Environmental control of seasonal variation in the butterfly Colias eurytheme: Effects of photoperiod and temperature on pteridine pigmentation

J. Insect Physiol., 1974, Vol. 20, pp.

1913 to 1924. Pergamon Press. Printed in Great Britain

ENVIRONMENTAL CONTROL OF SEASONAL VARIATION IN THE BUTTERFLY COLIAS EURYTHEME: EFFECTS OF PHOTOPERIOD AND TEMPERATURE ON PTERIDINE PIGMENTATION RICHARD

J. HOFFMANNt

Department of Biological Sciences, Stanford University, Stanford, California 94305, U.S.A. (Received 2 March 1974) Abstract-Photoperiod and temperature were examined for their effects on the four major pteridine components of the wings of Colias eurytheme. Orange individuals show reduced pteridine pigmentation in response to short photoperiods, but make no significant adjustment of pigmentation as a result of varying temperatures. ‘Alba’ females, on the other hand, respond to temperature, but levels of the coloured pteridines are apparently insufficient for there to be an observable effect of photoperiod. Low temperature causes an increase in the concentrations of the coIoured pteridines in these white females. The action of photoperiod in reducing resources committed to pigmentation in orange animals is consistent with the notion that it is advantageous to free nitrogen for other purposes under thermally stringent conditions. The response of the ‘alba’ females to temperature is more puzzling, but it may reflect the absence of selection for the canalization of this phenotype against the effects of temperature. INTRODUCTION

SINCE HOPKINS’ (1895) early work on pteridine pigmentation in the Pieridae, it has been shown that pteridines occur in most, if not all, pierid butterflies, and in few of the other families of Lepidoptera (FORD, 1947). The alfalfa butterfly, Colias ewytheme Boisd., an orange and black pierid, is a widely distributed and variable insect that shows both seasonal and geographic variation in the pigmentation of its wings. AE (1957) did the first controlled work that demonstrated that the melanic and pteridine variation in this butterfly is primarily controlled by photoperiod. It is now understood that the seasonal variation in the melanic pigmentation of the undersides of the hindwings is of direct adaptive significance (WATT, 1969; HOFFMANN,1973). In the cooler periods of the flight season, darker coloured forms, produced by short photoperiods, are more efficient at raising body temperature to about 35 to 38”C, the genus-wide optimum for voluntary flight, by the absorption of solar radiation. In the hotter periods, long-day, light-coloured morphs are adapted to prevent overheating and the resultant termination of flight. t Present address : Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543, U.S.A. 1913

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While earlier work has noted systematic variation in the pteridine pigmentation of the wings with photoperiod and with the season of collection (AE, 1957; HOFFMANN, 1973), and the developmental course of appearance of the four major pteridines found in this insect has been described (WATT, 1967), there has been no attempt to quantify the differences produced by seasonal environmental conditions. A study of this aspect of the variability of C. eurytheme was suggested by the observation (AE, 1957; HOFFMANN, 1973) that the red pigment erythropterin is reduced by short photoperiods in males and orange, male-like females. Further, during the late autumn, ‘alba’ females (produced by a sex-limited, autosomally transmitted, dominant gene yielding white females, instead of the usual orange coloration; REMINGTON, 1954) are collected in the field that show increased yellow and orange pigmentation. This phenotype can also be produced in the laboratory by allowing newly pupated animals to complete development and emergence at 15”C, suggesting that temperature also plays a r81e in pteridine variation while not affecting melanization (HOFFMANN, 1973). Previous work by WATT (1964) has shown that five major pteridines are present in C. eurytheme, but only four of these (xanthopterin, yellow; leucopterin, colourless; erythropterin, red; and sepiapterin, yellow) make a significant contribution to the coloration of the butterflies (WATT, 1967). Consequently, these four pigments were chosen for a quantitative study of the effects of environment on the pteridine pigmentation in these insects. While the ecological significance of pteridine pigmentation, and particularly its variation, is not well understood, it is anticipated that, through the accumulation of rigorous quantitative data concerning its variation, an adaptive understanding of the variation will emerge.

