Proc. S. Lond. Ent. Nat. Hist. Soc. 1943-44A: 69-79.
TEMPERATURE EXPERIMENTS ON THE PUPAE OF (1) HELIOTHIS PELTIGERA, SCHIFF., AND (2) PANAXIA DOMINULA, LINN.
WITH THE MANIFESTATION OF THE MENDELIAN LAW AT A GIVEN TEMPERATURE ONLY.
(With 2 Coloured Plates.)
By H. B. D. KETTLEWELL, M.A., M.B., F.R.E.S.
Read 11th December 1943.

I wish, first of all, to make perfectly clear the significance and definition of the meaning of these Temperature experiments. For the must part Temperature experiments can be divided into two types—the one is confined to experiments in which very high or very low temperatures are used and are of the nature of "shock" treatment. The other, which is the type to which I intend to refer in this paper, limits itself entirely to the use of normal temperatures properly controlled, as found within the normal limits of the range of outdoor temperatures in this country.

"Shock" temperatures tend to alter the normal pattern of the wings probably due to gross disturbance of the normal mechanism of pattern production, and they involve an extremely high mortality rate, in the region of 90%, and similar results can be obtained by other shock treatment such as electric shocks, X-ray application or mechanical shock due to dropping the pupa at the appropriate time. There is absolutely no evidence that any inherited factor plays a part in this.

On the other hand, the reactions of pupae to a constant, uniform, but normal temperature, whilst producing no shock, nevertheless subjects them to a condition of which the organism has no experience so that the genes which normally come into play in rotation in laying down the normal pattern in a normal and variable environment, are disturbed in relation to one another, with the result that areas of wing, which are normally flooded with pigment at a given period under natural varying conditions, find themselves at a different stage of development and un able to receive their normal pigment.

It is this variation in co-ordination between pigment deposit and stage of development of the scales of the wings which result in such phenomena as seasonal differences, summer and autumn forms, etc., and is merely the manifestation of the result of varying conditions on a sensitive gene-complex.

PLATE I. Proc. S.L.E. & N.H.S., 1943-4
Temperature experiments on the pupae of H. peltigera (see text)

(1) TEMPERATURE EXPERIMENTS ON THE PUPAE OF HELIOTHIS PELTIGERA, SCHIFF.

For the purpose of clearness this paper will be divided into two separate portions:—

A. Ultimate effects on pigmentation of imagines of H. peltigera.
B. Pupal reactions. These are a natural sequence to the result of experimenting with heat and cold on the pupae.

A. PIGMENTATION EFFECTS.

Since 1928, when larvae of this species were found commonly for the first time on the south-east coast of England feeding on Senecio viscosus and Convolvulus soldanella, large numbers, have been bred to the perfect state by many collectors, and under various conditions. Most of them have found a remarkable degree of variation in colour, which prompted a spate of papers on the subject in the following years.

In 1930 Dr E. A. Cockayne (1) wrote a full account of his results, and in conclusion stated "I have therefore very little doubt that light ground colour and reduced markings were the direct result of heat applied to the pupae, and since the two very dark ones developed during the coldest period in 1929 I have little doubt their dark colour was the result of cold applied to the pupae."

In 1931 I recorded (2) my own results and observations, which appeared to conflict with Dr Cockayne's conclusions. I attempted to tabulate the various factors which could possibly be responsible for the production of light or dark forms of peltigera. These included temperature, duration of pupal period, humidity, etc. I stated that I had bred uniformly darkish examples from a batch of pupae which I had forced, the pupae having first been kept in varying temperatures, and subsequently in heat, the whole period taking about 43 days in all. I therefore challenged Dr Cockayne's conclusions (previously stated) and asserted that, as the only difference in the method of forcing was one of humidity, "wet" heat was responsible for my dark ones and dry heat for his pale ones.

Later in 1931, A. J. Wightman (3) stated his views on his forcing of peltigera pupae. He allowed 10 days for the larvae to pupate, then dug them up, and after a further period of 10 days, "to allow the pupae to get hardened," he commenced forcing in dry heat by means of an oil lamp placed near them, to give a temperature of approximately 100° F. For the most part he produced medium-dark and dark examples, and he therefore pointed out that it in no way upheld the suggestions I had put forward earlier, on "wet" and "dry" heat producing dark or pale forms respectively.

