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1969

Journal of the Lepidopterists' Society

77

EFFECTS OF ENVIRONMENTAL FACTORS ON THE MARKINGS OF PIERIS RAPAE (PIERIDAE)

John M. Kolyer

55 Chimney Ridge Drive, Convent, New Jersey, U.S.A.

Introduction

The spring brood of Pieris rapae (Linnaeus) has the dark markings "somewhat reduced, sometimes entirely absent" in the United States (Klots, 1951) and "much less distinct" in England (Pugh, 1934). According to Comstock and Comstock (1943), the "typical form is dimorphic; the spring brood, which comes from the wintering chrysalids, is composed of smaller butterflies, which are of a duller white than the summer butterflies and with smaller black markings on the middle and tip of the wings." The spotless form of the spring brood male (Pieris rapae "immaeulata") is illustrated by the latter authors. Incidentally, the reduction in size of the spring brood noted by Comstock and Comstock (1943) was not found in a sampling of New Jersey specimens, in which forewing lengths (base to apex) averaged 22 mm for both males and females vs. 22 or 23 mm for summer brood specimens (Kolyer, 1966). Scudder (1899) notes the "more grimy under surface" of the spring brood.

In New Jersey, spring brood males are very often (over 50% of one sampling) of the "immaeulata" form, though the more heavily-marked specimens begin to resemble the summer brood, while spring brood females tend to lack the apical marking and have the basal area of the forewing dusky. The underside of both sexes shows more or less increased melanization. Examples are shown in Plate III, No. 26 (cf. Plate II, No. 17, for typical summer brood forewings).

The object of the present work was to study some of the factors, notably reduced temperature, which may be involved in the pronounced seasonal dimorphism of Pieris rapae. This paper is an extension of a previous publication (Kolyer, 1966) which was concerned principally with the effect on the markings of feeding larvae various chemicals affecting melano-genesis.

Experimental Procedure

The rearing procedure was the same as used in the earlier work (Kolyer, 1966); larvae were reared in cardboard boxes with gauze windows in the lids and were fed cabbage leaves from refrigerated heads. Data on the conditions of rearing are given in Tables 2-4. Before refrigeration, pupae were packed carefully along with facial tissue in fiber mailing cans, which were covered with polyethylene bags to exclude moisture.

78

Kolyer: Pieris markings affected

Vol. 23, no. 2

Table 1. Markings

Summary of Experimental Conditions and Effect on

Group No.

Special Conditions

Notable Adult Markings, with reference to specimen no. in Plates I-IV

1          Pupae at 40-45°F. for 3-7 weeks.

2          Pupae at 32-38°F. for 39 days.

3          Larvae reared in darkness at reduced temperature; pupae at 33— 68°F. for 8-26 days.

4          Larvae reared at reduced temperature (10 hr. photoperiod) to extend larval stage to 45-50 days.

5          Reared at room temperature with 10 hr. photoperiod (control for Group 4).

6          Pupae at 37-41°F. for 11-29 days, then 10-16°F. for 49 days.

7a, b Pupae (pupated at room temp, or

57-60°F.) at 34-39°F. for 25-32

days, then 12-16°F. for 40 days. 8a, b Pupae (pupated at room temp, or

57-60°F.) allowed to develop at

53-57°F. (13-29 day pupal stage). 9         No special conditions (control

for Groups 7-18). 10a, b Pupae (pupated at room temp, or

57-60°F.) at 34-39°F. for 61-67

days. 11         Pupae (pupated at 57-60°F.) at

53-57°F. for 24 days, then 34-

39°F.for37days. 12a, b Pupae (pupated at room temp, or

57-60°F.) at 34-39°F. for 151-

157 days.

13          New pupae exposed to 14°F. for 30 min.

14          New pupae exposed to 14 °F. for 2hr.

15          New pupae exposed to 18°F. for 1 hr., then 41 °F. for 15 min.

16          Pupae at 34-39°F. for 24-28 days, then left wingcase contacted with Dry Ice for 1 sec.

17          Like Group 16 but contacted with Dry Ice for 3 sec.

18          Pupae in oxygen atmosphere for 62 hrs.

Generally typical summer brood. Nos. 1

and 2 show extreme range of variation.

