Some contributions to the life cycle of Papilio zelicaon Lucas with special reference to the hardening of the wings

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SOME CONTRIBUTIONS TO THE LIFE CYCLE OF PAPILIO ZELICAON LUCAS WITH SPECIAL REFERENCE TO THE HARDENING OF THE WINGS

A Thesis Presented to the Faculty of the Department of Zoology University of Southern California

In Partial Fulfillment of the Requirements for the Degree Master of Arts

hy Bernard E. Brown August 1950

UMI Number: EP67188

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This thesis, w ritten by

Mr. Bernard Eldon Brown under the guidance of h..%3... F a c u lty Com m ittee, and app ro ved by a l l its members, has been presented to and accepted by the C o uncil on G raduate S tudy and Research in p a r t ia l f u l f i l l ­ ment of the requirements f o r the degree of

Master of Arts

Date.\

Faculty Committee

Chairman

TABLE OF CONTENTS PAGE INTRODUCTION

1

FEEDING

2

COLOR OF CHRYSALIS

5

WING HARDENING History

9

Experimental Material

11

Method

13

Experiments on the drying of the wings

14

Experiment on oxidative reaction

15

Experiment on severed wing

15

Experiment on killing the butterfly

16

Experiment with the anesthesia

16

Discussion

17

Conclusion

18

SUMMARY

19

TABLES

21

LITERATURE CITED

25

INTRODUCTION This is a study to expand the knowledge concerning Papilio zelicaon

Dueas, and with special reference to the

hardening of the wings upon emergence from the chrysalis. The study is divided into three parts. The first part concerning the feeding habits of Papilio zelicaon. and the second part relating to the color of the chrysalis are sec­ tions which deal specifically with this one species. This information is of interest and use to further work on this species, but probably has no general validity in the order Lepidoptera as a whole. The third section deals with the hardening of the wings upon emergence. The findings presented here probably are true for all Lepidopterans in general.

2 FEEDING Papilio zelicaon

Lucas is called the fennel swallow­

tail because the caterpillar is found typically on fennel. This is an example of food adaptation since fennel is a recent introduction from Europe. Parish (1920) in his paper on "immigrant Plants in Southern California" could not say just when fennel was introduced, but it was still "rare" in San Bernardino County in 1890, and only "casual" in Los Angeles County in 1898. It seems quite likely that the plant was introduced as a garden escapee in the period between 1880 to 1900. Comstock (1921) in his work on butterflies of California states that the Anise Swallowtail (P. zelicaon) is common throughout the state and that its favorite food is wild anise (Carum kelloggi Gray). This plant is not found south of Santa Clara County. The possibility arises that before the intro­ duction of fennel the butterfly was not in this area. There is no easy way to check on this* In this study the caterpillar in the field was found only on fennel (Foenlculum vulgare Hill). However it was found that they will eat carrot, celery, poison hemlock and two other umbells: Qenanthe and Leptotaena, and Citrus (orange)•

•»

Essig (1926:; 634) notes that they will eat wild carrot, wild parsley, water hemlock and dog fennel. He also

3 states they are found on Citrus (especially orange, but are not pests). In raising the caterpillars some difficulty was found in trying to get them to live on orange. They usually die, however one went into the chrysalis after an orange diet, and a small second stage larva ate all the rind off of an orange still green. Apparently some individuals can and will live on Citrus. Tests trying to get the caterpillars to feed on poplar and lilac were failures. Other species in the genus Papilio will feed on the plants at least in emergencies. Caterpillars in this study starved on these plants with no sign of ever having eaten any of the plants. This information supports a contention that this species will (in captivity at least) eat almost any plant in the Umbelliferae family, and some individuals will eat from at least one species in the Citrus genus, (Table I). Most of the caterpillars (but not all) which were fed upon Citrus and poison hemlock died. One might infer from this that these two plants are not always suitable as a food plant for these caterpillars. In the wild state the Umbelliferae plants chosen by the female probably depend upon other factors in addition to the suitability of the plant as food. Such factors as location and ecological niche undoubtedly influence the female which lays her eggs on a plant which for her is not a

