Int. J. Insect MorphoL & Embt3,ol., Vol. 12, No. 2,3, pp. 79 to 85. 1983. 0020 - 7322.83 $03 00 + 0 0 Printed in Great Britain. 1983 Pergamon Pre,~ l i d

E M B R Y O N I C D E V E L O P M E N T O F T H E O S M E T E R I A O F

P A P I L I O N I D C A T E R P I L L A R S , P A R N A S S I U S G L A C I A L I S B U T L E R

A N D P A P I L I O M A C H A O N H I P P O C R A T E S C. ET R. F E L D E R

( L E P I D O P T E R A : P A P I L I O N I D A E ) *

MASAHIRO TANAKA

Kano High School, Nany6-ch6, Kano, Gifu 500 Japan

and

YUKIMASA KOBAYASHI

Department of Biology, Saitama Medical School, 981 Kawakado, Moroyama, lruma-gun, Saitama 350-04, Japan

and

HIROSH1 ANDO

Sugadaira Montane Research Center, The University of Tsukuba, Sanada, Nagano 386-22, Japan

(Accepted 26 October 1982)

Abstract--Embryonic development of osmeteria of Parnassius glacialis and Papilio machaon hippocrates (Lepidoptera : Papilionidae) is described. After revolution of the embryo, a pair of rudimentary osmeteria is formed on the dorsal wall of the prothoracic segment. With the lapse of time, they invaginate deeply into the segment, and differentiate into the rudimentary gland and rudimentary protrusible sac. During the embryogenesis of Papilio, the gland grows and becomes a large ellipsoid with a central cavity, and well- developed protrusible sacs are formed, but they are poorly developed in Parnassius.

Index descriptors (in addition to those in title): Developmental stages, gland, protrusible sac, prothorax.

I N T R O D U C T I O N

THE PAPILIONID caterpillars have a pair of protrusible horns or osmeteria on the dorsum of the prothorax. Normally the osmeteria are invisible from outside but when the insect is irritated, they are everted and emit a pungent odour; therefore, the osmeteria are regarded as defensive glands (Eisner and Meinwald, 1965).

Many works on the morphology of the osmeterium and on the chemical nature of its secretory substances have been published (Schulze, 1911; Wegener, 1923; Eisner and Meinwald, 1965; Crossley and Waterhouse, 1969; Eisner etal., 1970, 1971; Burger et al., 1978; Suzuki et al., 1979; Honda, 1980a, 1980b). A large number of embryological studies of the Lepidoptera has been published to date, but only Kobayashi and Tanaka (1981) have reported that the rudimentary osmeteria originate as ectodermal invaginations of the embryonic prothoracic segment of Luehdorfia japonica.

The purpose of this paper is to provide detailed descriptions of the embryonic development of osmeteria of Parnassius glacialis and Papilio machaon hippocrates, and to compare the embryonic development of osmeteria among species or genera belonging to the Papilionidae, and to examine the phylogenetic relationship among them.

*Contributions from Sugadaira Montane Research Center, The University of Tsukuba, No. 64.

79

80 MASAHIRO TANAKA, YUKIMASA KOBAYASHI and HIROSHI ANDO

M A T E R I A L S A N D M E T H O D S

The eggs of Parnassius glacialis and Papilio machaon hippocrates were obtained from pregnant females that were captured in Gifu Prefecture, Japan, the former obtained during May of 1979 and 1980, and the latter during June- July of 1981. Freshly laid eggs were kept in the laboratory at room temperature (20- 23°C). The eggs at various ages were fixed in Carnoy's fluid for 30 - 40 min. After fixing, the chorion was removed, and the eggs were dehydrated and embedded in paraffin. About 8-I.tm thick sections were cut and stained with Delafield's haematoxylin and eosin.

O B S E R V A T I O N S

Format ion o f osmeteria o f Parnassius glacialis

The dura t ion of the deve lopmenta l period f rom oviposi t ion to comple t ion of the

embryo at room tempera ture ( 2 0 - 2 3 ° C ) was about 24 days in the labora tory . The eggs

were laid early in May and the larvae hatched out late in February o f the fol lowing year.

Therefore , the egg dura t ion was about 9.5 months in the field. Table l shows the outl ine of

embryogenesis o f P. glacialis.