( Note unusual asymmetric $ , no. 3).

Normal summer brood; some basal melani-

zation of 9 fore wings.

Four of 17 specimens rather lightly

marked; these are illustrated previously

(Kolyer, 1966).

Essentially summer brood markings; nos. 4

and 5.

Typical summer brood markings; no. 6.

One "immaculata" $ , one summer brood 9 ; nos. 7 and 8.

One "immaculata" 8 , 3 lightly-marked $ $ ; 1 summer brood 9 , 3 lightly-marked 9 9 (esp. no. 12); nos. 9-14. Summer brood markings with slight tendency to lightness, e.g. weak apical markings in a few 9 9 ; nos. 15 and 16. Summer brood markings, even for 4 dwarfed specimens; nos. 17 and 18. S $ with tendency toward basal melani-zation, 2 "immaculata"; one 9 with unusual melanization (no. 23); nos. 19-23. Summer brood markings (2. $ $ ); no. 25.

Of 21 $$,4 with weak spots, 5 "immaculata," tendency to basal melanization in others; of 16 9 9,4 very lightly marked, 1 with unusual melanization; nos. 32—35. 9 with weak markings and undersized fore wings; no. 27.

Essentially summer brood markings; no. 28. ( Note scales missing in spot 1 of 9 .) Asymmetric 9 with discal spot reduced on right forewing; no. 29. $ with light markings, 9 with apical markings almost absent, asymmetric $ with discal spot weak on left forewing; no. 30.

8 with light markings, asymmetric 9 with discal spot missing on left forewing; no. 31 Summer brood markings, like Group 9.

1969

Journal of the LepidopteHsts Society

79

Explanation of Graph 1 Frequency curves for forewing spot widths (spot 1 or upper spot for 9 $ ) for Group 1. Vertical axis = number of specimens; horizontal axis = spot width in mm.

In those cases in which larvae were exposed to reduced temperature under light (e.g. Group 4), a refrigerated compartment with a plastic window was used. Three layers (each 0.005 inch thick) of Aclar fluorocarbon film (Allied Chemical Corp.), which is highly transparent to light to a wavelength of 230 millimicrons, comprised the window separating the insects from the external light source. Pupae were similarly exposed in some cases (e.g. Group 8). Relative humidity and temperature in the tables are for the atmosphere external to the rearing boxes and do not describe the microenvironment at the surface of the cabbage leaf. Yields of adults generally are given as a fraction, e.g. 3/7 means that seven pupae yielded three adults. In Table 5, specimens were expanded normally unless otherwise noted. Incidentally, the photographs in Plates I-IV were taken by a published procedure (Kolyer, 1965) with specimens on either red (which appears as dark gray) or black velvet in Riker mounts.

Results and Discussion

Variability of markings. Discussion of the results must be preceded by emphasizing the great variability in the markings between individuals. This was pointed out in the earlier work (Kolyer, 1966), and another illustration is seen in Plate I, Nos. 1-3, and in Graph 1, which shows approximately normal distribution curves for the widths of the male spot

80

Kolyer: Pieris markings affected                   Vol. 23, no. 2

s*&

Am

^tutmm

ISSMWW I

Explanation of Plate I

Specimens of Pieris rapae (L.). 1-3, Group 1; 1, forewings of 6•$ $ showing range of variation of markings, especially the discal spot; the forewing just above the number 1 is a yellow form; 2, forewings of 6 $ 2 showing range of variation of markings; 3, unusual asymmetrically-marked $. 4-5, Group 4; 4, $, eclosed on day = 63; 5, $, eclosed on day = 117 and $, eclosed on day = 81. 6, Group 5 (control for group 4), forewings of 6 $ $ (right hand column) and 13 $ $. 7-8, Group 6; 7, unexpanded forewings of $; 8, $. 9^11, Group 7a; 9, unexpended forewings of $ (left) and $ (right); 10, unexpanded forewings of 2; 11, unexpanded forewings of £. 12-14, Group 7b; 12, $; 13, unexpanded forewing of $ ; 14, unexpanded forewing of $ .

