . A n u m b e r of aphids were reared in i so la t ion on leafdiscs. They were col lected as

virgin adul ts and c rowded toge ther in p las t ic tubes in which they were kep t at 20 °

in darkness and s tarved for per iods of f rom 4 hours to 3 days. Af t e r being s tarved

they were p l a c e d on fresh leafdiscs arid left overnight to reproduce . The results are

shown in Tab le I I . The aphids which were s ta rved for 4 hours and for 1 day

TABLE II

Effect on prenatal form control of exposing virgin adult aphids to periods of starvation

% Aphids Number which pro- Total % Alate

Period of Starvation of duced ~ 5 0 % Number of Progeny Adults alate progeny Progeny

4 hours 120 100% 1146 98% 1 day 61 96% 671 87% 2 days 69 47% 897 51% 3 days 26 0% 269 0%

218 BRUCE JOHNSON

produced almost exclusively alate progeny. After 2 days starvation many apterous progeny were produced while after 3 days starvation the aphids produced only apterous progeny. To determine whether the effect on form control was through desiccation, aphids were starved for 3 days in containers at high (99%) and at low (40%) relative humidities. Both lots of aphids produced exclusively apterous progeny when placed on leafdiscs, suggesting that the effect was not due to desic- cation alone.

Wilting of host plants has often been suggested as a factor which favours alate development. There is no doubt that wilting leads to restlessness among aphids feeding on a plant and it would thus have an effect of promoting alate develop- ment through increasing mutual stimulation of the aphids by one another. However, there is at present no evidence to indicate that it has any direct influence on the aphids. An experiment was conducted in which aphids were reared in isolation on leafdiscs which were removed from the water and allowed to dry out partially each day. The aphids went on to produce mainly apterous progeny when they became adults and reproduced on their original leafdiscs. It is possible that in this ex- periment the wilting of the host tissues was not severe enough to have any in- fluence, but if it had been any more severe it would almost undoubtedly have resulted in the aphids becoming restless and deserting the discs.

Postnatal Form Control

The course of development of larvae of A. craccivora can be influenced during the first two instars (JonysoN & BmKs 1960). The condition of the host tissues on which young aphids are feeding during this time can be an important factor in determining whether or not postnatal diversion from the alate to the apterous course of development occurs. This is shown in the following experiment.

First instar larvae were obtained from parent aphids which had been reared isolated on leafdiscs. The parents had then been crowded together in a 1" glass tube for 4 hours and left to reproduce ovemigh~ on a cutting. This ensured that their progeny were born as presumptive alates and had not already been prenatally diverted to the apterous course of development (see JOHNSON & BIRKS 1959). The larvae were distributed on several young bean plan'ts 20 cm high, which were then kept at 20 ° C. There was very little movement of the larvae once they had settled down and most of them remained on the same part of the plant throughout develop- ment. When they moulted into the fourth instar, they were collected and their form ,and position on the plants were recorded (Table III). Many more alates were present on the lower leaves than on the growing shoots suggesting that the con- dition of the host tissue the aphids were on may have influenced their course of development. !

As with the experiments on prenatal form-control, it is possible that the dif- ference in the proportion that developed into alates on different tissues when larvae were reared together may not have been due to a direct response of the larvae to the host. It may have been caused by a secondary effect resulting from greater

WING POLYMORPHISM IN APHIDS I n 219

TABLE I I I

Effect on postnatal form control of rearing larvae on different parts of intact bean plants

Position on No. of Total No. plant plants of larvae % Alates P

Shoots 25 1307 16 % ~ .001 Leaves 25 1935 49 %

restlessness of the aphids on the less favourable tissues, this in turn influencing the amount of interaction between individuals. It was therefore necessary to repeat the experiment with larvae reared in isolation and at controlled densities on dif- ferent hosts. Larvae were obtained in the same way as in the previous experiment and were then set out at one per leafdisc or ten per leafdisc. Three types of

disc were used; these were fresh and old discs from mature leaves and fresh discs cut from half-expanded leaflets of young seedlings. The larvae were placed on the discs and left to develop to the fourth instar before being counted (Table IV).

TABLE IV

Effec t on postnatal form control of rearing larvae at different densities on different host tissues

Host Tissue

Fresh discs cut from leaves of seedlings Fresh discs cut from mature leaves 7-day old discs from mature leaves

Rearing Density One/Disc Ten/Disc No. of No. of larvae % Alates larvae % Alates

82 52 116 97 78 83 107 96 60 90 93 98

Among larvae reared at one per disc, fewer developed into alates on discs from seedlings than on fresh or old discs from mature leaves. Crowding at ten larvae per disc during their development was effective in obscuring the response to discs of different kinds. Thus, as with prenatal form-control, so with postnatal form-

control the host can exert an important influence, and responses to the host can be obscured by interaction between aphids.