TABLE 1. TIME-TABLE OF EMBRYONIC DEVELOPMENT OF Parnassius glacialis AT ROOM TEMPERATURE (20- 23°C)

Approximate age of egg State of development

0-1 hr 2-16 hr

17-20 hr

1 - 2 days

3 - 4 days

5 - 7 days

8 12 days

13 -14 days 15 - 18 days

19- 21 days

22 - 24 days 9.5 months

Maturation and fertilization. Cleavage. Energids penetrate into periplasm and formation of blastoderm. Formation of ventral plate and serosa covers ventral plate. Formation of germ band. Formation and segmentation of inner layer. Completion of segmentation of germ band. Early embryo with minute appendages. Embryo attains most elongated stage. Formation of protocephalic and gnathal ectodermal invaginations, and spiracles. Shortening of length of embryo. Revolution. Embryonic moulting. Formation of setae of embryo. Pigmentation of head capsule and body wall. Completion of full-grown embryo. Hatching.

When the pro tocephal ic and gnathal appendages arrange themselves a round the

s tomodaea l opening, the anlagen of abdomina l appendages become vestigial, except those on the 3 r d - 6 t h abdomina l segment and anal appendages. During this process, the

embryo decreases in length and then it carries out revolut ion.

Af te r revolut ion, a pair o f elevations, which are rudiments of osmeter ia , appear on the

anterodorsa l wall o f the pro thorac ic segment (Figs. l , 10, 11). The rudiments , about 60 by 35 gm, consist of the anter ior and poster ior folds. The fo rmer is smaller and thinner than

the latter and is composed of a single cell layer, while the latter, composed o f lengthened

cells, is semicircular , about 35 by 25 ~tm in size (Figs. 1, I l). The fo rmer is the future

protrusible sac and the latter the future ellipsoid gland.

The Osmeteria of Papilionid Caterpillars 81

anterior posterior

rs r ~

r e

2 ~ rg rs

re

anterior posterior

r s "

7 rg

rg

rs tr

s e a

g

osm ~ g

FR;s. 1 - 9. F o r m a t i o n o f o s m e t e r i u m o f P. glacialis a n d P. machaon hippocrates ( d i a g r a m m a t i c ) . FIGS. I 4. Successive c h a n g e s o f o s m e t e r i u m o f P. glacialis ( long i tud ina l sect ion t h r o u g h p r o t h o r a x ) .

FK~. 5. 18-day-o ld e m b r y o o f P. glacialis ( long i tud ina l sect ion) . FR;S. 6 - 9 . successive c h a n g e s o f o s m e t e r i u m o f P. machaon hippocrates ( long i tud ina l sect ion t h r o u g h p r o t h o r a x ) , fg = fo regu t ; g = g l and ; hg - h indgu t ; m g = midgu t ; osm - o s m e t e r i u m ; re = r u d i m e n t a r y ep idermis ; rg = r u d i m e n t a r y g l and ; rs = r u d i m e n t a r y sac; s = sac; sea = seta:

tr = t r i chogen .

Soon after the 1st appearance of the rudiment, the groove between the anterior and posterior folds commences to invaginate posteriorwards. As its invagination deepens, the folds sink into the segment and the shape of the rudimentary gland changes from semicircle to lens-shaped (Fig. 2). At this stage, the anterior wall of the rudimentary osmeterium is the rudimentary protrusible sac and the posterior one is the rudimentary gland. The latter is about 50 by 25 ~m in size in longitudinal section. Nuclei of elongated ceils constituting the gland are found in their basal parts.

82 MASAHIRO TANAKA, YUKIMASA KOBAYASHI and HIROSHI AND()

When the embryonic moulting occurs, the rudimentary osmeterium is situated beneath the anterodorsal wall of the prothoracic segment, and becomes a sac with a central cavity. This sac connects with the dorsal surface of the prothoracic segment by the stalk of invaginated segmental wall. The concave, lens-shaped gland, composed of about 10 cells in longitudinal section, occupies the dorsal part of the rudimentary osmeterium (Figs. 3, 12). Nuclei of the elongated glandular cells are very clear and located in basal parts of cells. The cells are rich in cytoplasm and their boundaries are not distinct. The ventral part of the rudimentary osmeterium is composed of a single cell-layer, but in this stage the protrusible sac does not become membranous.

After embryonic moulting, the gland is about 45 by 25 ~m, and a thin cuticular membrane secreted by glandular cells detaches from the inner surface of the gland, and remains in the cavity.