In 1933 W. G. Wynn (4) experimented with dry heat on pupae which had been left to pupate for a period of a month. He obtained precisely the opposite effect to that which he had found in 1931 when he obtained pale examples. On this occasion he bred a varied assortment, but for the most part they were medium-dark.

At this point we were all so confused by the apparently conflicting results and views expressed that correspondence for the most part came to an end, leaving us very much where we started.

In 1932 I commenced a series of controlled experiments on the pupae of the species, in an attempt to take us a step further. These results were not published at the time because of the ever present and natural urge to reach final conclusions." These experiments are, in fact, far from complete, but on reviewing my notes recently I considered them worthy of report, if only to refute my own theory, previously advanced, of "wet" and "dry" heat.

Thanks to the kindness of Dr Garrod of St Bartholemew's Hospital, I had at my disposal reliable incubators, cool storage, etc., which offered the optimum conditions for experimenting.

The hundreds of pupae sacrificed on the altar of beat and cold all came from Dungeness larvae, which, unless stated otherwise, were always used within 24 hours of actual pupation. It is fundamentally important to note this. To ensure this, each larva was kept separately in a chipette box so that observation of pupal time was assured. This, of course, resulted in a high mortality rate.

Throughout the experiments (unless stated otherwise) the following temperatures were used, which varied to within three or four degrees:

Heat, 30° C. (=86° F.).
Cold, 6° C. (=42.8° F.).

EXPERIMENT 1 (as figured).

One dozen pupae were placed on dry cotton wool in a glass-topped tin box and put into heat. These produced 100% pale buff imagines with no marked development of markings or submarginal band. Similarly, the hindwings were white, with the black outer band in which the white mark near the margin showed up very clearly.

EXPERIMENT 2.

A repeat of Experiment 1, but the pupae were lying on cotton wool saturated with water, which was kept sprinkled from time to time so as to be kept constantly wet. They produced 100% pale buff examples indistinguishable from those of Experiment 1.

These two experiments, therefore, exploded the theory of "wet " and " dry " heat put forward by me a year earlier.

EXPERIMENT 3.

Had as its object to find out if cold, by its direct effect, would produce dark examples. It will be noted that the temperatures used in this experiment follow very closely the normal day and night temperatures to which pupae must be subjected in a state of nature late in the season.

Thirteen pupae were given alternate periods of beat and cold, in the proportion in hours of 20 to 4 respectively. They hatched in precisely the same number of hours of heat as those of Experiments 1 and 2. They were 100% pale buff specimens indistinguishable from those in Experiments 1 and 2.

That cold, as such, does not directly affect the colour of the resulting imagines is further borne out by the next experiment.

EXPERIMENT 4 (figured).

Of 60 pupae from larvae which pupated late in the year (in October), and which were placed in the cold, the majority showed no inclination to hatch. These were kept in the cool for four months, at times the temperature being just above freezing. Twenty of these were then placed in heat (in this case approximately 100° F.). They produced imagines of extremely light, colour, of both ground colour and pattern (including the reniform).

It can therefore be stated that neither humidity nor cold applied to the pupae produces any direct effect on the ultimate pigmentation of the imago. All these individuals show a uniformly pale ground colour with no accentuation of pattern. It would appear as if the actual speed of metamorphosis within the pupa prevents the maturing, or laying down, of the pigments to any great degree.

I wish here to make certain generalized statements about the pupal state. The life of every pupa can he divided into two phases: first a period of rest, when the anatomy of the pupa (with the exception of the nervous, circulatory, and respiratory systems) is in a state of disorganisation (following histolysis), and the second phase, which is constituted by the reorganisation of the imago from these broken-down larval tissues. The first phase may vary from a very short time to a length of time, measurable in years, during which the pupa is in a dormant state. The second phase is a period of activity." I can find no record of there being any attempt to differentiate these two phases, and as it appears to me to be all-important to be able to refer to them specifically in this paper I suggest the names of "passiphase" and "actiphase" be used respectively. The passiphase, then, is a period of inactivity which may be of great length; the actiphase, on the other hand, when once commenced must inevitably continue to its final conlusion with the development of the fully-formed imago, which, in the case of H. peltigera, must hatch immediately. In some species this is not the case (as in Eriogaster lamestris, L., and Brachionycho nubeculosa, Esp.), and a fully-formed imago, having completed the actiphase, remains dormant for a long period of time within the pupal shell. The actiphase, then, covers a comparatively short period, is progressive, and cannot be delayed beyond a certain time or death will take place. In peltigera, temperature effects during the passiphase in regard to ultimate pigmentation are nil. It is therefore with the actiphase part of the pupal state that I now wish to deal. It can be shown that when this phase is prolonged to the limit compatible with the life of the insect the darkest peltigera are produced. The pigment has had time to mature, and has been laid down to its maximum,