4 food plant* The factors inducing the female to oviposit on special plants would be interesting in themselves. In the early spring and late fall most of the cater­ pillars are found on young plants or fresh growth* During the season when the plants were dryer and more mature most of the caterpillars were found on the stem of the flower clusters. Although even then if young growth was available caterpillars were found upon it* A marked decrease in the caterpillars found in the field was observed during July. This could be correlated with other factors, but it is most probably due to a small gnat­ sized wasp. This wasp lays its eggs upon very young cater­ pillars. During the second larval stage the wasp larva emerges from the caterpillar, killing it. The wasp would form a cocoon and in about two weeks emerge as an adult*

5 COLOR OP CHRYSALIS Whatever the food, and In this study the caterpillars were found only on fennel, the egg is laid on the food plant of the caterpillar, which Is not a food plant for the adult# In the case of fennel, plants chosen are usually younger ones, isolated, hut near a large patch of fennel* Plowed fields that have broken stands of fennel, roadsides and railroad ways present good prospective locations for the caterpillars* A dense stand of fennel is usually completely barren of larvae. The reason behind this would be Interesting to know, since if scent is involved one would expect to find a dense stand as attractive as an isolated plant, especially since in most dense stands the individual plants are usually quite acces­ sible to the butterfly. The egg, laid on these plants, hatches in four days. The caterpillar emerges, eats the egg case, then starts feeding on the plant. There follow four caterpillar stages. The first stage is a small (0.5 cm.) black caterpillar with a white girdle. This is followed by a larger stage (3/4 cm.) also black and white. The third stage still has the white girdle but adds reddish dots and white spots over the feet. The final caterpillar stage is a large (4 cm.) handsome green animal with black and red or yellow bands on it. This large caterpillar fattens up and then in nature leaves the food plant to seek a spot to become the pupa. For the chrys-

alls a web mat is made on some plant or surface to hook the cremaster into when it is developed. A life-line is then formed around the thorax and the caterpillar settles down and becomes the chrysalis, this forming under the skin. The old skin is then shed, and the chrysalis hardens. The pupal changes then take place. -It is interesting to note that there are two different colored chrysalids: green and brown. These two shades may vary somewhat but are quite distinct and rather good camou­ flage, If brown, the chrysalis resembles the dead petioles of fennel, and if green, fit in well with the green foliage. It first appeared that there is a tendency to match the background, that is, brown chrysalis on brown, green on green. Of the chrysalids used in this study most were of necessity on green fennel (although in each case brown paper backgrounds were also available). A record kept of the number of chrysalids on various backgrounds showed no consistent correlation, namely of 79 chrysalids on a green background 54 were green and 25 were brown. On a brown background 5 were green and 4 were brown. The tendency to match is. appar­ ently a matter of chance, although matching the background could possibly have survival value, (Table II). In the chrysalid coloration however, there was noticed a tendency for the percentage of green ones to become more prevalent as summer came on, (Table II). This might be be-

7 cause of some seasonal factor, such as temperature, induces the color of the chrysalid since the green-brown ratio in­ creased from 2:1 to about 4}1 and finally all green. Of the various seasonal factors temperature would seem most obvious. Chilling animals about to enter the chrys­ alis apparently had no effect. All of the July first lot were chilled and all turned out green. Four other chrysalids of various age were also chilled with no color change elicited* The next explanation that came to mind suggested that the pattern might be set by chilling the larvae at various times during their growth. Two lots of five larvae were placed in similar jars and given similar treatment except that one jar was chilled overnight in a refrigerator on five different occasions. The result was that in both jars the entire lot was green in color. It would be desirable to be able to control the ratio of brown to green colored chrysalids, so as to insure that the bulk of them be green. It would therefore be useful to know what factor or factors control chrysalids to be brown, since these emerge at unpredictable times. The color of the chrysalis has a definite relation to the time of emergence, (Table III). In general the green ones emerge in about two weeks• The brown ones remain dormant for an unpredictable length of time. Of 34 chrysalids kept over winter 31 were brown.

8 Various other lots recorded showed that the green ones were usually hut not always emerged within two weeks, whereas the brown ones were usually still dormant, (Table III).