At about 20 days after oviposition, the full-grown embryo has setae. A pair of glands is situated in the centrodorsal part of the protrusible sac just behind the head capsule (Fig. 5). Each gland retains the same size since embryonic moulting, but has changed its form. The gland is composed of regularly arranged secretory cells, and bears a cytoplasmic, finger-like projection into the cavity. Each nucleus of the cells is very clear, but the boundaries of cytoplasm are not distinguishable at the basal region. In this stage, the protrusible sac is found under the dorsal wall of the prothoracic segment and upon the brain, and is 75 by 30 lam in size in longitudinal section.

Formation of osmeteria of Papilio machaon hippocrates The total duration of embryonic development of Papilio was about 8 days at room

temperature (20-23°C) . At about 5 days after oviposition, the embryo undergoes revolution. After revolution, a pair of rudimentary osmeteria appear on the dorsal wall of the prothoracic segment. They consist of anterior and posterior folds, about 100 by 70 ~m (Fig. 6). The former is the future protrusible sac and the latter, about 65 by 35 lam in size, the future gland, as observed in Parnassius.

In later development, a change occurs in the rudimentary gland, which is composed of about 20 cells and about 100 by 40 I.tm in size, namely, the wall of the gland quickly thickens and becomes a glandular structure (Figs. 7, 8, 14). In sagittal section, the gland shows a C-shaped structure. With the lapse of time, the developing gland gradually enlarges and finally becomes an elongated ellipsoid with a lumen having an opening. The rudimentary protrusible sac also develops quickly, so that the ventrobasal wall of the invagination becomes a single-cell-layered sheet complexly folded beneath the gland.

At the time of embryonic moulting, the ellipsoidal gland, about 170 by 75 I.tm in size, is situated between the dorsal wall of the prothoracic segment and the foregut, and shows a typical glandular structure. The wall of the gland is uniformly thick, and is composed of 2 5 - 30 columnar cells in longitudinal section (Figs. 9, 15) and about 15 in cross section (Fig. 16). Each glandular cell has a large amount of cytoplasm, and has a nucleus located

in its basal part. After embryonic moulting, the cuticular membrane detached from the inner surface of

the gland is found in the lumen. During later developmental stages, the ellipsoidal gland enlarges and the lumen also becomes larger.

At about 7 days after oviposition, when the full-grown embryo bears setae and its head is pigmented, the gland located under the dorsal wall of the prothorax is about 250 by 75 I.tm in size and connects with the protrusible sac on its ventral side. In Papilio, the stalk

The Osmeteria of Papilionid Caterpillars 83

Fro. 10. Longitudinal section of 14-day-old embryo of P. glacialis. FI~;. 11. Rudimentary osmeterium of P. glacialis, longitudinal section through prothorax of 14-day-

old embryo. Fic;. 12. Osmeterium invaginated into prothorax of P. glacialis, longitudinal section through

prothorax of 15-day-old embryo. FIc;. 13. Osmeterium of full-grown embryo of P. glacialis, longitudinal section of prothorax of 18- day-old embryo, am = amnion; br brain; ed - epidermis; g = gland; rg = rudimentary gland; ro = rudimentary osmeterium; rs = rudimentary sac; s = sac; se = serosa; sog = suboesophageal

ganglion; sto - stomodaeum; y - yolk.

connecting the gland with the protrusible sac is not formed. The lumen of the gland is large

and its opening is retained as at embryonic moul t ing. The wall of the gland is uni formly

thick, about 30 Ixm, and consists of about 3 0 - 4 0 co lumnar cells in longi tudinal section. At this time, the inner surface of the g landular cells shows a rough structure, but

does not have distinct f i laments or microvilli . This morphological condi t ion of osmeteria persists until hatching of the embryo.

When the osmeteria of newly hatched larvae are thrust out, the gland locates in the distal part of the protrusible sac (Fig. 17).

D I S C U S S I O N

After revolut ion, the rudiment of the osmeter ium makes its appearance as a pair of

minute elevations on the dorsal wall of the prothoracic segment of the embryo, and consists of the anter ior and the posterior parts. With the lapse of time, the rudiment

invaginates into the segment. The process of the osmeter ium format ion in Papilio machaon hippocrates and Parnassius glacialis is the same as in l,uehdorfia japonica (Kobayashi and Tanaka , 1981). The original size of the rudiments of Parnassius, however, is smaller than those of Papilio and Luehdorfia.

