The biology of (: Arctiidae) with special reference to the androconial system Downloaded from https://academic.oup.com/zoolinnean/article/96/4/339/2658339 by guest on 01 October 2021

MICHAEL BOPPRE

Forstzoologisches Institut der Universitat Freiburg, Fohrenbuhl 27, 0-7801 Stegen- Wittental, Federal Republic of Germany

AND

DIETRICH SCHNEIDER

Max-Planck-Institut fur Verhaltensphysiologie, 0-8131 Seewiesen, Federal Republic of Germany

Received January 1987, revised and accepted for publication October 1988

The reproductive biology of the arctiid and C. gangis exhibits a novel ontogenetic phenomenon, the morphogenesis of male coremata size being directly controlled by the quantity of hostplant-derived pyrrolizidine alkaloids ingested by the larvae. In addition, the same alkaloids directly control the quantity of male pheromones synthesized and available for deployment by these scent organs. Reproduction in these thus shows striking linkages between ecological, behavioural, chemical and morphological features. This paper presents an account of the general biology of the moths, and reviews the literature on Creatonotos as a basis for reports on experimental work.

KEY WORDS:- - Creatonotos transiens - Arctiidae ~ Lepidoptera - androconial

organs ~ sexual behaviour - chemical communication - pyrrolizidine alkaloids - reproductive biology.

CONTENTS Introduction ...... 340 Material and methods ...... 340 Notes on the life history ...... 341 General notes on the biology ...... 342 Morphology ofcoremata ...... 342 Variability of corematal size ...... 345 Corematal secretion ...... 347 Behavioural use of coremata ...... 347 Discussion ...... 350 Morphological and chemical aspects ...... 350 Functional aspects ...... 351 Ecological aspects...... 353 Concluding remarks ...... 354 Acknowledgements ...... 354 References ...... 355 Note added in proof ...... 356 339 00244082/89/080339 + 18 $03.00/0 0 1989 The Linnean Society of London 340 M. BOPPRB AND D. SCHNEIDER

INTRODUCTION The arctiid Creatonolos Hiibner exhibits striking phenomena relevant to chemical ecology, sociobiology and developmental biology. The males possess inflatable androconial organs which vary in size in relation to the amount of pyrrolizidine alkaloids (PAS)ingested by the larvae from their hostplants. These hair-bearing scent organs emit a pheromone which is synthesized from PAS. While in the Lepidoptera male scent organs are usually expanded briefly during a final phase of courtship behaviour, males of Creatonotos display their coremata for long periods with or without the presence of females. Downloaded from https://academic.oup.com/zoolinnean/article/96/4/339/2658339 by guest on 01 October 2021 Here we present an account of what is known about the biology of Creatonotos, describe the morphology of the androconial system in some detail, discuss the scant and scattered literature on this genus, and provide some general ecological and behavioural information in order to supply a basis for earlier reports on experimental studies (e.g. Boppri. & Schneider, 1985; Wunderer et al., 1986) and further work in preparation.

MATERIAL AND METHODS The genus Creatonotos presently comprises 15 or so from Asia, , and Africa. Our studies concern Creatonotos gangis (L.) and C. transiens (Walker) (Fig. 1 ) from , where both species occur sympatrically. Adult moths were collected at a light near Dolok Merangir, North Sumatra, and laboratory cultures were established from field-caught females shipped to Germany. We kept the larvae, in groups of 1&50, in clear plastic containers of various sizes (depending on the size of the caterpillers) and, after having tested a variety of European (substitute) foodplants, we raised them routinely on leaves of Taraxacum ojicinale Web. and/or on a semi-artificial diet (see below). For morphological and behavioural observations we also used moths from larvae

Figure 1. Males and artificially inflated coremata of C. gangis (left) and C. trunsiens (right). Note the different shape of the coremata which is typical for the species. Natural size. BIOLOGY OF CREATONOTOS MOTHS 341 which had ingested different amounts of pyrrolizidine alkaloids (PAS),either by feeding on PA-containing plants (see below), purified PAS extracted from plants and dispensed on Taraxacum leaves, or such PAS mixed into the diet. Adults were kept in plastic containers (520 x 280 x 250 mm) for mating; for observations on their behaviour larger cages (usually 980 x 650 x 570 mm) and red dark-room lights were used. Morphological studies of the coremata were made by dissection and by artificial inflation of the organs. For the latter, we introduced a plastic tube (3 mm in diameter) into the severed abdomen of a freshly killed male and gently applied air pressure through the tube by means of a syringe. Using this method, Downloaded from https://academic.oup.com/zoolinnean/article/96/4/339/2658339 by guest on 01 October 2021 coremata can be gradually inflated and deflated. For scanning electron microscopy (SEM), air-dried coremata were glued on aluminium stubs, coated with gold and observed with a Zeiss Novascan 30, operated at 15 kV acceleration voltage. To assess the different sizes of coremata, the organs were photographed and/or cut off and weighed on a micro-balance (Mettler H20T).