1969                                Journal of the Lepidopterists' Society                                     81

Table 2. Origin of Eggs and Chronology for Groups 1-6

Chronology (days from arbitrary starting point)

Group                                                      ------------------------------------------------------------------

No.            Origin of Eggs                      Oviposition Hatching Pupation Eclosion

1       U.S.D.A.1                                             -                  0              9-17     approx. 36-72

2       Third generation of culture2            0-2              4-6          24-28 71-75

3       Same                                                 Same           Same         32-50 62-87

4       Adults collected at Morristown       0-2              3-5          48-55 63-117

and Berkshire Valley, New Jersey, August 6 and 7, 1966

5       Same                                                 Same           Same         20-26           27-31

6       U.S.D.A.3_________________________-_________0-2          16-34________103

1 Eggs from field-collected adults were reared at U.S. Department of Agriculture, Ag. Res. Service, Entomology Res. Div. lab at Charleston, S.C., under the direction of W. J. Reid, Jr. Larvae were fed collards or cabbage.

2 The culture (at Convent, N.J.) was started July 2, 1964 with eggs from adults taken at Morris-town and Flemington, N.J.

3 Eggs were from N. R. Spencer, U.S. Department of Agriculture, Entomology Res. Div., Columbia, Missouri.

and female spot 1 (the upper discal spot). The intensity of the markings also varies greatly. This variability noted for the summer brood also occurs in the spring brood. For example, among the eight forewings in Plate III, no. 26, will be noted a male and a female wing that might pass for somewhat lightly-marked summer brood specimens. The result is that conclusions must be drawn with caution, especially where only a few specimens are involved.

Possible factors determining spring brood markings. In New Jersey, conditions for the spring brood differ from summer brood conditions in (1) reduced temperature during the larval period (in September and October), (2) shortened photoperiod (causing the diapause necessary for overwintering), (3) reduced temperature at the time of pupation, (4) reduced temperatures, often well below freezing, during the winter, and (5) reduced temperature during completion of pupal development (typically in April) when diapause has ended. Number 4 may seem to be the most notable difference, but the others must be considered.

Another way to look at the problem is to consider the point in development at which spring brood markings are determined, i.e. the larval stage, the early part of the pupal stage (pupation and shortly thereafter), the dormant part of the pupal stage (when the pupa has hardened but has not begun final development), or the final portion of the pupal stage when the imago develops and wing pigments are deposited.

The experiments were intended to give some idea of the relative importance of the above factors and of their time of operation.

Besults discussed in terms of possible factors. The experimental conditions and results, given in detail in Tables 2-5, are summarized for

82

Kolyer: Pieris markings affected                   Vol. 23, no. 2

A 6 <*# r il mm

^^ ** <£m

I J& mm "B»

^g ^gp ^KP

^3 j^Bm jBm

dBSm wKmm Wm& ~"               __ ra*.

. « 41

^jgj2 ^^^h^ ^sprif

•»* 4*^1

SSMS M

mm* Am

^S ^a ^^g 0-gA

^^^ #M* ^^» 4BP ^w

mm* *' r m*

**_%*

Explanation of Plate II

Specimens of Pieris rapae (L.). 15, Group 8a, forewings of 12 # $ (first column and top four in second column) and 21 9 9; the four $ $ in the second column eclosed after day = 46, as did the five 9 9 at the bottom of the last column. 16, Group 8b, forewings of 25 $ $ (first three columns) and 13 9 9; the two $ $ at the bottom of the third column eclosed after day = 46, as did the 9 at the bottom of the last column. 17-18, Group 9 (control for Groups 7-18); 17, forewings of 16 S $ (first two columns) and 13 9 9; 18, $ (lower left) and 3 9 9, undersized specimens (forewing length 16 mm vs. 17-21 mm for remainder of Group 9). 19-21, Group 10a; 19, unexpanded forewing of 9 ; 20, 9 ; 21, $ .

1969

Journal of the Lepidopterists' Society

83

convenience in Table 1. The following discussion covers, by number, the five factors enumerated above.