9 WING- HARDENING History The literature has nothing to offer that is directly relevant to this subject; however there are several papers on hepidoptera wings, the findings of which will he reviewed to present the setting for the problem of wing hardening* In the development of the Lepidopteran wing Mercer (1896) states that it is formed in the very first larval stage as an invagination of the hypodermis. Until the

fifth

larval stage it merely grows larger within the body cavity* By the fifth larval stage the wing becomes defined* The hypodermis becomes covered with cuticle (developed from the outer hypodermal layer). Underneath the hypodermis prolif­ erates a layer of hypodermal cells called the basement membrane* At this time the basal membrane cements the wing as a single layer. This is done by means of certain cells, the hypodermal pillars. When the wing is expanded, the basement membrane layers slide over one another and allow the blood to enter, but these fused pillars prevent the wing from blowing up like a balloon* At emergence, Bering (1926rll) states that there is a pumping of blood and air into

the wing* The pumping of

blood is easily observed but it

is doubtful as to how air

could be pumped into the wing although as the tracheae expand, air would naturally flow into them.

10 Snodgrass (1935:215) in his "Principles of Insect Morphology" states merely that the wings are expanded by blood and that after hardening blood circulation is restricted to the basal portion of the wings or at least wings once hardened no longer bleed if the tips are cut off. Concerning the actual cause of hardening there is some controversy as to just what happens. Guignon (1936) states that the post-nymphal development of the wing is neither an unfolding nor an unplaiting. He states that the post-nymphal development cannot take place if: 1. The wings cannot be agitated. 2. Pulsation organs of the thorax are not intact. 3. There is no accumulation of blood in the pendant wings. 4. There is no possibility for air to enter by the • mesothorax stigmata. All of these points are doubtlessly sound; however concerning point three, Moffat (1900) states that one can cut off the tips of the wings so that pressure forces fluid to escape and the wings still reach normal size. Continuing with M. Guignon*s (p. 720) observations, he states that as the wing expands the chitin of the wing undergoes a progressive dessication and its elasticity diminishes.

(. . . "mais, aufur et a mesure qu*elle se

distend, la chitine de l*aile subit une dessication pro-

11 gressive et son elasticite diminuerT.) It must be confessed that it appears that the butterfly is drying its wings during the inactive hour after emergence. Also for the dead specimen which requires straightening of a bent wing one has only to put the specimen in a humidity chamber, allow the.wings to go limp, straighten out the de­ fect, and dry in the new position. The experiments described below would tend to contradict this idea of drying, and suggest that humidity is not a factor in the hardening of the wings. Wiggleworth (1947:29) in his "Principles of Insect Physiology" notes that . . . "Little is known of the actual chemistry of hardening; probably (like blackening) it is an oxidative change in the existing secretions, catalyzed by oxidative enzymes, rather than an impregnation with other substances; for neither hardening nor darkening takes place normally in an atmosphere of nitrogen." Experimental Material. The only species used in this study was Papilio zelicaon Lucas. The location of this species in the field has already been discussed under feeding. The specimens were collected about once a week from September 1949 until August 1950. There was, however, a period when there were none to be found, from about the middle of December until the middle of March.

12 No special collecting schedule was followed. Early in the spring one stand of fennel was thoroughly cleared of all available caterpillars. T?hether due to this or other factors, no further finds were made until late July. In other stands a practice of taking one-half of all caterpillars was made. These stands were never then completely devoid of maturing animals. It is not known to the writer how far this butterfly wanders from the original stand to lay her eggs. The time involved in raising a specimen from collected egg to emer­ gence is only five weeks if the chrysalis is green. The brown chrysalids can last at least for several months. The caterpillars were kept in gallon jars. A piece of newspaper was dampened and put into the bottom of the jar, to maintain humidity, and for ease in cleaning. The high humidity prevented too rapid drying of the fennel. For young caterpillars up to twenty could be put into one jar and have adequate food for oneyweek. For the caterpillars in the last stage the food was eaten in about four days by five caterpillars, and thus had to be replenished about twice a week. Young caterpillars may be left uncovered, but the older ones wander when they prepare to pupate, and must be sealed in the jar* Method. In general during the summer season the green colored chrysalids last about two weeks, and then the