NOTES ON THE LIFE HISTORY The larvae of both species are brown and very hairy (for descriptions see, for example, Piepers & Snellen, 1905; Kalshoven, 1981 : 323). They look very similar and the only obvious difference between them is their coloration (black with white pattern in C. transiens, dark brown with yellowish pattern in C. gangis). Under our breeding conditions (temperature 20-25°C; light-dark cycle 12 : 12 h), the development from egg to imago took 3945 days (egg: 6-7 days; seven larval stages: 3-4 days each; prepupa: 2-3 days; pupa: 8-10 days, females 1 day less than males). The young larvae appear to be gregarious and assemble in groups, at least for moulting. Nevertheless, cannibalism occasionally occurred, particularly in the last instar. Before starting to feed (about 24 h) after a moult, they consume their old skin. For pupation they spin a light cocoon, which is very hygroscopic and contains larval hair. Adult moths emerge in the middle of the photophase. There are few reports on larval foodplants of Creatonotos in the literature. For C. gangis, Fletcher ( 19 14) names coffee, groundnut, lucerne, and other low growing plants; further records are Brassica sp. (Brassicaceae) and Ipomoea reptans Poir (Convolvulaceae) (Anonymous, 1965), Imperata Cylindrica (Poaceae) (Robinson in Varley, 1962), sugar cane (Saccharum oficinarum, Poaceae), “Gramineae (Poaceae) and other plants’’ (Kalshoven, 1981), and

sp., Asteraceae; Crotalaria sp., Fabaceae; Heliotropium sp., Boraginaceae) were also Downloaded from https://academic.oup.com/zoolinnean/article/96/4/339/2658339 by guest on 01 October 2021 readily accepted. However, if the caterpillars were given a choice between PA- lacking and PA-containing plants, both were eaten and no obvious preference was observed (cf. Discussion).

GENERAL NOTES ON THE BIOLOGY In the laboratory, the moths sit motionless when there is light and become active at dusk. First, they fly about for a while and eventually settle. Males then display their coremata (see below), while a little later females start emitting pheromones; this can be recognized by rhythmic movements of the abdomen. Mating is readily achieved in confinement; it occurs during the first 3-4 h of the night and takes 3-6 h. In coitu, the female wings always rest on those of the male, and multiple matings are not uncommon in captivity. Females start laying eggs in clusters on any substrate the night following insemination; a female produces about 800 eggs. The moths have a rather short proboscis, used to imbibe moisture. They live for 5-7 days in confinement. When disturbed, the moths feign death (Fig. 2A) while displaying the aposematic coloration of the upperside of the abdomen, which is red in C.gangis and yellow in C. transiens. Simultaneously, small droplets of haemolymph may be exuded from a pair of prothoracic glands (Fig. 2B).

MORPHOLOGY OF COREMATA The coremata of the two species in question, C. gangis and C. transiens, exhibit minor morphological differences only: in C. transiens, the larger species, the

Figure 2. Male Creatonotos transiens. A, Feigning death. B, Exuding froth from prothoracic glands. BIOLOGY OF CRE4 TONO TOS MOTHS 343 organs can also be larger. In C. transiens all four corematal tubes are curved (and form two ovals) but in C. gangis the outer tubes are virtually straight (cf. Fig. 1). However, the basic morphology is identical and the following information is valid for both species unless otherwise stated. The fully expanded coremata of Creatonotos (if well developed, cf. below) consist of four cuticular tubes clothed with hairs (Fig. 1). The tubes (the inner two being longer than the outer pair if the organ is small or medium-sized) are air-filled and arise from an air-bladder located at the ventral side of the abdominal tip, formed by the intersegmental membrane between the 7th and 8th sternites. Downloaded from https://academic.oup.com/zoolinnean/article/96/4/339/2658339 by guest on 01 October 2021