(1)   In certain Colias species, the effect of a slowed development rate is said to resemble that of reduced temperature (Hovanitz, 1945). In Polygonia c-album, the length of the larval period is supposed to control production of light and dark forms (Klots, 1951). In Group 4, the larval period was extended by reduced temperature from the normal summer length of about three weeks to 45-50 days. Only three individuals were obtained, none of which had spring brood markings. This demonstrates that larvae reared at reduced temperatures can give summer brood markings but, considering variability, does not show that such larvae cannot give spring brood markings. In Group 3, in which the larval stage lasted about 28-44 days due to reduced temperature, there was some reduction in markings in four of 17 individuals, but this might be attributed to exposure of the pupae to reduced temperatures for up to 26 days. In conclusion, reduced temperature during the larval stage had no dramatic result such as a high incidence of reduced markings or production of "immaculata" males.

(2)   Photoperiod may determine spring and summer forms in some butterflies. For example, variation of the photoperiod can produce the seasonal forms of Ascia monuste (L.) (Pease, 1962). In Pieris rapae, the photoperiod is known to be responsible for diapause when the temperature is sufficiently low. For example, almost all pupae diapause when larvae are reared at 17-20° C. with a 10 hr./day photoperiod (Barker, Cohen, and Mayer, 1964), while at 20° C., but not at 24° C., pupae diapaused when larvae were given photo-periods of under 11 hrs./day (Barker, Mayer, and Cohen, 1963). At summer temperatures in New Jersey, normal summer brood markings resulted when larvae were reared in darkness or with 18 hrs. light/day (Kolyer, 1966). Normal summer brood markings also were given by 10 hrs. light/day at summer temperatures (Group 5; Plate I, No. 6) or at reduced temperatures (Group 4). However, spring brood markings were given with photoperiod provided by diffuse summer sunlight during rearing when the pupae were held for over two months at reduced temperatures, as will be seen. In summary, all that can be said is that photoperiod gave no striking results in the experiments, which does not mean that it might not have considerable effect with high-intensity fluorescent light or direct sunlight, for example. As will be seen, a reduced photoperiod (or diapause) is not necessary for experimental production of spring brood markings, although it may have an influence in nature.

(3)   Aberrations in butterflies may be caused by chilling recently-

84

Kolyer: Pieris markings affected                   Vol. 23, no. 2

sss

• ~ &

Ss3

& + &

M*6

Explanation of Plate III

Specimens of Pieris rapae (L.). 22-23, Group 10b; 22, forewings of 14 $ $ (first two columns) and 8 9 2 (one unexpanded); 23, forewing (above) and underside of heavily melanized 2 . 24, underside of normal summer brood 2 for comparison. 25, Group 11, 2 $ $. 26, forewings of 4 $ $ (left column) and 4 2 2, typical spring brood (taken at Flemington, N.J., May 1, 1965), and spring brood ("immaculata") $ (below) taken at Southold, Long Island, N.Y., circa 1947. 27, Group 13, 2. 28, Group 14, $ (bottom) and 2 2 2. 29, Group 15, 2. 30, Group 16, 2 $ $ and 2 2 2 . 31, Group 17, 2 $ $ (above) and 2 .

1969

Journal of the Lepidopterists' Society

85

Table 3. Larval and Pupal Conditions for Groups 1-6

1 Starting about 24 hrs. (occasionally 3-4 days) after pupation, pupae were refrigerated for approx. 21-49 days. The pupae received perhaps 1 hour light/week during sorting.

2 Starting about 0.5-4 days after pupation, on day-28, pupae were refrigerated for 39 days. Then the pupae were left at room temp, with 18 hrs. light/day.

3 Starting on day-12 (Oct. 19, 1964), the larvae (3-8 mm) were removed from room temp, conditions and reared in the dark at reduced, fluctuating temperature (unheated building, Convent, NJ.). The average of 38 random temperature readings was 53°F.

4 For day-32-58 (unheated building). The average of 48 random temperature readings was 51 °F. Pupae were in darkness in the unheated building for 8-26 days. On day-58 (Dec. 4, 1964) they were transferred to the rear room (67-77°F., 36-52% rel. humidity, 18 hrs. light/day). All pupae were brown.