84 MASAHIRO T,XNAKA, YL/KIMASA KOBAYASHI and HIROSHI ANt)()

f:J<,. 14. lnvaginated rudimentary osmeterium of P. m. hippocrates , longitudinal section through prothorax of embryo of post revolution stage.

Fl~. 15. Rudimenlary osmeterium of P. m. hippocrates , ~ongiludinal section through prothorax of embryo of embryonic moulting stage.

tqc,. 16. Rudimentary osmeterium of P. m. hippocrates , cross section through prothorax of embryo of embryonic moulting stage.

FIci. 17. Everted osmeterium of newly hatched larva of P. m. hippocrates , longitudinal section through prothorax, br = brain; ct cuticle; ed = epidermis; g - gland; os - opening of sac; re -

rudimentary epidermis; rg = rudimentary gland; rs = rudimentary sac; s sac.

The Osmeteria of Papilionid Caterpillars 85

The rudimentary glands of Papilio and Luehdorfia become large ellipsoids with central cavities in the full-grown embryo. That of Papilio is an open ellipsoidal body, while that of Luehdorfia is a closed one. In Parnassius, those types of glands are not formed. The well-developed protrusible sacs are formed in Papilio and Luehdorfia, but that of Parnassius is poorly developed.

In the embryo with setae and pigmented head, the gland of Luehdorfia has many microvilli or filaments on its inner surface. In Papilio, however, such structure is not yet formed.

To summarize, the manner of the embryonic osmeterial formation in these 3 species is essentially the same in their early stages, but the shapes of osmeterial glands in the full- grown embryos greatly differ among various species or genera. We believe that the differences in osmeteria formation, obtained in the present study, might have phylogenetic significance among the species or genera of the Papilionidae.

R E F E R E N C E S

BURGER, B. V., M. ROTH, M. LE Roux, H. S. C. SPIES, V. TRUTER and H. G~ERTSEMA. 1978. The chemical nature of the defensive larval secretion of the citrus swallowtail, Papilio demodocus. J. Insect Physiol. 24: 8 0 3 - 5.

CROSSLEY, A. C. and D. F. WATERHOUSE. 1969. The ultrastructure of the osmeterium and the nature of its secretion in Papilio larvae (Lepidoptera). Tissue Cell 3:525 - 54.

EmNER, T. and Y. C. MEINWALD. 1965. Defensive secretion of a caterpillar (Papilio). Science ( Wash., D.C. ) 150: 1733-35 .

EISNER, T., T. E. Pt.lSKE, M. IKEDA, D. F. OWEN, L. VAZQUEZ, H. PEREZ, J. G. F~ANCI.EMONT and J. MEINWALD. 1970. Defensive mechanisms of arthropods. XXVII. Osmeterial secretions of papilionid caterpillars (Baronia, Papilio, Eurytides). Ann. Entomol. Soc. Amer. 63: 9 1 4 - 15.

EtSNER, T., A. F. Kt_UGE, M. 1. IKEDA, Y. C. MEtNWALD and J. MEtNWALD. 1971. Sesquiterpenes in the osmeterial secretion of a papilionid butterfly, Battus polydamas. J. Insect Physiol. 17:245 - 50.

HONDA, K. 1980a. Volatile constituents of larval osmeterial secretion in Papilio protenor demetrius. J. Insect Physiol. 26: 3 9 - 45.

HONDA, K. 1980b. Osmeterial secretion of papilionid larvae in the genera Luehdorfia, Graphium and Atrophaneura (Lepidoptera). Insect Biochem. 10:583 - 88.

KOHAVASHI, Y. and M. TANAKA. 1981. Embryonic development of the osmeteria of Luehdorfiajaponica Leech (Lepidoptera, Papilionidae). Konty~ 49:344 - 50.

SC'HULZE, P. 1911. Die Nackengabel der Papilionidenraupen. Zool. Jahrb. Anat. 32:181 -244 . SUZUKh T., J. UEHARA and R. SUGAWARA. 1979. Osmeterial secretion of larvae of papilionid butterflies

(Lepidoptera : Papilionidae). 1. Identification of myrcene from Luehdorfia japonica and L. puziloi inexpecta. Appl. Entomol. ZooL 14: 3 4 6 - 4 9 .

WEGENER, M. 1923. Die biologische Bedeutung der Nackengabel der Papilionidenraupen. Biol. Zentralbl. 43: 292 - 301.