EXPERIMENT 5 (figured).

Twenty fresh pupae were placed in the beat for 24 hours with the object of stimulating the commencement of the actiphase. They were then taken out and placed in the cold. A certain number continued to develop very slowly, and after four to six weeks it appeared that some of the survivors were nearly ready for hatching. However, they appeared too weak to complete this, so the remainder were put into heat and in n short time five hatched, which were by far the darkest peltigera I have ever seen. All are very dark. Two (Nos. 1 and 2) have both hindwings and abdomen uniform grey-black, forewings dark chocolate brown, with darker brown markings showing indistinctly on top of this.

I now attempted to take the subject n step further by investigating in which part of the actiphase the pigment was actually laid down.

It will be appreciated that there is no outward visible sign of the change over from passiphase to actiphase. I therefore tried to find out the earliest definite sign of the commencement of the actiphase. Pupal colour, trans-illumination, etc., all failed to help, and eventually I was driven to accept the earliest definite sign as being the commencement of the darkening of time pupal eye-area.

Furthermore, at higher temperatures with fresh pupae the passiphase is practically eliminated, so that one may consider the commence meat of actiphase and the introduction of the fresh pupae into heat as coinciding. By doing this it was therefore possible to get a unit of comparison for the temperature, 86° F. From the commencement of actiphase (=C) to the time of hatching (=H) it took an average of 8,1 days (200 hours) of heat. All imagines (with one exception, No. 5, Exp. 1, vide infra) in Experiments 1, 2, and 3 hatched in this time.

There is a remarkable uniformity in this with certain gross exceptions, hereafter to be discussed (under B, Pupal Reactions). At this temperature a darkening of the eye-mark (E) appears on an average on the 5th day (117 hours). This can be shown thus:

At 86° F. CH = 8 1/2 days (200 hours).
CE = 5 days (117 hours).
EH = 3 1/2 days ( 83 hours).

My first experiments, then, were to try to find out whether the pigment was laid down before or after the appearance of the eye-mark (i.e. in CE or EH).

EXPERIMENT 6 (figured).

I attempted to prolong the EH period of the actiphase for as long as possible. Thirty fresh pupae were put into heat and left there until the eye mark was developed. They were then taken out and placed in cold. The earliest hatched in 13 days (No. 1), and the last in 29 days (No. 5). These insects are a graded series, the darkest being those which took the longest time to hatch, which are definitely darker than those which took the shortest time, but by no means could they be considered really dark peltigera. The darkest are a uniform dull brown-buff, conspicuous for the fact that all the normal markings (sub-marginal band, etc.) are in distinct and lost, submerged in the slight degree of darkening of the ground colour.

It will appear then that, firstly, there is no darkening of the normal markings of the forewings during the EH period. Secondly, there is only a small degree of general darkening of the ground colour laid down during this period. Conversely, it was suggestive that the darkening of the normal pattern would be entirely laid down during the earlier CE period. To test this out I attempted to prolong the CE period to its maximum.

EXPERIMENT 7 (figured).

It will he shown later that during a succession of broods there appear to be certain pupae which have an inability to pass into the dormant state but must enter upon the actiphase regardless of a moderately low temperature.

From a number of such pupae placed in cold, 10 were picked out on the appearance of the eye-mark. They had been in the cold then from the commencement. The CE period was approximately 3 weeks.

Four of these pupae, showing very faint eye-marks, were put in heat, and all hatched in between five to six days (3 figured, Nos. 1, 2, 3). All the normal markings are greatly darkened and stand out on a normal pale buff ground colour. The sub-marginal band is dark and the scallop mg internally stands out in striking relief against the pale ground colour.