Figure 3. Progressive stages of artificial expansion of large coremata in C. transiens in ventral (v), lateral (1) and dorsal (d) views. 344 M. BOPPRE AND D. SCHNEIDER In their retracted state, the coremata are invisible, being entirely hidden within the abdomen. Their pneumatic inflation (see below) can be simulated by introduction of air into the abdomen (see Material & methods). Progressive stages of coremata inflation are pictured in Fig. 3. First, between abdominal segments 7 and 8, two brush-like bundles of hair appear at the ventral side (Fig. 3A). While most of the hair is of a greyish colour, the inner (ventral) side of the hair bundles consists partly of yellowish hairs. With further injection of air, the hair bundles are protruded and flap ventrally, at right angles to the long axis of the abdomen (Fig. 3B). The two bundles then separate into four, the outer (dorso-ventral) pair simultaneously fanning out (Fig. 3C-F) . An air-bladder Downloaded from https://academic.oup.com/zoolinnean/article/96/4/339/2658339 by guest on 01 October 2021 appears ventrally. At this stage the inner hairs stay as bundles, and they do not inflate and fan out until the outer (lateral) tubes have almost entirely expanded (Fig. 3G-I). The process of expansion can be likened to the action of camera bellows: the cuticle of the tubes does not stretch, it unfolds. The tubes initially unfold perpendicular to the abdomen but eventually, if the organ is maximally expanded, all four tubes come to lie in the lateral plane of the abdominal axis (Fig. 3L), through the effect of the air-bladder. At this stage, the yellow hairs of the inner tubes point ventrally. Note that the alignment of the coremata depends on the degree of protrusion of the air-bladder, that is on the pressure applied to this organ. Although artificial corematal inflation seems to match the natural process very closely (see below and Fig. 9), reduction of applied air pressure only permits the coremata to deflate to a stage depicted in Figs 3B and 9D. The entire retraction of the organ cannot be simulated-probably the hair bundles are eventually retracted into the abdomen by muscles. Microscopically, the cuticle of the tubes appears runkled and the hairs arise from prominent sockets; there is no obvious difference between the two species. The dimensions of the hairs vary according to corematal size; the hairs can be 3- 6 mm long; in medium-sized organs, their basal diameter is about 23 pm and 12 pm in grey and yellow hairs, respectively. Towards their tips, the hairs become slender. The grey hairs have rough surfaces over their entire length (Fig. 4A, B, E), closely resembling the typical “lattice structure” of lepidopterous wing scales (Downey & Allyn, 1975). The yellow hairs have a rather smooth general surface structure, but show longitudinal ribs at their base and at their tips they are quite spiny (Fig. 4C, D, F). Internally the hairs are composed of interconnected ribs with lacunae. Underneath each hair a prominent sac-like

Figure 4. SEM of surface of corematal hairs of C. trunsiens (A-D) and C. gangis (E-F). A, B, E, Grey hair. C, D, F, Yellow hair. Scale bars: A-E=5 pm, F=2 pm. BIOLOGY OF CREATONOTOS MOTHS 345 glandular cell, visible even with a dissection microscope, reaches into the air- filled lumen of the corematal tube.

VARIABILITY OF COREMATAL SIZE In the field, males with coremata varying in size from minute to very large can be found at a single location at the same time. Figure 5 shows examples; in Fig. 6 the weights of individual coremata are plotted for each species. There is no relation between body size and size of coremata, as demonstrated by weighing dried moths and their coremata (Fig. 7), and we did not discover structural Downloaded from https://academic.oup.com/zoolinnean/article/96/4/339/2658339 by guest on 01 October 2021 differences apart from the coremata. The latter vary in all their features (length of tubes and hair, thickness of tubes and hair, number of hairs). Thus, although the size of expanded coremata can exceed the wingspan of the insects and make up 5% of the dry body weight, these organs can also be almost invisible, or any intermediate size. This variation appears continuous, without modality for particular sizes, although a peak in distribution is recognized in our samples. As can be seen from Fig. 6 (and as we also found with smaller samples from other habitats), this variation is related neither to habitat nor season, but appears to be typical for the species.