In this experiment very few of the larvae which were reared at ten per disc developed into apterae regardless of whether the discs were cut from mature leaves or seedlings. Yet on the shoots of whole intact seedlings in the previous experiment many larvae had developed into apterae although they were sometimes packed together at a high density. This would suggest that either discs cut from seedlings rapidly lose their ability to induce apterous development, or the larvae which were on discs were "crowding" one another more than were the larvae on whole plants. Nearly half of the larvae reared at one per disc cut from seedlings developed into apterae suggesting that the discs d o still favour apterous development. The greater

2 2 0 BRUCE JOHNSON

production of alates may have been due, at least in part, to a difference in the behaviour of the larvae; those on discs being more active and so stimulating one another more than those on whole intact plants. This may have been because on leaf discs the aphids were confined on top of the leaf, whereas on whole plants they rarely settle in this position. This factor, combined with some deterioration of the leaf tissue, could account for the difference in response of the aphids when reared urLder crowded conditions on seedlings and on leafdiscs cut from the leaflets of seedlings.

Effect of Starvation Starvation of larvae can result in ~heir switching f rom the alate to apterous

course of development. The period,s of starvation involved are necessarily much shorter than those needed to produce an effect in adults, since young larvae die if kept off their host for long periods. It was shown in a previous paper (JOHNSON 1965) that, when first-instar larvae were starved in isolation for several hours, there was a small increase in the percentage which developed into apterae compared with unstarved controls. On the other hand, when the larvae were starved together in a tube, the starvation effect was counteracted by interaction between the larvae so that there was no increase in aptera production.

Crowding can be effective in counteracting the effects of starvation even when the period of starvation is long enough to cause a high mortality of larvae and al- most complete ap.terous development among the survivors. Larvae were obtained in the same way as in the previous experiments. Some were transferred directly to fresh leafdises at one per disc, some were starved 24 hours in isolation and then transferred singly to leafd~scs on which they were reared in isolation; others were starved while crowded together in a 1 cc container for 24 hours and then transferred to leafd~scs on which they were reared at a density of ten per disc. There was a mortality of almost 50% among the starved aphids. Tl~e results (Table V) indicate that prolonged starvation strongly favours apterous development and that its effect can be almost completely counteracted by keeping the larvae

TABLE V

Effect on Postnatal Form Control of starving first instar larvae for 24 hours

Treatment Number of surviving larvae % Alates Direct transfer: not starved 89 84% Starved together in a tube for 24 hours 181 89% Starved in isolation for 24 hours 64 5 %

crowded together. This is in contrast to the effect of crowding in starved adults where the effects of prolonged starvation were not able to be counteracted by crowd- ing (page 217).

D I S C U S S I O N

The experiments described in this paper suggest that the condition of the host

WING POLYMORPHISM IN APHIDS III 221

plant can be an important factor influencing the control of form in Aph i s cracci-

vora both before and after birth. There are several ways in which the host can affect form. With prenatal control, the parent aphids can be influenced both by the host on which they fed as larvae and by the host on which they are currently feeding while reproducing. It is tempting to think of the first of these effects as nutritional and the second as a response to taste, but further evidence is required to resolve whether this is in fact so. The host can also produce an indirect effect through influencing the behaviour of the aphids on it; on wilting and unhealthy plants, aphids tend ,to be more restless and so move about and stimulate one another more than they do on healthy plants and this can lead to alate productio n (JOHNSON 1965).

Host and "crowding" can be either supplementary or antagouistic to one another. Old host tissues and crowding both favour alate production and so supplement one another. Seedlings and crowding, on the other hand, have opposite effects. With prenatal form control, whether alate or apterous progeny are produced a.t any particular time will depend both upon the previous experience of the parent and upon its current experience, both of these with respect to both host and stimulation by other aphids. It will be shown in a further paper that temperature and photo- period can also exert an important influence on form control.

Exposure of aphids to periods of s.tarvation was shown to favour apterons development both when the parents were starved and also when young larvae were starved. This appears superficially to contradict the evidence that poor condition of the host plant favours alate production. However, it must be remembered that in the starvation experiments the aphids were prevented from taking any food at all, in the case of the adults for 2---3 days and in the case of larvae for one day, whereas aphids feeding on old leafdiscs were able to imbibe sap, albeit sap which was perhaps low in some essential nu~-ients.