The remainder of these pupae were kept in cold until the eye-marks were fully developed. When put in heat they hatched in 3 days, producing individuals with increased dark markings on a mahogany-red ground colour, quite unlike the ground colour of those individuals which were taken out of cold at the earliest sign of E (Nos. 4 and 5).

The following points can therefore be elicited:

The degree of darkening of the normal pattern is decided at an early stage, before the earliest signs of the appearance of the pupal eye-marks.

The maximum degree of ground colour pigment is laid down at approximately the sanie time that the eye-marks are developing, and to a lesser degree after they are fully present.

The following formulae can be postulated, using the two temperatures (42.8° and 86° F.):

  Pattern Ground Colour
Exp. 1, 2, 3.   CH 8 1/2 days. Pale buff. Pale.
Exp. 5.   CH 35 days. Dark. Very dark.
Exp. 6. { CE 5 days. Pale. Dull brown-buff.
EH 29 days.
Exp. 7. [ { CE 21 days. Dark. Pale.
EH 5 days.
{ CE 30 approx Dark. Mahogany-red.
EH 3 days.

Lastly, there remain to be done a large number of experiments for those temperatures between 42.8° and 86° F.

It will be appreciated that, as always, this particular range of temperature is the most difficult to keep accurately because the normal daily temperatures vary both above and below the particular degree wanted. Both heat and refrigeration may therefore be needed to either increase or lower the temperature to the desired figure. I have myself tried out two temperatures within this range, namely, 54° F. and 70° F. In each case my technique was to introduce pupae into a large thermos container in an atmosphere where the temperature was at the desired figure. The temperature within the thermos remained remarkably constant.

EXPERIMENT 8.

Half-a-dozen pupae were introduced into a thermos at 54° F., following 5 1/2 hours of heat (86° F.) to stimulate the commencement of actiphase. E appeared in approximately 13 days, and hatching took place in 24 days. The only non-crippled insect was a dark example of a beautiful ruddy-brown ground colour, and with normal markings not particularly well-marked. All the cripples were obviously dark individuals.

EXPERIMENT 9 (figured).

Pupae were introduced into a thermos at 70° F. E was reached in 6 days, and imagines appeared in 17-20 days. They are all insects with intermediate light ground colour, but with very well developed markings.

It would be unwise to draw conclusions from these incomplete experiments, but it does suggest that the laying down of the pigment, both of the pattern and the ground colour, may not be entirely a matter of length of time, but that the actual temperature itself may also play a part, and that the pigmentation of the pattern may be laid down at a higher temperature (=70° F.) than that of the ground colour.

In view of these findings we will now analyse the original results of my own and others investigations, results which appeared so conflicting.

There is a common error, allowing for discrepancies, running through all our original experiments. It was our failure to recognize that pupal changes had already taken place in much of the material we eventually subjected to heat, and, secondly, our inability to realize that the degree of pigmentation may be decided at a comparatively early stage of pupal life, approximately the middle third of the actiphase.

My own, Wightman's, and Wynn's pupae were 30, 10 and 20 (approximately) days old (allowing for time for the larvae to pupate) before being given heat. It must also be pointed out that the difference in temperature between air-heat and that of 3 or 4 inches underground is considerable. Pupae dug up and left for 10 days (such as Wightman's) are subjected to great fluctuations of normal day and night temperatures, and the actiphase will have been well on the way before he commenced experimenting. Similarly, Dr Cockayne's 1929 examples were merely the expression of the varying temperatures they had experienced during individual actiphase, so that those which hatched in "a heat wave" had had their colour decided days or weeks previously. If this fact is understood and guarded against, results will be found remarkably constant. The pupal period can be divided into two parts, the passiphase and the actiphase. Temperature effects during the passiphase have no effect on subsequent pigmentation of imagines (in H. peltigera). Temperature during the actiphase is indirectly (and possibly directly) responsible for the degree of pigmentation laid down. It is the duration of the actiphase which is mainly responsible.

Pigment is laid down in two separate periods of the actiphase. The earlier one, which occurs before the appearance of the pupal eye-mark, is limited to the normal brown pattern of the wings, and the later one, which coincides with the darkening of the eye-mark and continues to some degree afterwards, is limited to the ground colour. These two periods are distinct, though there may be an overlap which takes place during the early appearance of the eye-mark. No amount of subsequent heat or cold eau alter the colour when once a given period is passed.