Figure 5. Examples of artificially expanded coremata of C. tmnsiens (A-C) and C. gangis (D-K) collected in the field. The respective weights are: A, 10 pg; B, 180 pg; C, 260 pg; F, 130 pg; G, 80 pg; H, 160 pg; I, 150 pg; K, 70 pg; D and E not weighed-natural size. 346 M. BOPPRE AND D. SCHNEIDER

IIII t I I 11111111111 ill Ill I I I It I I I Downloaded from https://academic.oup.com/zoolinnean/article/96/4/339/2658339 by guest on 01 October 2021 -

0. I I 2 Corematal weight (mg) Figure 6. Weights of coremata of field-caught C. transiens (A, B) and C. gangis (C).Vertical bars mark dry weights of individual coremata, dots give average weight in sample, figures in () = sample size, and horizontal bars indicate standard deviation. Males for A originate from Paden Panjang (West Sumatra), those for B and C come from Dolok Merangir (North Sumatra).

As reported elsewhere (Schneider & Bopprtt, 1981; Schneider et al., 1982), the ontogenetic development in size of the coremata (as well as the biosynthesis of pheromone; see below) is directly related to the amount of pyrrolizidine alkaloids (e.g. heliotrine, Fig. 8A) larvae ingest from their host plants (details in BopprC & Schneider, 1985).

00 80 00 'i O 0 00O 0 O00

0

1 I 30 50 80 Dry body weight (mg) Figure 7. Coremata weights plotted against dry body weights of field-caught C. trunszens to demonstrate independence of body and corematal sizes. BIOLOGY OF CREATONOTOS MOTHS 347

Figure 8. Molecular structures of A, pyrrolizidine alkaloid heliotrine and B, pheromone hydroxydanaidal. Downloaded from https://academic.oup.com/zoolinnean/article/96/4/339/2658339 by guest on 01 October 2021 COREMATAL SECRETION Chemical analyses of corematal extracts revealed, in both species, 7-hydroxy- 6,7-dihydro-5H-pyrrolizine-1-carboxyaldehyde (‘hydroxydanaidal’; Fig. 8B) (Schneider et al., 1982), the very same compounds secreted by androconial organs of Euploea and Utetheisa moths (cf. Discussion). Both grey and yellow hair contain this chemical (T. W. Bell, personal communication). Despite repeated attempts to find additional components of corematal secretions, no other volatiles have yet been found (T. W. Bell, J. Meinwald, S. Schultz, W. Francke, personal communication). The major non-volatile component of coremata extracts is cholesterol (Bell & Meinwald, 1986). In extracts from field-caught males, amounts of hydroxydanaidal varying between zero and 450 pg were found, and laboratory-raised specimens contained this substance only if their larvae had access to PA-containing plants (Schneider et al., 1982). Thus, PAS are used as precursors for the biosynthesis of hydroxydanaidal, and the amount of pheromone produced relates to the amount of PA ingested by larvae (details in Bopprt & Schneider, 1985). Surprisingly, biosynthetic conversion of PA into hydroxydanaidal can proceed with inversion of the configuration at the asymmetric center (2-7, that is, the insects always secrete R( -)-hydroxydanaidal (Bell et al., 1984; cf. Bell & Meinwald, 1986).

BEHAVIOURAL USE OF COREMATA In the literature, there are only two reports on the behavioural use of the coremata in the field. Pagden (1957) was the first to write on the huge coremata of C.gangis and their natural extrusion. By chance, he observed a displaying male sitting on the wall of his study in Penang, Malaysia, and he made photographic records. E. W. Diehl (personal communication) has also occasionally observed male C. gangis sitting with expanded coremata near the verandah light of his house at Dolok Merangir, North Sumatra. So far, the only observations under natural conditions are those made by Robinson (in Varley, 1962): “The males and females of Creatonotus gangis are active for an hour only after sunset. The female climbs up a stem of the foodplant, the tussocky grass ‘lallang’ (Zmperata cylindrica (L.) Beauv.), and extrudes a small scent organ from the end of her body. The males assemble and hang by their front legs within a yard or two of the female. When a male is settled two coremata appear from the end of his body and may either extend symmetrically to full size (when they are as long as the rest of the ) or perhaps get tangled up . . . Whilst a number of males are hanging about with coremata extended another male may arrive and 348 M. BOPPRE AND D. SCHNEIDER Downloaded from https://academic.oup.com/zoolinnean/article/96/4/339/2658339 by guest on 01 October 2021