5  Starting on day-6, the larvae (3—6 mm) were removed from room temperature conditions and reared at 10 hrs. incandescent light/day and 52-58°F. until day-25. Then the larvae were refrigerated (darkness, 34-37°F.) until day-29. From day-29-39, the larvae were returned to 52-58°F. for 10 hrs. incandescent light/day while being refrigerated (darkness, 34-37°F.) for the remaining 14 hrs./day (object was to slow growth). From day-39-55, the larvae were again exposed to 10 hrs. incandescent light/day at 52-58°F. Light was provided by a 25 watt bulb 30 inches from the larvae.

6 Light was provided by a 25 watt bulb 25 inches from the pupae. Of new pupae, four were brown and three more or less green.

7 Light was provided by a 25 watt bulb 30 inches from the larvae.

8 April 8-May 13, 1967 at Convent, N.J.

9 The pupae were refrigerated (37-41°F.) about 12 hrs. after pupation. On day-45, the pupae were transferred to a freezer (10—16°F.), left for 49 days, and then transferred to a refrigerator (37—41°F.) for 1 day before removing (on day-95) to rear room conditions (76-84°F., 52-57% rel. humidity, diffuse sunlight).

formed pupae. For example, the wing pattern of Nymphalis anitopa (L.) and other species is particularly susceptible to change by chilling at 12-48 hours after pupation (Kiihn, 1927). Newly-formed pupae of Zerythnia hy-permnestra Sc. were chilled at -10° C. to give adults with an extensive black pattern (Racz, 1967). The frequency of melanic and albinic butterflies in certain parts of Pomerania has been attributed to very severe temperature changes (Mecke, 1926). When recently formed Pieris rapae pupae

Kolyer: Pieris markings affected

Vol. 23, no. 2

IPP % p I

Explanation of Plate IV

Specimens of Pieris rapae (L.). 32, Group 12a, 2 $ $ (above) and 2 2 $ ; the undersides are shown in the right-hand photograph. 33, Group 12b, 2 $ $ ; the undersides are shown in the right-hand photograph. 34, Group 12a, $ and forewings of 6 8 S (above; two unexpanded) and 5 2 2 (one unexpanded). 35, Group 12b, $ underside with upper surface of forewing to the left, and forewings of 8 $ $ (above; three unexpanded) and 7 2 2 (three unexpanded).

1969                                Journal of the Lepidopterists' Society                                     87

were chilled (Groups 13-15), some variations in markings were noted, but there was no indication that the spring form might be produced in this way. Some reduction of markings was noted when one wing case was contacted briefly with solid carbon dioxide (dry ice) (Groups 16 and 17), but the symmetrical effects are suspected to be due to the refrigeration preceding dry ice treatment. In one female (Plate III, No. 31) the discal spots were obliterated on the chilled wing, but the overall results suggest that extreme chilling of new pupae be discounted as the predominant cause of spring brood markings. In any event, it is hard to imagine sudden, extreme temperature changes as the prime natural causative factor. Pupation at 57-60° F., a rather mild variation, had no noticeable effect on markings vs. pupation at room temperature in Groups 7, 8, 10, 11, and 12. The only notable effect was on pupal color, which generally was darkened. The green pupae (estimated at 40-50% of the total) formed at 57-60° F. were heavily speckled with black vs. solid light green for green pupae formed at room temperature. Of course, differences in light composition and intensity, and in photoperiod, were involved as well as reduced temperature.