MATERIALS

AND METHODS

Laboratory strains of Co&s eurytheme were established from wild caught females from Los Banos, California. Rearing procedures for larvae have been previously described (HOFFMANN, 1973). For the photoperiod experiments, animals developed under a variety of photoperiods from L16 : D8 to LlO : D14 at 25°C. For the temperature experiments, larvae were allowed to develop on 16 hr light per day at 25°C. When the pupae had hardened, the insects were placed at random by sex in temperature-controlled chambers to complete development and emergence, so that wing pteridine synthesis took place under different thermal Once the adults had emerged, they were spread on pinning boards regimes. to dry. Specimens were stored in darkness until needed for pteridine quantification. Pteridines were extracted from one forewing and one hindwing of each adult in O-5 ml of 0.05 M carbonate-bicarbonate buffer, pH 10, for 1 min at 100°C. The wing residues, still bearing melanin, were removed, and 100 ~1 of each extract were applied in 1 in. streaks to each of two 2 in. wide strips of Whatman No. 3MM chromatography paper. One strip from each individual was separated by electrophoresis in the above buffer for 3 hr at 1000 V in a Savant Instruments, Inc. flat

191.5

PTERIDINEVARIATIONINCOIJAS

plate electrophoresis apparatus. The other was separated by descending chromatography in a mixture of 1-propanol-distilled water, 2 : 1 (v/v). Blank strips were run each time. After the strips were dried, pteridines were located by their fluorescence under U.V. light (principal emission: 254 nm). Spots containing the four major pteridines were cut out and eluted in 2 ml O-1 N NaOH for 2 min at lOO”C, with the exception of erythropterin, which was extracted for at least 20 min at room temperature to avoid alkaline attack on the side chain. Because of unsatisfactory behaviour of leucopterin and sepiapterin in the chromatography solvent, these pigments were eluted from the electrophoresed strips, while erythropterin and xanthopterin were taken from the chromatographed ones. Paper fragments were centrifuged down and the eluants were measured with a Zeiss PMQ II spectrophotometer against appropriate blank eluants using the wavelengths and extinction coefficients in Table 1. The extraction procedure quantitatively converts sepiapterin to its pterin-6-carboxylic acid derivative, so this was TABLE l-PHYSICAL

Pteridine Xanthopterin Leucopterin Erythropterin Sepiapterin*

CHARACTERISTICS OF THE FOUR MAJOR PTERIDINES EXAMINED IN C. eurytheme Absorption peak (nm)

Extinction coefficient

392 345 470 262

6,500 10,250 13,400 20,800

Electrophoretic migration (anodal) (cm) 23 18 17.5 5

% 0.32 0.20 0.23 -

Absorption peaks and extinction coefficients are taken from Watt (1967), unless otherwise noted. See text for chromatography and electrophoresis procedures. * Peak absorption and extinction coefficient listed are for pterin-6-carboxylic acid, the derivative of sepiapterin formed during the elution procedure. Values taken from BLAKLEY (1969).

measured in its place. Results were converted to total pmoles pteridine per insect for the temperature studies. Since photoperiod has been shown to affect size directly (HOFFMANN, 1973), results for the effects of photoperiod were converted to nmoles pteridine per mg dry wing weight. Standard analysis of variance (SOKAL and ROHLF, 1969) was used to test the significance of the effects of photoperiod or temperature on each pteridine examined. RESULTS

Effects of photoperiod In addition to causing changes in body size and hindwing melanization (HOFFMANN, 1973), short photoperiods also act to reduce the wing area covered by

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erythropterin in males and orange females. The red pigment recedes to basal areas of the forewings and is somewhat reduced in area of distribution on the.hindwings as well. This effect is illustrated in Fig. 1. Further, the intensity of orange coloration, where it does remain, is reduced in short-day individuals, leaving the appearance of a much yellower insect. In contrast, the wing surfaces of long-day insects are usually brilliant orange overall. Photoperiod produces no visually

FIG. 1. Effect of photoperiod on erythropterin distribution in male C. eurytheme. Left, long-day animal. Right, short-day animal. Stippled areas represent the presence of the red pteridine erythropterin. Orange females respond in a similar fashion. ‘Alba’ females do not respond to photoperiod with respect to their pteridine pigmentation. Scale, 0.5 cm.