Figure 9. Different views and stages of display of corernata by C. gangis (A-N) and C. transiens (0). See text. BIOLOGY OF CREATONOTOS MOTHS 349 mate at once without preliminary display! When a female mates she withdraws her scent organ and within a minute or two all the males withdraw their coremata, fold their wings and remain at rest until the next night”. In our laboratories, the insects became active at dusk. Illumination with red darkroom lights did not cause any apparent disturbance. They flew (and ran) about for a while (particularly at the top of the cages) and after a while some of the males ‘settled’. Usually utilizing their front legs only (Fig. 9), they hung from the (gauze) top or walls of the cage, or on twigs provided. Then the wings were spread to the ventral side of the body, thus exposing the abdomen (Fig. 9B). Rhythmic longitudinal contraction and telescoping of the abdomen was then Downloaded from https://academic.oup.com/zoolinnean/article/96/4/339/2658339 by guest on 01 October 2021 observed, which apparently served to increase the pressure within the body. Eventually two bundles of hair appeared at the ventral abdominal tip (Fig. 9C, D). Following the protrusion of the androconial organs (Fig. 9D; taking 5- 20 min), the corematal tubes were expanded (Fig. 9E-0; taking up to 5 min). This looked identical to the artificial expansion described above. The males may stay in this ‘display posture’-during which continuous rhythmic pumping movements of the body continue to occur-for up to 4 h; however, this activity usually declines after 1-2 h. Although the coremata seemed to be maximally inflated, this was often not actually the case: gentle mechanical stimulation (vibration of the substrate, a breath of wind) made the males apply more pneumatic pressure to the organ while, at the same time, the abdomen was repeatedly thrust towards the stimulus source (Fig. 9M). However, strong disturbance caused retraction. Up to the stage when all hairs formed two bundles (Fig. 9D), this was done by release of air, then presumably activity of retractor muscles returned the apparatus to the inside of the abdomen. While the release of air from the coremata happened quite quickly, a male required several minutes to stow the organ fully. Coremata can be inflated and deflated repeatedly during one night and on successive nights. The males in our laboratory populations never all displayed coremata at the same time. Corematal display is apparently influenced to a certain extent by environmental factors; one is humidity (facilitating display), others may be chemical and/or visual stimuli. However, display behaviour can usually be induced artificially by decreasing the light intensity. If males are deprived of a dark phase, they display their coremata more readily next evening and become active at higher light intensities than usual. We assume a strong drive for display behaviour. Also, corematal display is not restricted to males possessing large or medium-sized coremata: males with small organs also engage in display behaviour, but we cannot state if there is a relation of organ size and display frequency/duration. Calling behaviour of females, recognized by pulsing movements of the abdominal tip, was also observed in captivity (see also Wunderer et al., 1986). However, we never observed extrusion of a scent organ as stated by Robinson (see above). The pheromone glands of female Creatonotos become visible only by strong squeezing of a female’s abdomen, which causes them to evert; then they appear as a pair of antler-shaped tubes (Fig. 10). The number of branchings and their size exhibit significant variation but this is due neither to larval diet nor the pressure applied when expanding them. Although under our laboratory conditions matings occurred frequently, we rarely observed the final stages of mating behaviour, which are rather 350 M. BOPPRE AND D. SCHNEIDER Downloaded from https://academic.oup.com/zoolinnean/article/96/4/339/2658339 by guest on 01 October 2021

Figure 10. Artificially protruded female pheromone glands of C. transicns, demonstrating their variability. Scale bar=2 mrn. inconspicuous and rapid. It is noteworthy that the insects do not require a large amount of room for mating: it often took place in a 250 ml container. In confinement, females show no obvious mating preference for males with large coremata. Also, there appear to be no other differences between insects the larvae of which had or did not have access to PAS. Although we often kept both species together in one cage, they never interbred.

DISCUSSION The similarity of both species needs to be emphasized strongly. The wing patterns and body colours of C. gangis and C. transiens are distinctive for the species, but with respect to their biology no significant differences are yet apparent. The outstanding aspect in the biology of Creatonotos is the coremata, and the following discussion concentrates on them.