(4) Pugh (1934) stored Pieris rapae pupae, presumably in diapause, at 20° C. and in a "cool cellar." After two months the pupae at 20° C. began to emerge to give eight specimens all "intermediate between typical spring and summer broods" in markings. The pupae in the cool cellar were removed to room temperature after six months to give eight specimens with "typical spring markings." In the present work, nondiapause pupae were refrigerated at 40-45° F. for 3-7 weeks (Group 1) to give summer brood markings, at 32-38° F. for 39 days (Group 2) to give no effect except some basal melanization of female forewings, at 34-39° F. for 61-67 days (Group 10) to give two or three "immaculata" out of 16 males (Plate II, No. 21; Plate III, No. 22) and a tendency to basal and underside melanization and loss of apical markings in females (especially Plate III, No. 23), and at 34-39° F. for 151-157 days (Group 12) to give five "immaculata" out of 21 males and four of 16 females with extremely light markings (Plate IV, Nos. 32 and 33, shows specimens with spring brood markings). When pupae were exposed to 34-41° F. for 11-32 days and then to 10^16° F. for 40-49 days (Groups 6 and 7) there was a pronounced reduction in markings; of 5 males, 2 were "immaculata" (one shown in Plate I, No. 7), and of five females, three were more or less lightly marked (most notably No. 12, in Plate I). The significance of the above reduction in markings by storage at reduced temperature can be seen by comparing with the control forewings (Group 9) in Plate II, No. 17).

88

Kolyer: Pieris markings affected                   Vol. 23, no. 2

Table 4. Larval and Pupal Conditions for Groups 7-18

Laboratory site—Rearing was done at Convent, N.J. Origin of Eggs—Adults collected at Morristown, N.J., June 18, 1967. Oviposition—0-3 day (June 20-23, 1967). Hatching—3-6 day (June 23-26, 1967).

Temperature and Relative Humidity During Larval Stage—73-87°F. and 41-55%, except that on day-17 (July 7) the larvae (final instar) of Groups 7b, 8b, 10b, 11, and 12b were transferred to 57-60°F. and 47-55% rel. hum. The pupae were refrigerated when less than 24 hours old.

Light During Larval Stage—Diffuse sunlight (approx. June 23-July 14, 1967) for Groups 7a, 8a, 9, 10a, 12a, and 13-18. For the other Groups, diffuse sunlight (approx. June 23-July 7), then (after day-17) 10 hrs. light/day (50 watt daylight fluorescent bulb 24 inches from larvae).

Chronology

(days from                                                                                                 Yield

June 20, 1967)                                      Pupal Conditions                          Adults

Group----------------------------------------------------------------------------------------------from

xt            "T).,~^j-:----T7^1„^:^„                     T :~Uj-                              TV.^^ otp u ^1 u,----- erf            tti

1969

Journal of the Lepidopterists' Society

89

The conclusion is that storage at reduced (but not necessarily sub-freezing) temperatures, without diapause, causes a significant incidence of spring brood markings, e.g. five "immaculata" males of total of 21 after 5 months at 34-39° F. In New Jersey, the pupae are exposed for 6-7 months to generally low temperatures, often far below freezing (daily lows of 10-20° F. are not uncommon in midwinter, and a record low of -34° F. was recorded in New Jersey in 1904). Intuitively, one might expect these subfreezing temperatures to influence the markings. However, delayed pupal development itself, without reduced temperatures, seems to cause reduced markings since Pugh (1934) reported specimens "intermediate between typical spring and summer broods'7 by storage of diapause pupae at 68° F. Certainly, the present work demonstrates that subfreezing temperatures are not necessary to obtain the spring form. Thus, in the production of spring brood markings in nature, it is not obvious what contribution is made by reduction of temperature below that level of cold needed to arrest pupal development.

(5) In Group 8, nondiapause pupae were allowed to develop at 53-57° F. About 13-29 days were required for development, presumably more or less simulating natural conditions in New Jersey in April. (Typical time for development in the summer is 5-10 days, e.g. Group 9). The result (Plate III, Nos. 15 and 16) was a slight tendency toward reduction of markings, but there was no pronounced effect such as formation of

<—

1 Within 24 hours after pupation, the pupae were refrigerated (34-39°F.) and left to day-50. Then the pupae were left in a freezer at 12—16°F. for 40 days. To reduce possible temperature shock, the pupae were kept at 28°F, for 12 hours and then 34°F. for 1 day before removing (on day-71) to 68-82°F. and 48-56% R.H. (diffuse sunlight).

2 On day-25, the pupae were removed from refrigeration (34—39°F.) and exposed to continuous light (50 watt daylight fluorescent bulb 24 inches from larvae) at 53-57°F. and approx. 30% R.H.