13 butterfly emerges. The wing coloring starts showing through the chrysalis case about a day before emergence. Emergence is usually in the morning, thus by chilling the chrysalis a day or so one can retard development. This allows one to remove the chrysalis from the refrigerator in the morning, confident that emergence will occur in about one hour. The chrysalis case is broken back of the head case and along a seam between the antennae and wing covers. This whole section lowers under pressure from the feet, and the i

butterfly emerges and may crawl as high as possible. Finally the animal comes to rest, hangs upside down, the wings hanging as flabby sacks• The first experiment described below tested the theory that the wings harden as a result of drying. To test this idea the butterflies upon emergence, or better slightly before, were placed in a humidity jar. This was an ordinary large mouthed jar. This jar was fixed with a screen across the bottom about two inches from the bottom. About one and one-half inches level of, hot water was then placed in the jar, which was then sealed with lard. As the water cooled the relative humidity would become 100% and condensation would take place on the walls of the jar. A crumpled paper was in the jar to allow the emerged butterfly to hang upside down, free from obstructions. There was an observation window made by rubbing a section inside the jar with "Chrystal Mist,"

14 a salve used to prevent fogging of spectacles* In the open a butterfly would normally attempt flight in about one and one-half hours. One test of the effect of the humidity chamber on the hardening of the wings was to release the butterfly after a similar time and see if it could fly at once* A more direct effect of the humidity of hardening was observed by placing the chrysalis inside a perforated box {4J- by 2\ by li inches). In this box the butterfly could hang upside down, but the wings touched the bottom and curved, thus when hardening took place the curved tips would be a permanent feature of the wing. Other experimental methods used were simple, and need no special explanation. These methods will be described or apparent from the experiments. Experiments on the drying of the wings. On ten different occasions (listed by date in Table IV) a butterfly was put into the humidity chamber before emergence. Upon emergence the butterfly climbed up the paper, pumped out its wings-and allowed them to harden. On an average, the butterflies were removed in about one and one-half hours. It was considered that the wings were' normally hard if the butterfly could fly when tossed into the air. This time of one and one-half hours was derived from a series of obser­ vations in which butterflies were allowed to harden their

15 wings normally, and fly when startled by waving a hand at them. A second method was to place the butterfly in a small box so that a crumpled wing would result if permanent hardening took place. Thirteen specimens were used for this purpose. It was removed at the end of from one and one-half hours to five hours. The longer time was sometimes necessary since one could not always assume the chrysalis just put into the chamber would emerge in one hour, and in the box there was no way to see at exactly what time the butterfly did emerge. If there was a permanent bend in the wing it was considered that hardening had taken place despite 100$ relative humidity. Experiment on oxidative reaction. Only one experi­ ment was run to test Wigglesworth*s suggestion that hardening was due to oxidation. In this case a butterfly, newly emerged, was drowned in boiled water (cooled and presumably free from oxygen). The wings on this butterfly failed to harden. Also severed wings put into the boiled water failed to harden. This experiment was not repeated because it killed the butterfly, and as the next experi­ ment shows, dead butterflies fail to harden their wings. Experiment on the severed wings.

Three experiments

were run by severing the wing of a newly emerged butter­ fly and a wing partially expanded. In all three cases these

16 severed wings remained soft and pliable, even after normal exposure to air (oxygen)* Experiment on killing the butterfly* Two experiments were run in which the butterfly was killed by decapitation immediately after emergence with the observed result that the wings did not harden* Experiment with the anesthesia* These experiments started as an accident. In an effort to kill the newly emerged butterfly with ether and thus avoid a badly bled dead butterfly, a newly emerged butterfly was put for one hour into a jar with some ether* At the end of the hour the butterfly was removed* It revived, but the wings, previously pumped up failed to harden, although this butter­ fly lived for five days. This experiment was repeated on three occasions with the same results, although the butter­ fly did not always live for five days —

one died in two

days. In all these cases the wings were incapable of supporting flight when the butterfly was thrown into the air.