Morphological and chemical aspects Androconial organs in Lepidoptera show great differences in their morphological expression between species (even amongst those that are closely related), a fact recognized since Muller’s ( 1877) original investigations. However, although considerable work has since been done on androconial morphology (see Bopprk, 1984a, for refs), there is no report on morphological variation within individuals of a given population, which makes Creatonotos unique. Apart from the individual variation, the coremata of Creatonotos match the expected requirements for androconial organs: they exhibit a huge surface area promoting evaporation of chemical signals, and they are hidden when not in use, thus preventing uncontrolled pheromone release. Although the precise mechanism of coremata expansion is unknown, it is evident that they are inflated by air pressure. The bladder at the base of the four tubes appears to serve as a terminal air reservoir; nevertheless, a complex valve system must be involved. Most likely, the Creatonotos system resembles that of another arctiid, Estigmene, studied by Willis (1982). BIOLOGY OF CREA TOArOTOS MOTHS 35 1 Fundamentally, the gross morphology of the coremata of Creatonolos is very similar to comparable organs found in other Lepidoptera, particularly many Arctiidae. This appears to be true for the fine structure of the corematal hairs as well as for their glandular bases; and again, there is great similarity to Estigmene (Willis, 1982). Our preliminary observations (using a transmission electron microscope) of the large secretory cells resulted in micrographs similar to figures published by Nielsen (1979, 1982), who conducted a detailed investigation of the coremata of the European arctiid Phragmatobia fuliginosa. We do not know of any significance for the structural difference between the two corematal hair types in Creatonotos. Downloaded from https://academic.oup.com/zoolinnean/article/96/4/339/2658339 by guest on 01 October 2021 The coremata can provide a very large surface area for pheromone release. Assuming a hair to be 10 pm in diameter and 5 mm long and that there are 3000 hairs per male, the hair surface would total 4.5cm2. In fact, the area is even larger because of the lattice surface structure of the hairs (Fig. 4). If the coremata of a male produce 500 pg hydroxydanaidal, each glandular cell would synthesize more than 0.15 pg pheromone. Other Lepidoptera also utilize PAS as pheromone precursors. Dihydropyrrolizine pheromones and the PA-dependency of their biosynthesis was originally recognized in Danainae, where the adult males search for withered PA-containing plants and ingest PAS (for summary and ref5 see BopprC, 1984a). While in danaines PAS are thus gathered by adults, in Utetheisa (Arctiidae)-as in Creatonotos-the larvae obtain PAS from their hostplants (Culvenor & Edgar, 1972; Conner et al., 1981). Since all host plants of Utetheisa contain PAS, individual males produce roughly equal amounts of hydroxydanaidal. However, with respect to the amounts of pheromone found in Creatonotos and in danaines, we are faced with great variation depending on the amount of PAS gathered by larvae or adults, respectively. Thus, Creatonotos is not exceptional with respect to individual variation in pheromone amounts, nor with respect to the precursor dependency. However, Creatonotos does represent the first case in the kingdom in which organ-specific morphogenesis is recognized to be regulated in a dose-dependent way by a particular secondary plant substance (for further discussion see Bopprk & Schneider, 1985; Boppri., 1986). Thus, Creatonotos might be a model for explaining polymorphic expressions of secondary male characters in other insects, and it seems to be a potent study object for developmental biology with respect to the mechanisms of growth regulation.