Finally, there remains for us to review these conclusions in the light of Genetics.

In the experiments here demonstrated, it can be assumed for simplicity that the internal genetic formula of each individual is the same as regards colour and pattern. That is to say that each insect would produce the same form under identical treatment. It is the ENVIRONMENT WHICH HAS BEEN VARIED. The light forms, the dark forms, and the banded forms merely represent the minimum and maximum expressions of pigment deposit as the result of variation in the timing of the flooding of the wings with pigment and the readiness of the scales to incorporate it. They are the expressions of the same gene-complex which controls this, under a varying environment.

As there is no reason to believe that the actual pigment of the ground colour and the pigment of the markings differs chemically, it is most probably the state of readiness of the scales themselves to receive the pigment which decides the colour, the scales in the normal bands and pat tern being ready to receive this pigment at an earlier period than the scales of the rest of the wing responsible for the ground colour. At higher temperatures neither is ready to receive any pigment.

B. PUPAL REACTIONS.

During the course of investigations into the effects of temperature on the imagines of H. peltigera, certain interesting observations were made on the reactions of the pupae.

In general the fresh pupae reacted extraordinarily uniformly to the moderate temperature used. As has already been stated, however, there appeared from time to time certain gross exceptions. It was found that approximately 1% would fail to respond to heat at 86° F., and would show no signs of onset of the actiphase even after all the others had hatched. These individuals appeared refractory to normal heat, and in spite of it remained dormant in the passiphase. In Experiment 1 there was one such pupa. All the rest had hatched in an average of 8 1/2 days (200 hours), yet this pupa remained inactive and showing no sign of the eye-mark at the end of 14 days at this temperature. It was therefore transferred to cold for 21 days before being returned to heat. It then reacted in precisely the same way as the others had done, hatching within 200 hours and producing a similar pale huff imago (No. 5). It would therefore appear that this pupa had an inhibitor to heat which was satisfied by a short period of cold, after which it acted normally. I suggest that the presence of this inhibitor may be due to an inherited factor.

I wish now to turn to other pupal reactions. During a rapid succession of broods in the summer months there appears to be a tendency to rapid metamorphosis, and there is some evidence that in some pupae this urge to hatch is independent of temperature effect. In Experiments 4 and 7 certain pupae, which had been placed in cold, failed to face a dormant state but passed slowly into the actiphase in spite of the moderately low temperature, and subsequently hatched. It would appear as if these pupae were unable to remain in the passiphase, and that the machinery for undertaking hibernation was absent. It is to be assumed that there must be some factor present enabling the other pupae to hibernate. This also may well be an inherited factor.

It now appears possible to designate certain formulae for various groups of pupae of this species:

"H" represents presence of Hibernating Factor.
"h" represents absence of this factor.
"T" represents Direct Temperature (Heat) Control (=Absence of Inhibitor).
"t" represents presence of Inhibiting Factor to Heat.

The majority of the pupae have the formula "TH." They will respond to moderate heat; nevertheless, they are capable of undertaking hibernation in the absence of heat. "tH" represents those insects which are refractory to heat and are not stimulated to undertake the actiphase by it (Experiment 2). "Th" represents those pupae which are unable to go into hibernation, and must therefore develop into imagines or die.

It would appear that the valency of "H" varies markedly in the individual both as regards the duration and the degree of cold necessary to satisfy it. Earlier on it is little developed and easily satisfied. When once the hibernating stage has been undertaken and several months have passed, the pupal reactions to moderate heat are entirely different and far from uniform.

In Experiment 4 the high temperature of 100° F. was used on pupae which had been in hibernation for 5 months. It is true they all hatched, but they took much longer than summer pupae to commence the actiphase and in hatching. This is even better seen in more moderate temperatures as in Dr Cockayne's 1929 insects, from larvae collected in the autumn of 1928. Of 23 pupae which survived the winter 4 of the re suiting imagines did not hatch until September and October (the 22nd) the following year, having " successfully resisted the extremely hot weather of 14th-21st July."

It would appear, then, that after wintering the formula of pupae is "th" for moderate temperatures, and that the date of hatching is decided by the time taken for the "H" factor to become "h", and that during this time the pupa is unaffected by moderate heat.