17 DISCUSSION This investigation, which began as an effort to determine which ecological factors induce the wings to harden, has developed into a problem that will probably require the efforts of a biochemist to completely unravel. First of all the ecological process of drying, as suggested by Guignon, does not appear to be the best answer# The results of the experiments in this study makes it appear probable that the wings harden independent of relative humidity. If It is not a drying process the next suggestion in the literature is that some sort of an oxidative process causes the wings to harden. Wigglesworth notes that the wings do not harden in nitrogen. If as seems likely, the nitrogen kills the butterfly this is not too surprising, since the wings of a dead butterfly will not harden even in air. This would not eliminate Wigglesworth*s observation that hardening is quite likely "an oxidative change in existing secretions, catalysed by oxidative enzymes. The experiments with the anesthetized butterflies would tend to confirm this idea. Enzymes are generally produced for.'a limited time in response to some physical or chemical body change. Since wing hardening takes place during the first hour after emergence, it is plausible to assume that the enzyme is also produced only during this time. If that be

18 true it would follow that if the butterfly were knocked out (but not killed) so that blood and air circulation stopped during this critical hour the enzyme would never be carried to the part of the wing where it was needed. Even if the- butterfly revived later there would not be the necessary catalyst needed to cause the wings to harden. A severed wing would not harden, apparently for lack of the same catalyst* There is one other possibility, that of a deposition of calcium precipitates or some other precipitation, and an impregnation of the cuticle and supporting tracheae with this substance. Wigglesworth (1947) discounts this possibility, but gave no specific reasons. He, of course, believed in the oxidative catalyst. Workers such as Mercer, who studied the wing structure, but with no especial regard for the problem of hardening, seem to think the wing is covered with chitin before the pupal stage, and note no change in this structure later; that is they make no mention of any sort of change due to impreg­ nations • CONCLUSION The hardening of the wing of Papilio zelicaon Lucas is not due to drying. It is possible that stiffening is due to impregnations of calcium or some other substance, brought in at the time the wing is expanded by the blood. It is more

19 probable that the wing chitin hardens by some oxidative process under control of an enzyme produced during the hour while hardening taxes place and is carried into the wing by the blood which expands the wing sack* SUMMARY The feeding of Papilio zelicaon lucas is not limited entirely to the family Umbelliferae, since the larvae will eat at least one species of Citrus. In the Umbelliferae the larvae are found to eat any member of the family so far tested. Only the poison hemlock (Qonium maculaturn L.) was found often to prove unsuitable as food, but even on this plant at least half of the larvae lived to complete pupation. In this area the caterpillar is found typically on fennel (poeniculum vulgare Hill) which is a plant native to Europe* The native plant stated to be the usual food of R* zelic&on is not found in the area south of Santa Clara County. It was found that there are two colors of chrysalis. The green colored chrysalis ordinarily completes the pupal stage in about two weeks, the browrn chrysalis nay remain in the pupal stage for months.

It was observed that there is an

increase in the ratio of green to brown colored chrysalids from about Z:1 in the early spring to almost entirely green by the last of July. Efforts to correlate this change with

cooling (or cool weather) were unsuccessful* The hardening of the wings of the butterfly was found to be independent of drying. Other possibilities for the cause of hardening seemed to be best explained by an enzyme which during the first hour after emergence cause changes, probably of an oxidative nature, in the chitin covering and tracheae of the wing that makes then hard and rigid.

21 TABLE I FEEDING HABITS OF PAPILIO ZELICAON LUCAS

PLANTS TESTED

RESULT

CATERPILLARS USED

Poplar (Populus Sp.)

3 large 3 medium 3 small

dead in 8 days dead in 6 days dead in 4 days

Lilac (Syringa vulgaris)

1 large 3 medium 3 small

dead in 6 days dead in 2-5 days dead in 3-5 days

Poison Hemlock (Conium maculatum L.)

5 large4 small-

All would feed upon the plant but usually died within 3 days, one reached the pupal stage.

Celery (Apium gravelolens L.) 6 mixed

Would feed and grow to pupate.

10 mixed

Would feed and grow to pupate.