detectable effects in the pteridine pigmentation of the ‘alba’ females. Since orange females respond to photoperiod the same way as males and ‘alba’ females make no visually obvious response, the effects of photoperiod on pteridine variation were examined quantitatively in males only. Photoperiods over the ecologically relevant range (HOFFMANN, 1973) of L16 : DS to LlO : D14 were examined for their effects on the pteridine content of adult wings. Five specimens were chosen at random from each photoperiod (except from Lll : D13, where only four were available) and a total of twenty-nine insects was examined. The results of the quantitative analysis of the effect of photoperiod on weight specific pteridine pigmentation in C. eurytheme are presented in Fig. 2. Analysis of variance for an effect of photoperiod on weight specific pteridine (see Table 2) shows a significant effect only for the red pigment erythropterin. Nonetheless, it is striking that all curves are lower on short days than on long days. In fact, when

PTERIDINE

IN COLIAS

VARIATION

1917 J

160X 140 ,130 150; c = I 70

;,

+f+---+

E&J_ 1 i 50B 40

1

30:fl;

L-&5-; lot I

-

d

0

- 10

11

12

13

14

15

Hours Light/Day

16

FIG. 2. Effect of photoperiod on the four major pteridines (weight specific) in males. 0, Xanthopterin; 0, leucopterin; cl, sepiapterin; q, erythropterin. Photoperiod produces a statistically significant effect only on erythropterin (see Table 2). Bars indicate f 1 S.E.

TABLE%-PROBABILITY SIGNIFICANCE (WEIGHT

OF

VALUES AN

EFFECT

UNCORRECTED)

OF

PTRRIDINE

FROM

THE

ANALYSIS

PHOTOPERIOD

ON

PIGMENTATION

OF

WEIGHT OF THE

VARIANCE SPECIFIC

WINGS

FOR AND

OF C.

THE TOTAL

eurytheme

MALES

Pigment

N

Weight specific

Total

Xanthopterin Leucopterin Erythropterin

29 29 29

O*lO
O-01
Sepiapterin

24

0~05
0.025
total weight specific pteridine content is analysed, there is a very nearly significant effect (0.05
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RICHARDJ.HOFFMANN

concerted effects of photoperiod on size and pigmentation brings about a reduction in the amount of resource committed to all of the major pteridine components found in C. eurytheme. ESfects of temperature Five specimens of each sex and phenotype were selected at random from each treatment group. Pupae were incubated at 15, 20, 25, and 30°C. Sixty insects were examined. The effects of pupal development temperature on adult C. eurytheme males are plotted in Fig. 3. From Fig. 3 and Table 3 it is apparent that there is no significant effect of temperature on the pteridine content of males. Fig. 4 and Table 3 demonstrate that the situation in orange females is not

FIG. 3. Effect of temperature on the four major pteridines in males.

Symbols as in Fig. 2. Temperature produces no significant effect on any of these pteridines. Bars indicate If:1 S.E.

TABLE 3-PRoBABILITy VALUES FROM THE ANALYSIS OF VARIANCE FOR THE SIGNFICANCE OF AN EFFECT OF TEMPERATURE ON THE PTERIDINE PIGMENTATION OF C. eurytheme

Pigment Xanthopterin Leucopterin Erythropterin Sepiapterin

Males

Females (orange)

O*lO
O*lO
Females (‘alba’) P< 0.001 0.5O
* Sepiapterin was not detectable in high temperature ‘alba’ females, but was barely detectable by fluorescence on the electrophoresed strips from low temperature individuals. Quantities were not suthcient for measurement by the techniques employed.

PTRRIDINE

VARIATION

IN

COLIAS

1919

different from that in males. However, there seems to be a trend towards greater amounts of the yellow pigment xanthopterin in the 15°C individuals. Nonetheless, temperature has no statistically significant effect on any of the four major pteridines in orange females.

FIG. 4. Effect of temperature on the four major pteridines in orange females. Symbols as in Fig. 2. There is not significant effect of temperature on the pteridines of orange females. Bars indicate + 1 S.E.