Functional aspects In general, male Lepidoptera bring their androconial organs into play during the final phases of courtship behaviour, and for a short time only-that is, after a male has approached a female by means of chemical or visual signals. For several species it has been established that stimulation with male pheromones is required by females before they will mate. Thus, male pheromones have often been thought to be 'aphrodisiacs'. However, in the past there has been little more than speculation on the information coded by the chemical signal. The subject has recently been reviewed and discussed by Boppri: (1984a) who suggested that we should abandon considering male scents mainly in the context of recognition 352 M. BOPPRfi AND D. SCHNEIDER but rather assume multiple functions in intra- as well as in inter-sexual communication; the widespread analogous development of androconia in Lepidoptera is apparently matched by a variety of functions. In contrast to other Lepidoptera, many of which expand androconial organs hydraulically or mechanically, the pneumatic inflation mechanism, together with the large size, of the androconial organs of Creatonotos does not permit their rapid expansion. Also, they are displayed for long periods regardless of the presence of a female. Thus, the coremata of Creatondos definitely serve some function(s) other than those usually attributed to androconial organs (cf. Bopprt., 1984a). Downloaded from https://academic.oup.com/zoolinnean/article/96/4/339/2658339 by guest on 01 October 2021 The coremata obviously release a pheromone, and it is understandable that large coremata would release large amounts of pheromone more effectively than small ones, and also that it would be a waste to build large coremata if no pheromone was available (it appears economical to relate organ size and amount of pheromone). The major question, however, remains: what is the significance (if any) of the individually differing hydroxydanaidal amounts emitted by the different sized coremata? According to our laboratory experience (see above, cf. Schneider, 1983; Wunderer et al., 1986) it is not unlikely that mate-finding in Creatonotos follows a dual strategy, similar that described for Estigmene. In Estigmene, Willis (1982) and Willis & Birch (1982) demonstrated in field experiments that early in the evenings males display their coremata which not only attract conspecific males (which then commence displaying, too), but also both virgin and previously mated females. In these aggregations (‘leks’), consisting ofup to 22 males (ten on average), matings take place. Temporally separated from mating that occurs as a result of this male display behaviour, is mating initiated by female calling, which takes place later in the night. Thus, Estigmene exhibits a dual mating strategy, the advantage (and evolution) of which remains puzzling, however. Unfortunately, the release of pheromones by the coremata in Estigmene (although most likely) is only assumed. Further studies should involve chemical investigations at both the species and individual levels, as well as experiments with purified male and female secretions, in order to reveal the environmental and individual conditions under which either strategy is applied. It should also be investigated whether the pheromones are synthesized de nouo or depend on a dietary precursor: Willis ( 1982) found indoor-raised males and females (although they exhibited luring behaviour) to be unattractive to conspecifics of either sex in the field, but he did not use many specimens for this test. Within the Lepidoptera there are two more known examples which, because of potential analogy, need to be considered in the context of sexual behaviour of Creatonotos. A large amount of literature on Hepialus humuli (Hepialidae) reports that the males assemble in huge aggregations (involving up to 2000 moths). Several papers suggest-although the discussion is controversial-that (assumed) pheromones emitted by the spectacular male tibia1 ‘scent brushes’ attract females for mating (for review, see Mallet, 1984). To date, few experimental investigations have been made, the androconial secretion is not known, and the (likely) involvement of visual cues also requires thorough study. For Eldana saccharina (Pyralidae), Atkinson ( 1981) reported males luring females while sitting in groups of 3-6. Again, there are no more than a few observations. In contrast to Estigmene and Creatonotos, in Hepialus and’ Eldana there is no BIOLOGY OF CREATONOTOS MOTHS 353 evidence for the presence of female-luring pheromones (although they might ultimately prove to be involved). With respect to Creatonotos, we have not yet succeeded in observing the ’s behaviour in the field, or in conducting ethological experiments under natural conditions. Therefore, the idea of a dual mating strategy in Creatonotos, comparable to Estigmene, is not fully established. Nevertheless, it seems to be a reasonable hypothesis, supported by Robinson’s field-observations and by various laboratory findings (see also Wunderer et al., 1986). Since mating behaviour in Creatonotos appears particularly complex, special care is required in further behavioural studies. In contrast to many other Lepidoptera, particularly Downloaded from https://academic.oup.com/zoolinnean/article/96/4/339/2658339 by guest on 01 October 2021 butterflies, Creatonotos can mate in confinement without performing a stereotyped behaviour pattern. This does not, however, necessarily imply the lack of complex behaviour or peculiar mate-finding and mate-selection strategies. As stressed, field studies are indispensible to determine the principal roles of male and female scents in Creatonotos. In addition to information on mate- finding, there is the question of the adaptive significance of the variation in the morphological and chemical expression of the androconial organs: is it an advantage or disadvantage for individual males to gather PAS, and thereby have prominent scent organs rich in pheromones? According to the above hypothesis, a male releasing a large amount of pheromone might attract more conspecifics than a pheromone-poor male, but does it have a higher reproductive success? Perhaps PA-containing and PA-lacking males follow different strategies and we should be prepared to find an even more complex situation than in Estigmene.