F. V. L. Jarvis has shown (5) that the effect of continued heat on pupae of P. brassicae, which were of a hibernating strain, was to in crease the time of hibernation up to 2 years, and ultimate death. He explained this by assuming that heat stimulated the production of "H", and that those cells in the body of the pupa which produced the sub stance only cease to function (or atrophy) after a period of cold. It appears that a similar state may exist in some peltigera. This is certainly a great provision of nature to ensure the survival of certain individuals for the following year no matter what are the weather conditions.

It is common knowledge that the progeny of later broods in certain seasons frequently come to nothing due to sudden changes in weather conditions.

It will be very interesting to see if this theory can he applied to those peltigera which come from the West of England. Here in Devon and Cornwall this insect appears to be indigenous, recurring each year regularly quite unlike its Kentish confreres. They differ also in other respects. There is no evidence of the same degree of multi-broodedness; the larvae feed practically entirely on Ononis; the imagines come regularly to sugar, whereas the Kentish ones do not, even though sugaring is regularly undertaken on the spot where it is known to occur in thou sands. Furthermore, I believe I am able to recognize these Western peltigera in collections. The forewings are a golden-yellow quite unlike any bred by me from Kent; the outer margin of the forewings ap pears more rounded, and they probably have a different origin. There is evidence that our Kentish ones are the progeny of immigrants from Southern Germany. These Devon ones may have arisen originally from Western European peltigera but are now indigenous.

I should expect to find in these a much higher percentage of pupae refractory to heat (=tH) so that the majority are adapted for a twelve month life history, though there is evidence that in some seasons it is at least double-brooded. This provision would obviously adapt the species much more satisfactorily for a resident, because more pupae would be encouraged to undertake hibernation quite unaffected by extrinsic temperature conditions (=T).

In Kent and Eastern England this would be of little help as the pupae appear to be unable to withstand the winter here anyway, so that these pupae would be merely wasted. The repopulation of this species in Eastern England is therefore from fresh immigrants from Southern Europe, where multi-broodedness is the rule, and where a high pro portion of pupae with a temperature inhibiting factor (t) would he a disadvantage.

PLATE II. Proc. S.L.E. & N.H.S., 1943-4
The effect of a constant temperature (75° F.) on the pupae of a particular strain of P. dominula (For explanation see text)

SUMMARY ON PUPAE.

The pupae of Kentish peltigera fall into four groups representing a combination of the presence or absence of two factors which may he inherited, and by which the duration of the pupal state is decided. The two factors are: 1. An inhibitor to heat (=t). 2. An ability to undertake hibernation (=11). The majority of fresh Kentish pupae are capable of hibernating, yet will respond rapidly to heat (=TH). A small minority are refractory to heat unless first satisfied by a short period of cold (=tH). A proportion are unable to pass into hibernation and cannot survive a winter (=Th). It appears that after a long period of cold (=hibernation) pupae re act differently on subsequent application of moderate heat (=th). It would appear that there is a balance between the degree of heat and the valency of the hibernating factor in deciding the duration of the pupal period in the pupae that have once undertaken hibernation.

REFERENCES.

  1. E. A. Cockayne. Ent. Rec., xlii, 1939.
  2. H. B. D. Kettlewell. Ent. Rec., xliii, 1931.
  3. A. J. Wightman. Ent. Rec., xliii, 1931.
  4. W. G. Wynn. Ent. Rec., Vol. 45-46, 1933-34.
  5. F. V. L. Jarvis. Proceedings of S. Lond. Ent. and N.H. Soc., 1941-42, Pt. I.

(2) TEMPERATURE EFFECTS ON THE PUPAE OF PANAXIA DOMINULA, LINN.

In the spring of 1943 I decided to take temples of larvae from known broods of P. dominula, with the twofold purpose, firstly, of discovering, in advance of the main hatching, the results of the previous year's pairings, and, if possible, to force through a second generation in the year, and, secondly, of seeing if any differences were noticeable between those bred at a constant warm temperature and those (in the main brood) bred at varying out-of-door conditions. The method employed was as follows: In early March approximately 20 larvae, which were just coming out of hibernation, were taken from each brood. These were placed in a temperature of 70° F. (varying + or - 5°). They fed up rapidly, and pupated at the end of the month and produced imagines in April. With the exception of one brood which showed certain gross changes hereafter to be described, the following minor variability was noted: There is a definite tendency at 70° F. for all dominula of normal pattern to have a reduction of thee size of the forewing spots, in particular the subapical blotch is narrowed and hooked internally. The apical spots are small or partly absent.