It has recently been suggested that the use of artificial feeding techniques may facilitate a study of the influence of nutrition on wing polymorphism in aphids (MITTLER & DADO 1962)' Although apterous development can result from aphids feeding on plant tissues that provide a rich source of nutrients, such a seedlings or growing shoots, it is also favoured by severe starvation. In studies on the effect of artificial diets on form control, care must be taken to determine whether any apterous development that takes place is due to a factor in the diet promoting wingless development or to severe malnutrition.

The failure of some authors in the past to demonstrate an influence of the condition of the host plant on wing polymorphism requires comment. In A . crac-

civora wing polymorphism can be influenced by a variety of factors including temperature, photoperiod, contacts with other aphids, severe starvation and atten- dance by ants as well as by the condition of the host plant. In experiments designed to demonstrate an effect of the host plant it is essential to control all of these factors rigidly and particularly contacts between aphids. If this is not done, any effect of the host could be obscured or negated. In the past the way in which the

222 BRUCE JOHNSON

various factors interact to influence form, and the unidirectional nature of form control have not been appreciated, and it is probable that where no effects of host condition have been detected, the insects have been subjected to a combination of factors which have prevented the influence of the host from becoming manifest.

ZUSAMMENFASSUNG

FLtJGELPOLYMORPHISMUS BEI APHIDEN 111. DER EINFLUSS DER W1RTSPFLANZE

Der EinfluJ3 des Zustandes der Wirtspflanze auf den Fltigelpolymorphismus yon Aphis craccivora Koch wurde unter kontrollierten Bedingungen untersueht. Zur Untersuchung des praenatalen Einflusses auf die Fo rm wtwden die Mutterl~iuse bei 20 ° und 10 Stunden Licht je Tag isoliert auf Puffb~hnen-Keimlingen und auf Blattsoheiben yon Puffbohnen gehalten, die auf N~ihrlSsung schwammen. Die Blattl~use wurden dann als jutr~ge Adulte auf frische Blatt- scheiben iJbertragen und Wiihrend der folgenden 24 Stunden die Fo rm der produzierten Larven festgestellt. Es lieB sieh zeigen, dab die Form tier Nachkorrmaen sowohl yon der Wirtspflanze beeinflul3t werden kann, auf der die Mutterliiuse ihre Larvalentwicklung durchgemacht haben, wie v o n derjenigen, auf der sie sich zur Zeit der Larvenproduktion ernShrten. Es wird vermutet, dab das erstere einen NahrungseinfluB darstellen k6nnte, wShrend das letztere eine Gesohrnacks- reaktion sein k6nnte. Blattl~iuse, die auf Blattscheiben aufgezogen wurden, brachten mChr ge- fliigelte Nachkommen hervor als diejenigen, die sich an Keimlingen entwickelten. Unte r t )ber- v61kerungsbedingungen war der Prozentsatz der Gefliigeltenproduktion vergr6Bert und zwar st~irker bei Liiusen, die auf Blattscheiben aufgezogen worden waren, als bei L~iusen, die auf Keimlingen heranwuchsen.

Erwachsene Eiiuse, die gefliigelte Naehkommen erzeugten, konnten durch eine dreitiigige Hungerperiode zur Produktion ungefliigelter Nachkommeu veranla~t werden. L~ingeres Hun- gern bestimmte aueh Larven des ersten Stadiums, die urspriJnglich zur Entwicklung yon Fliigeln determiniert waxen, sich zu Uugefliigelten zu entwickeln. Die Wirktmg des Hungers konnte bei Larven, abet nicht bei Erwachsenen durch 13bervblkerung unterbunden werden.

REFERENCES

BONNEMAISON, L. (1951). Contr ibut ion h l '6tude des facteurs provoquant l 'apparit ion des formes ail6es et sexu6es chez les Aphidinae. Ann. @iphyt. 2 : 1--380.

JorrNSON, B. (1965). Wing polymorphism in aphids II. Interactio~ between aphids. Ent. exp. & appl. 8 : 49--64.

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.

MITTLER, T. E. & DADD, R. H. (1962). Artificial feeding and rearing of the aphid, Myzus persicae (Sulzer), on a completely defined synthetic diet. Nature, Lond. 195, 404.

Received for publication: 22 November 1965.