The effect of temperature is quite different for the ‘alba’ females. The quantitative results are presented in Fig. 5 and Table 3. There is no effect of temperature on leucopterin, the pigment contributing most to the ground colour of this form. But the other three pigments are significantly affected by the temperature at which pupal development takes place. The effect is visually most striking in the case of xanthopterin, the yellow pigment which makes the greatest contribution to the orange phenotype. There is at least a threefold increase in the amount of this pigment with low temperature. The yellow pigmentation is distributed over the surfaces of both the fore- and hindwings, giving the ‘alba’ females so treated an overall yellow cast. Erythropterin also increases dramatically with lowered temperature, there being nearly a fourfold increase between 30 and 15°C. The visual effect in whole animals is not as striking as with xanthopterin because levels are still rather low at 15”C, and this red pigment is mainly confined to the upper sides of the forewings. While sepiapterin was not present in any individuals in sufficient quantities to measure with these techniques, there was barely detectable fluorescing material at the appropriate mobility on the electrophoresed strips from the low-temperature

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animals, while there was no detectable fluorescence from high-temperature insects. This suggests that there is some increased synthesis of this yellow pigment as well with low temperature, leaving leucopterin as the only major pteridine component unaffected by temperature in ‘alba’ females. It is important to note in this context, however, that the levels of the coloured pteridines do not approach the amounts

on the four major pteridines of ‘alba’ females. produces a significant change in all three coloured pteridines. While sepiapterin was not measurable by these techniques in any ‘alba’ females, it was present as barely detectable fluorescence on the electrophoresed strips from low-temperature animals. Bars indicate + 1 S.E. FIG. 5. Effect of temperature

Symbols as in Fig. 2. Temperature

found in the orange phenotypes. Even at 15°C the level of xanthopterin does not reach 50 per cent of that found in orange females at any temperature (Fig..4), SO that temperature does not act to bring about a complete recovery of the coloured pteridines to the levels found in orange phenotypes. DISCUSSION

The biosynthetic pathway of the four major pteridines found in C. eurytheme is known (WATT, 1967). Pteridines are synthesized from a precursor pool of guanosine, probably a guanosine phosphate. The pathway is bipartite, one branch leading to 6-alkylated pteridines (sepiapterin in this study), and the other leading to the other three pigments by cleavage of the 6-alkyl group of the initial pteridine product. Xanthopterin is then the precursor for colorless leucopterin and for red erythropterin by alkylation at the 7-position of the pteridine ring. That the side chain in erythropterin is derived from Krebs cycle intermediates is demonstrated by incorporation of radioactive label from malate, pyruvate, and lactate into the side chain. TUSKES and ATKINS (1973) studied the effects of temperature on orange C. eurytheme and claimed to have found a second generation effect of temperature on the degree of orange pigmentation. That is, they report that temperature has no

PTERIDINE VARIATION IN COLIAS

1921

effect on pteridines in the F, generation, but there is an effect on the F, and subsequent generations when the line is continuously reared at the new temperature. Low temperatures produce an effect similar to that produced by short photoperiods in orange individuals reported in the present study. If the effect that they observed was a reduction of erythropterin pigmentation, their data suggest that there may be some sort of selection by low temperature for different quantities of mitochondria that are not expressed unless applied to the parents (perhaps by affecting the developing germ line). As discussed above, the side chain of erythropterin is known to be related to Krebs cycle intermediates, and low temperature could be acting to select varying proportions of mitochondria in the cells and reduce the pool of available precursors to add the side chain to the 7-position of xanthopterin to make erythropterin. Whether such an effect would be cytoplasmic or an inductive effect on the nuclear genetic material remains a question. Further, TUSKES and ATKINS used an intermediate photoperiod (L12 : D12) which could place the photoperiodic system sufficiently close to the critical photoperiod, and hence in an unstable state, that temperature could perturb it. It is not clear, either, whether second generation effects would be important in the field, since it is clear that photoperiod alone can produce the entire range of phenotypes without any thermal modification at all, and second generation effects may be of minimal importance in a seasonal environment. The only time conditions are constant for long periods is during the sumer when levels of erythropterin are maximal. WATT (1967, 1973) has previously examined pteridine levels of C. emytheme from eastern United States stocks. His insects were reared on 16 hr photoperiods at about 27°C. The results of this study of C. eurytheme froIn the central valley of California, when insects were reared under similar conditions, are in close agreement with those of WATT, with the notable exception that erythropterin content of California insects is less than that from eastern stocks. The lower level of erythropterin in California stocks may be related to the absence in the west of C. eurytheme’s close relative, C. philodice Latreille, with which it hybridizes extensively, although not at random, in areas of sympatry (TAYLOR, 1972). Since it is known that C. ewytheme is visually sensitive to red light (POST and GOLDSMITH, 1969)) the elevated levels of erythropterin in eastern populations may represent character displacement to increase ease of species recognition between yellow C. philodice and orange C. eurytheme in areas of sympatry. There are presently no C. philodice in California’s central valley as far north as Los Banos where these stocks were obtained. There are reports (TAYLOR, personal communication) of C. philodice beginning to appear from southern California, and one would expect that increased orange pigment levels will follow this invasion if the above hypothesis is correct. The phenotype-specific difference in responsiveness of C. eurytheme pteridine pigmentation to temperature is curious. Males and orange, male-like females do not respond to temperature (although they do respond to photoperiod), while ‘alba’ females respond strongly to the influence of temperature. (The lack of an apparent response of this form to photoperiod probably results only from the lower levels of the coloured pteridines, rather than from an intrinsic difference in ability to