In those broods which contained the ab. medionigra strain the expression of this gene appeared to he much accentuated at this temperature in particular in regard to the hindwing pattern.

Of 13 insects bred at 70° F., 3 showed complete joining of the black band of the hindwing from the costal spot to the inner angle. A further 6 had the costal spot joined to the central spot of the hindwing, to make a "dagger-shaped" mark. 4 showed no marked increase of markings.

On the other hand, of a series of 61 from the same brood, but bred at varying out-of-door temperatures, none had the complete band and only 14 had the costal and central spots joined. I am unable to state any constant differences of the forewing spots due to temperature.

The results of one brood are worthy of recording in detail: The parents, both from wild Deal larvae in 1942, were bred out of doors and were both normal for the factor to be discussed (Nos. 7 and 8). Actually the ♂ was referable to ab. crocea and the ♀ was asymmetrical, with the subapical blotch, of one forewing only, merged into the apical group of spots.

23 insects were bred at 70° F. Of these, 6 were normal dominula (as No. 5), 5 were of an entirely new form with the forewings practically all black (glossy blue or glossy green) and with only the remains of the spot at the inner angle present, along with minute traces of the lower end of the subapical blotch. The hindwings showed a great increase of all the black markings (as Nos. 1, 2, 3, 4). The remaining 12 were inter mediate between this latter form and normal dominula, varying from individuals with only the upper basal spot split into two to those with extensive breaking up of all normal spots present with a definite tendency for the basal spots to be represented by small horizontal streaks (as Nos. 9-16).

It therefore appeared that the brood segregated into a simple Mendelian ratio 1:2:1 at 70° F., with a variable heterozygote.

Twenty insects of the same brood were later bred in a conservatory facing West with temperatures varying from 45° F. to 80° F. All were normal dominula (as No. 6).

The new homozygous recessive form in some respects resembles ab. paucimacula, Schultz, but in the absence of further evidence must not be accepted as this. Furthermore, certain of the heterozygotes show a likeness to ab. diluta, which I described and figured in these Transactions for 1942, only the hindwings are not so pink.

It appears then that we have here a gene-complex which is able to express itself only in an abnormal environment, in this case the abnormality being the absence of temperatures lower than 70° F., or alternatively the speed of the metamorphosis produced a condition whereby the flooding of the wings with pigment took place before the wing scales were ready to receive, or reject, it in such situations respectively as would produce the normal pattern. I think this is the first case of this type of heredity to be proved in the Lepidoptera, although it has been demonstrated on many occasions in other orders of life.

Collins in 1927 found an albino strain of barley. It was a simple recessive and controlled by the action of a single factor, but the albino type was only able to express itself when the plants grew at a temperature below 6.5° C., whilst above 18° C. they developed the full amount of chlorophyll and could not be distinguished from normal plants.

A similar gene temperature control has been demonstrated in the Amphipod Gammarus chevreuxi, also in Siamese cats and Himalayan rabbits. We shall expect to find it in many more instances in the Lepidoptera and even at present one has suspected it in certain other cases: L. camilla, ab. nigrina, and A. paphia, ab. melaina, but so far with no experimental proof.

FINAL SUMMARY OF CONCLUSIONS.

There remains for me to review the results of experimenting with these two widely separate species under consideration in regard to temperature. In H. peltigera it has been demonstrated that a particular temperature during a critical period decides the ground colour and depth of pattern of the wings, the temperature acting on a normal uni form gene-complex. In P. dominula the same point is taken a step farther because the particular characteristic, increased pigment de posit, can only manifest itself in the presence of a particular gene acting in an abnormal environment which is recessive to the typical dominula which are the homozygous dominants of the brood and retain their normal pattern in both environments.

Once again the concept of Professor Goodrich has been clearly shown that "no single part or character is completely 'acquired' or due to inheritance alone. Every character is the product both of the factors of inheritance and of environment and can only be reproduced when both are present."