Ecological aspects As pointed out already, knowledge on the ecology of Creatonotos is extremely scant. We have observed the species mainly in cultivated areas but we have no idea if this represents their typical biotope. Ecological studies-not only with respect to mating behaviour-have to be a major aim for further research on these insects. Both the published foodplant records and our experience demonstrate that Creatonotos gangis and C. transiens are polyphagous. However, we only used European plants, that is substitute food and, with respect to field records (see above), it has to be questioned if the caterpillars develop properly on all recorded plant species; many records (those on agricultural plants in particular) may only be accidental, in the sense that single larvae were only found on these plants by chance. Thus, a discussion on food preferences and the natural hostplant range of Creatonotos must await proper field studies. In the light of our knowledge of the dependency of corematal morphogenesis and hydroxydanaidal biosynthesis on pyrrolizidine alkaloids (Schneider & BopprC, 1981; Schneider et al., 1982; BopprC & Schneider, 1985), one might expect adaptations in egg-laying behaviour and/or food choke by larvae to ensure that all individuals (the males in particular) obtain PAS, that is one would assume that (as in Utetheisa) egg-laying as well as larval feeding behaviour would have evolved towards host specialization. However, Creatonotos is polyphagous and, in the laboratory, fails to show a clear preference for PA- containing plants, resulting in the great variation with respect to androconial size and pheromone quantities. Nevertheless, Creatonotos larvae are 354 M. BOPPRE AND D. SCHNEIDER pharmacophagous (Bopprt, 1984b), that is they eagerly feed on glass-fibre discs and other substrates if they have been soaked with PAS. With present knowledge we cannot solve this apparent paradox: we have to assume that males do not depend on PAS for their reproductive success, even though they appear to invest energy in morphogenesis and biosynthesis if they do happen to obtain the alkaloids, In this context, the defensive potency of PAS has to be considered. Pagden (1957) recognized that Creatonotos gangis appears distasteful to the common house gecko. Recent studies show that PAS are stored and render Creatonotos unpalatable for various potential predators. The defensive use of PAS for insects Downloaded from https://academic.oup.com/zoolinnean/article/96/4/339/2658339 by guest on 01 October 2021 has been suggested repeatedly to be their original role for insects, and has then led to the evolution of a multiplicity of associations of insects with PA-containing plants (BopprC, 1986). The PA-mediated linkage between chemical defence and chemical communication might prove to be the key for understanding the complex mating behaviour of Creatonotos, as well as of insect-PA relations in general. As pointed out by Eisner (1980), Conner et al. (1981) and Brown (1984), the amount of PAS gathered by a male might be assessed by the female on the basis of PA-derived pheromone, which might then be a measure for the amount of PAS the female can expect to receive with the spermatophore. This could be decisive in female mate-choice (Bopprt, 1986). Last but not least, it has to be stressed that C. gangis and C. transiens often occur sympatrically, which poses the question of their sexual isolating mechanism(s) or specific mate-recognition system. Since sexual communication in both species seems to be purely chemically mediated, one has to expect differences in the chemical composition of the attractive secretions of both sexes. With respect to corematal odours, so far, chemical analyses have failed to reveal any difference; for female scents see Wunderer et a/. (1986) and Bell & Meinwald (1986).

CONCLUDING REMARKS The relationship of Creatonoto, to pyrrolizidine alkaloids appears ambivalent-the larvae are pharmacophagous with respect to these secondary plant substances, PAS drastically affect the morphology of the androconial organs as well as the male pheromone biosynthesis, and they provide chemical protection, but nevertheless, Creatonotos does not appear at all ‘dependent’ on PAS for reproduction. We hope to explain this paradoxical situation by further ethological studies in the field, particularly on the function of hydroxydanaidal in the life of these insects. The quantitative relations between the amount of PA ingested, the size of the coremata and their pheromone content (BopprC & Schneider, 1985) allows us to breed for behavioural studies insects with precise, predictable pheromone contents. This property, together with the challenging developmental questions, renders Creatonotos an experimental animal of unusual value.

ACKNOWLEDGEMENTS The major part of this study was undertaken in 1979-1981 when M.B. was at the Max-Planck-Institut fur Verhaltensphysiologie, Seewiesen, and continued at the Institut fur Zoologie, SFB 4, Universitat Regensburg; M.B. acknowledges BIOLOGY OF CREATONOTOS MOTHS 355 support by the Deutsche Forschungsgemeinschaft (SFB 4/B6). We thank Dr E. W. Diehl for the supply of livestock and H. Sochting-Mayr, E. Roth and M. Bairov for assistance in the laboratory. Also, we are most grateful to R. I. Vane- Wright for revising the manuscript linguistically and for his stimulating criticism.

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