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respond.) The orange phenotype is strongly canalized against the action of temperature, at least during the pupal and pharate adult stages, whereas the ‘alba’ females are not. This suggests that the pool of pigment precursors leading to the coloured pteridines in orange individuals is sufficiently large to overcome any physiological effect of temperature by mass-action effects. Apparently enough precursor is synthesized, regardless of temperature, to yield near maximal levels of coloured pigmentation. Alternatively, cold may induce compensatory synthesis of additional pigment-producing enzymes. Short photoperiod acts in orange individuals to reduce the total amount of resources committed to pteridine pigmentation, both directly and by its action on wing size (Fig. 2, Table 2). A clue to the significance of this observation may lie in the work of WATT (1973) on the ‘alba’ variant of the genus. He suggests that the adaptive significance of this curious allele is to reduce the amount of resources committed to pigmentation during pupal and pharate adult development, releasing nitrogen to other functions. This advantage might be balanced by the apparent preference of males for mating with coloured females. The present study suggests that the orange phenotype, at least in this highly multivoltine species, also commits less total resource to pigmentation in cooler seasons when short photopheriods obtain. This observation is consistent with the notion that a redistribution of nitrogen resources is advantageous when thermal conditions are relatively stringent. In the ‘alba’ females, which have similar levels of leucopterin to those of the orange phenotypes (WATT, 1973; this study), the situation with respect to temperature is quite different from the orange insects. The effect of the ‘alba’ gene is to reduce the amounts of the coloured pigments without appreciably altering the levels of the colorless leucopterin. There is apparently insufficient coloured pteridine left for there to be an observable difference in their levels on On the other hand, the levels of coloured pteridines are different photoperiods. dramatically affected by the temperature at which pupal development takes place. It may be that ‘alba’ females accomplish a general reduction in pigmentary nitrogen genetically, while orange animals do so in cooler weather physiologically by cuing to photoperiod. The effects of temperature in the ‘alba’ females are more puzzling. It is not clear why there has not been selection for the canalization of the ‘alba’ form to prevent the partial reversal of the advantages accrued to this phenotype by increasing the amount of nitrogenous resources committed to pigmentation in cold weather, if it is indeed an adaptation to thermally stringent conditions. A clue may lie in the observation that the yellow and orange tinged forms of ‘alba’ females are not so commonly observed in other North American members of the genus (WATT, personal communication). C. eurytheme almost certainly was primarily more southern in its distribution before the advent of large monocultures of alfalfa (Medicago s&m), the larval foodplant in the field. In fact, the species has never been observed to diapause, meaning that large numbers are killed annually by the onset of winter at the northern ends of its current distribution, which suggests that the species has not yet completely adapted to its northern distribution. The appearance of coloured ‘alba’ females in the autumn in more