3 Within 24 hours after pupation, the pupae were refrigerated (34—39°F.). On day-86 the pupae were removed to rear room conditions (68—83°F., 45—55% R.H., diffuse sunlight) and allowed to develop. Group 10a was under refrigeration for 63—67 days, Group 10b for 61—65 days.

4 Having shown no sign of development at 53—57 °F., the two pupae were again refrigerated (34-39°F.) on day-49. On day-86 they were removed to rear room conditions (68-83°F., 45-55% R.H., diffuse sunlight). Thus, the pupae spent about 24 days at 53-57°F. and 37 days at 34-39°F.

5 Within 24 hours after pupation, the pupae were refrigerated (34—39°F.). On day-176, they were removed to rear room conditions (67-74°F., 30-42% R.H., diffuse sunlight). Group 12a had been refrigerated for 153-157 days, Group 12b for 151-155 days.

6 Still-soft pupae (less than 8 hours old) were placed in a freezer at 14°F. for 30 minutes and then returned to the rear room.

7 As in footnote 6, but at 14°F. for 2 hours.

8 Pupae (less than 9 hours old) were placed in a freezer at 18°F. for 1 hour, then transferred to a refrigerator at 41 °F. for 15 minutes, and then returned to the rear room.

9 On day-46, the pupae, which had been refrigerated within 24 hours after pupation, were removed from the refrigerator. A rounded piece of dry ice was pressed against the left wingcase for 1 second, and the pupa then was left at ambient conditions (74-89°F., 45-63% R.H., diffuse sunlight).

10 As in footnote 9, except that dry ice exposure was 3 seconds.

11 A one-liter flask containing the pupae (less than 24 hours old) was well-flushed with oxygen (Linde Co.) and stoppered tightly. For humidification, solid potassium carbonate hydrate in contact with the saturated solution was included; this gives 43% R.H. in air at 72°F. (humidity level imparted to oxygen unknown). After 62 hours the pupae were removed (day-24) to the rear room.

90

Kolyer: Pieris markings affected                   Vol. 23, no. 2

Table 5. Data on Adults

(See Table 1 for illustration numbers in Plates I-IV)

1 Unlike any of 57 d" d" or 49 ? $ in a control brood reared at <

2 Including dwarfed specimens (1 cf, 3 ? ? ).

SB = summer brood markings, like No. 17 in Plate II AM = apical marking

S = spot(s) LM = lightly-marked BM r= basal melanization of forewing UEx = unexpanded

-81 °F. and 35-61% R.H.

UM = underside melanization FW = forewing I = "immaculata" E = expanded normally UEcl = unable to eclose

1969                            Journal of the Lepidopterists' Society                                 91

Table 5. Continued

"immaculata" males. Two of the pupae which had formed at 57-60° F. (10 hrs. light/day) failed to show signs of development (whitening of the wingcases) after 24 days at 53-57° F. and were suspected to be diapause pupae (Group 11). After 37 days refrigeration, these were allowed to develop at room temperature (7-9 days required) and gave two males with summer brood markings (Plate III, No. 25). The conclusion is that any slight effect on markings by moderately reduced temperature during completion of development of the adult wings would be overshadowed in nature by the effect of reduced-temperature storage, which in Groups 10 and 12 gave a significant portion of spring brood markings even though the pupae completed development at 67-83° F.

The above results suggest that the point in development during which the principal factor (extended reduced temperature) is effective is the dormant part of the pupal stage. A purely speculative explanation for the results is that the chromogen which is finally oxidized to give the markings (Onslow, 1916) is distributed only in the final stage of development after diapause has broken (or the pupae have been removed from refrigeration). Then, the normal movement of chromogen to the upper surface and out from the base toward the apex may be restricted by unknown changes which have occurred in the wing during prolonged dormancy. This would explain the loss of apical and discal markings and the concentration of melanin at the base of the forewing and on the undersides of the wings.