Oenanthe sarraentosa Presl.

6 mixed

Would feed and grow to pupate.

Leptotaena dissecta Nutt.

6 mixed

Would feed and grow to pupate.

Orange (Citrus sp.)

8 mixed

Six died,xone small one ate the rind off of a green orange, one pupated.

Carrot (Daueus carota L.)

*

22 TABLE II PUPA AMD COLOR OF BACKGROUMD

DATE

OM GREEN BACKGROUMD No. green brown - colored chrysalis

OH BROWN BACKGROUND green brown

12 May

6

8

—

—

31 May

6

2

-

1

3 June

5

5

-

1

8 June

3

3

-

-

11 June

7

0

-

-

12 June

8

1

1

-

16 June

5

0

1

-

20 June

6

3

1

1

26 June

10

3

2

1

1 July

7

0 (all chilled)

-

-

26 July

15

0

-

—

Also chilled were green chrysalis from 2 3 June

1

20 June

1

26 June

2

23

TABLE III NUMBER OP CHRYSALIDS REMAINING IN A LOT AFTER TWO OR MORE WEEKS

Brown Green colored chrysalids

Remaining on 12 June

Overwinter lot (34)

3

31

12 May lot (6 green. 8 brown;

1

6

'3 June lot (5 green, 6 brown)

1

6

Lot

On 29 June green brown

12 May (6 green. 8 brown)

On 5 July green brown

On 26 July green brown

1

6

1

6

1

6

3 June

(5-6)

0

5 .

0

5

0

5

8 June

(3-2)

0

2

0

2

0

2

11 June (7-0)

0

0

-

-

-

-

12 June

(9-1)

0

1

0

1

0

1

20 June (6-3)

5

2

0

2

0

2

12

4

10

2

1

3

31

26 June

(12-4)

Over winter (3-31)

1 (3 died)

24 TABLE IV TIME REQUIRED FOB HARDENING OF THE WINGS

Late

Released from humidity chamber after:

19 Nov* 1949

1 hour

30 minutes

26 Nov.

1 hour

25 minutes

3 Le c «

1 hour

11 Dec*

1 hour

45 minutes

17 May 1950

1 hour

30 minutes

23 May

1 hour

20 minutes

24 May

2 hours 40 minutes

25 May

2 hours

25 May

1 hour

30 minutes

26 May

1 hour

30 minutes

5 minutes

(Table continued on next page)

25

Date

Released from perforated box in the humidity chamber after:

28 May

1 hour

30 minutes - not yet emerged, r.eusad next day*

31 May

1 hour

30 minutes ~ two specimens* one not yet emerged.

11 June

5 hours

two specimens.

16 June

5 hours

two specimens.

19 June

1 hour

20 June

4 hours

two specimens.

2 9 June

3 hours

two specimens.

30 June

2 hours

30 June

1 hour

30 minutes - not yet emerged* used next day.

30 minutes

LITERATURE CITED Comstock, J.A., Butterflies of California.

Bull. So*. Calif.

Acad. Sci., 20(1): 5-6, 1921. Essig, O.E., Insects of Western North America.

N.Y.

Macmillian Co., 1035 pp., 1926. Guignon, Gabril, Sur le development Post-nyraphal des ailes des Lepidopteres. Hering, Martin,

C.R. Acad. Sci. Paris. 204:720-721, 1936.

Biologie der Schmetterlinge.

Berlin, 480 pp*,

1926.. Mercer, William Fairfield,

The Development of the Wings in the

Lepidoptera. Jour. New York Entom. Soc., 8:1-20., 1896. Moffat, J.A., Remarks on the Structure of the Undeveloped Wings of the Saturniidae.

Entom. Soc. of Ontario,

25:63-65., 1894. Parish, S.B.,

The Immigrant Plants of Southern California.

Bull. So. Calif. Acad. Sci., 21(4):3-30., 1920. Snodgrass, R.E., Principles of Insect Morphology. McGrawHill N.Y. 667 pp., 1935. Wigglesworth, V.B., Principles of Insect Physiology. Methuen and Co. Ltd. London

434 pp., 1947.