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1923

northern regions may simply reflect little selection in the insect’s recent past for strong canalization of the ‘alba’ phenotype against temperature fluctuations. Field collections of ‘alba’ C. ewytheme usually contain the orange and yellow tinged variants only in the latter part of the flight season (HOFFMANN, 1973 ; SHAPIRO and SHAPIRO, 1973). S ince the form can be produced in the laboratory simply by varying pupal and/or pharate adult temperature, even within a single split brood, it is curious that the variant forms are not usually found in the early spring when the insects first begin to fly. Thermal conditions are relatively cool then, too, in the northern portions of its distribution, although not as cool as in the autumn in California where insects fly from March or April to December. There may be other factors operative here, in addition to temperature. For example, some broods of ‘alba’ females do not show the same amount of elevation of the coloured pteridines with low temperature as shown in this study. Further study will be required to clarify the nature of this variability in ability to respond to temperature. It is also possible that spring-captured females are derived from larvae that developed in more southern, warmer regions. The adults then invade more northern regions to establish the populations annually. This would be consistent with the apparent inability of the species to overwinter in the north by diapause. Unfortunately, our understanding of the adaptive significance of these aspects of the seasonal variation in C. eurytheme is still limited by our relative lack of information on the details of the functional significance of the ground coloration. It is almost certainly involved in species recognition, but until we have a detailed knowledge of the important signals involved, it will be unclear which aspects of the phenotype should be free to vary. A further complication is presented by U.V. reflectance patterns that are also important in this and other species of C&V (SILBERGLIEDand TAYLOR, 1973). These patterns are produced by structural modifications of the scales (GHIRADELLA et al., 1972), rather than by the direct effects of the pigments. Until we know the relative contributions of these two kinds of colour, our understanding will probably remain limited. There is a mounting body of evidence, though, that suggests that pteridine pigmentation may be reduced as an adaptation to cold climates when other selective forces permit. Further study of the ‘alba’ variant and its adaptive significance should shed additional light on the importance of this aspect of the variability of the genus C&as. Acknowledgements-This study was supported in part by Grant No. GB 18704 from the National Science Foundation to W. B. WATT. I thank W. B. WATT and C. SA~SAMAN for helpful discussions and commentary on a draft of the manuscript. REFERENCES AE S. A. (1957) Effects of photoperiod on ColiQs eurytheme. Lepid. News 11, 207-214. BLAKLEYR. L. (1969) The Biochemistry of Folic Acid and Related Pteridines. John Wiley, New York. FORD E. B. (1947) A murexide test for the recognition of pterins in intact insects. Proc. R. ent. Sot. Lond. (A) 22, 72-76.

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GHIRADELLA H., ANESHANSLEY D., EISNERT., SILBERGLIED R. E., and HINTONH. E. (1972) Ultraviolet reflection of a male butterfly: interference color caused by thin-layer elaboration of wing scales. Science, Wash. 178, 1214-1217. HOFFMANNR. J. (1973) Environmental control of seasonal variation in the butterfly Colias ewytheme-I. Adaptive aspects of a photoperiodic response. Evolution 27, 387-397. HOPKINSF. G. (1895) The pigments of Pieridae: a contribution to the study of excretory substances which function in ornament. Phil. Trans. R. Sot. (B) 186, 661-682. POST C. T. and GOLDSMITHT. H. (1969) Physiological evidence for color receptors in the eye of a butterfly. Ann. ent. Sot. Am. 62, 1497-1498. REMINGTONC. L. (1954) The genetics of Colias (Lepidoptera). Adv. Genet. 6, 403-450. SHAPIROA. M. and SHAPIROA. R. (1973) The ecological associations of the butterflies of Staten Island. J. lies. Lepid. 12, 65-128. SILBERGLIED R. E. and TAYLOR 0. R. (1973) Ultraviolet differences between the sulphur butterflies, Colias ezwytheme and C. philodice, and a possible isolating mechanism. Nature, Lond. 241,406-408. SOKALR. R. and ROHLF F. J. (1969) Biometry. W. H. Freeman, San Francisco. TAYLOR 0. R. (1972) Random vs. non-random mating in the sulfur butterflies, Colias eurytheme and Colias philodice (Lepidoptera: Pieridae). Evolution 26, 344-356. TUSKESP. M. and ATKINS M. D. (1973) Effect of temperature on occurrence of color phases in the alfalfa caterpillar (Lepidoptera: Pieridae). Environ. Ent. 2, 619-622. WATT W. B. (1964) Pteridine components of wing pigmentation in the buttertly Colias eurytheme. Nature, Lond. 2Q1, 1326-1327. WATT W. B. (1967) Pteridine biosynthesis in the butterfly Colias ewytheme. J. biol. Chem. 242, 565-572. WATT W. B. (1969) Adaptive significance of pigment polymorphisms in Co&as butterfliesII. Thermoregulation and photoperiodically controlled melanin variation in CoZias eurytheme. Proc. nut, Acad. Sci. U.S.A. 63, 767-774. WATT W. B. (1973) Adaptive significance of pigment polymorphisms in Colias butterlliesIII. Progress in the study of the ‘alba’ variant. Evolution 27, 537-548.