Other factors investigated. Hot, moist air is reported to produce in-

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creased black pigmentation in Junonia coenia (Hbn.) (Schrader, 1926). In the present work, Pieris pupae were allowed to develop at 69-78° F. and a constant 87% relative humidity (measured at 75° F.); the adults had normal summer brood markings. Development of pupae at an unnaturally-high barometric pressure (825-830 mm), 43% rel. humidity, and 69-78° F. also gave summer brood markings. Four dwarfs were produced by some of the last larvae to pupate in Group 9 (see Plate II, No. 18). These had forewings 16 mm in length (measured from base to apex) vs. generally 21 mm for the remainder of Group 9, but the markings were essentially normal. In Pieris brassicae, the small form "minor" can be produced by starvation (Gardiner, 1963), but the above dwarfs were produced in the presence of an ample food supply. At the time the dwarfed pupae were formed mortality among both larvae and pupae was very high, presumably due to an unidentified disease; this factor may have been related to the observed dwarfism.

When Gonepteryx rhamni pupae had been exposed to pure oxygen, the adult butterflies were much paler than normal (Becker, 1937). On the other hand, atmospheric oxygen is responsible for the dark markings of Pieris brassicae by oxidation of localized chromogen on the wings of the maturing pupa (Onslow, 1916). Though this condition is not found in nature, Pieris rapae pupae were exposed to oxygen for various lengths of time in the present work. When nine pupae (less than 24 hours old) were left in pure oxygen (humidified as described in Table 4, Note 11) at 73-87° F. for one week, the adults (eclosed on the last day) failed to expand in all cases. The markings in the unexpanded wings seemed not unusual. The same result was given with five pupae (four eclosed) similarly exposed to oxygen for 6 days. When the time of exposure (10 pupae) was reduced to 62 hours (Group 18), nine adults eclosed, eight of which expanded normally. The markings were neither more nor less intense than those of the control (Group 9).

Summary

Experiments were conducted to evaluate the relative importance of some environmental factors in determining the spring brood markings of Pieris rapae (L.), including reduced intensity of the apical and discal markings and increased melanization of the base of the forewing and the undersides of the wings.

Normal summer brood markings were produced by rearing the larvae at reduced temperatures to extend the larval period from the normal three weeks to 45-50 days.

Chilling of newly-formed pupae gave some aberrations in markings but was not indicated as a factor in seasonal dimorphism.

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Only a hint of spring brood markings was obtained by allowing pupae to develop at 53-57° F. or by refrigeration (32-38° F.) of pupae for 39 days,

A significant proportion of specimens with spring brood markings, including the male form "immaculata" and females with no apical markings and heavy melanization of the basal area of the forewing, was produced by refrigeration of pupae at 34-39° F. for about 65 days (two or three of 16 males were "immaculata") or about 155 days (five of 21 males were "immaculata"). Spring brood markings were also produced by refrigeration pupae at 34-41° F. for 11-32 days and then at 10-16° F. for 40-49 days (two of five males were "immaculata"). None of the pupae in the above experiments were in diapause.

High relative humidity or high barometric pressure had no effect on markings when pupae developed at summer temperatures. In an incidental experiment, pupae exposed to pure oxygen for 6-7 days gave adults unable to expand their wings. Briefer exposure (62 hrs.) allowed normal expansion, but markings were typical of the summer brood.

The conclusion is that subfreezing temperatures, diapause, shortened photoperiod, and reduced temperature during the larval stage, time of pupation, or completion of development preceding eclosion, were unnecessary in producing significant proportions of spring brood specimens experimentally. However, the possibility cannot be excluded that the above factors may play a role in nature.

The present work and the literature suggest that the dominant factor causing reduction of markings might be expressed as extended dormancy of the pupa, necessarily accompanied by temperatures sufficiently reduced to prevent completion of development. The role of degree of temperature depression, beyond that needed to arrest pupal development, is not clear from the available data.

Acknowledgments The author acknowledges the valuable aid of Mr. W. J. Reid, Jr., U. S. Department of Agriculture, Entom. Res. Div., Charleston, South Carolina, for supplying pupae and accompanying data, and the assistance of Mr. N. R. Spencer, U. S. Department of Agriculture, Entom. Res. Div., Columbia, Missouri, for supplying eggs from his Pieris rapae culture.

LITERATURE ClTED

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Zeichnung von Vespa im besonderen. Zeitsch. Morph. u. Okol Tiere, 32(4):

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