Chemoecology (2009) 19:155–168 DOI 10.1007/s00049-009-0016-8
CHEMOECOLOGY
RESEARCH PAPER
Structure and function of the eversible organs of several genera of larval firefly (Coleoptera: Lampyridae) Xinhua Fu Æ V. Benno Meyer-Rochow Æ John Tyler Æ Hirobumi Suzuki Æ Raphael De Cock
Received: 1 January 2009 / Accepted: 12 May 2009 / Published online: 26 May 2009 Ó Birkha¨user Verlag, Basel/Switzerland 2009
Abstract Larval defensive behaviour and eversible organ morphology were compared in the firefly species Luciola cruciata Motschulsky 1854, L. lateralis Motschulsky 1860, L. leii Fu et Ballantyne 2006, Lampyris noctiluca Linnaeus 1767, Pyrocoelia analis Fabricius 1801, P. pectoralis Oliver 1883, P. sp. and two Diaphanes species. In all cases the eversible organs are located on the pleural cuticle, dorsal to the spiracle, but there is considerable variation between species in their number, size and shape. In La. noctiluca they are confined to the abdomen, whereas in the other species they also occur on the meta- and mesothorax. In La. noctiluca and the Pyrocoelia species the organs are
X. Fu (&) College of Plant Science and Technology, Huazhong Agricultural University, 430070 Wuhan, China e-mail:
[email protected] V. B. Meyer-Rochow Faculty of Engineering and Sciences, International University Bremen (IUB), 28725 Bremen, Germany e-mail:
[email protected] V. B. Meyer-Rochow Department of Biology (Ela¨inmuseo), University of Oulu, 90014 Oulu, Finland e-mail:
[email protected] J. Tyler 5 Woodfield, Lacey Green, Buckinghamshire HP27 0QQ, UK H. Suzuki Research and Development Division, Olympus Corporation, 2-3 Kuboyama-cho, Hachioji, Tokyo 192-8512, Japan R. De Cock Evolutionary Ecology Group, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium
columnar in shape, contrasting with the forked organs of the Luciola and Diaphanes species. The eversible organs of the Luciola species, which are all aquatic, are remarkably larger (relative to body length) than those of the other, terrestrial species. On organ eversion the Luciola species emit an odour resembling pine oil, the Diaphanes species smell weakly of mint and L. noctiluca and the Pyrocoelia species produce no discernible scent, though ants are nonetheless repelled by contact with everted L. noctiluca organs. The organs of all the species studied support protuberances on their external surface, though these too vary in shape, size and ornamentation between species. In all species except L. noctiluca each protuberance is connected to a well-developed globular body via a long, thick stalk. Circular foveae are located in the centres of these globular bodies. The globular bodies consist of secretory cells characterized by the presence of numerous mitochondria and an extensive system of cisternae and tubular endoplasmic reticulum. Behavioural tests revealed that small larvae run away instead of becoming immobile and glowing, whereas large larvae start to glow when disturbed. The study shows that the eversible larval organs form an important part of a defensive arsenal in the Lampyridae. Keywords Aposematic Defence Eversible organs Evolution Firefly Larvae
Introduction The adults and larvae of many firefly species are well known to be distasteful to vertebrate and invertebrate predators and possess the ability to bleed reflexly when attacked (Blum and Sannasi 1974; Knight et al. 1999; Tyler and Trice 2001; Tyler 2001a; Trice et al. 2004; Ohba and
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Hidaka 2002; Fu et al. 2006a). In fact, ingesting fireflies can be lethal to lizards and frogs (Sexton 1960; Lloyd 1973). In the blood of adult Photinus fireflies, Eisner et al. (1997) discovered lucibufagins (LBGs) that act as emetics when ingested by predators. Some Photuris fireflies evidently acquire LBGs by preying on the noxious Photinus fireflies, and pass these compounds on to their eggs and larvae (Eisner et al. 1997; Gonza´lez et al. 1999a, b). Adults of most firefly species glow or flash, but bioluminescence in the larvae is common to all species without exception. The larvae glow when stationary, crawling or when disturbed (Christensen and Carlson 1982; Sivinski 1981; Branham and Wenzel 2003). Their pulses of light represent a conspicuous signal that has been shown to function as an aposematic defence, which warns predators of distastefulness and unprofitability (Underwood et al. 1997; De Cock and Matthysen 1999, 2001, 2003) quite like the freshwater limpet Latia neritoides does with its luminescent mucus when attacked (Meyer-Rochow and Moore 1988). However, remarkably little is known about the chemical, morphological, physiological or behavioural components of larval firefly defences. Tyler (2001b) briefly described the eversible organs in L. noctiluca Linnaeus 1758 and Trice et al. (2004) compared the pleural defensive organs of the larvae of three genera of fireflies, namely L. noctiluca, L. cruciata and a Nyctophila species. Eversible organs were first described in the aquatic larvae of L. lateralis and L. cruciata 80 years ago (Okada 1928) and more recently in Luciola owadai Sato et Kimura (Ohba et al. 1994). However, the studies on these eversible organs were not complete. Fu et al. (2007) studied the external morphology and function of the eversible organs of the aquatic firefly L. leii in detail. Observations by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) revealed that there are numerous, almost spherical protuberances on the surface of each organ. Each protuberance is connected to a well-developed secretory cell via a 0.1-lm thick and 0.2-lm long stalk and bears three to six 7-lm long spines on its apex. By means of a thorough investigation of larval defence reactions, we discovered that the larvae of two genera of terrestrial firefly, Diaphanes and Pyrocoelia, also possessed the eversible organs. Through years of preparation, we finally obtained enough material from around the world to compare the fine structure of eversible organs in different genera of both terrestrial and aquatic firefly larvae in order to explore their function. This paper presents new results on the morphology and function of the eversible organs in the aquatic larvae of the firefly species L. leii, L. lateralis, L. cruciata as well as the terrestrial larvae of the fireflies L. noctiluca, P. pectoralis, P. analis, Pyrocoelia sp., Diaphanes sp. 1 and Diaphanes sp. 2.
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Materials and methods Nine firefly species, representing four genera, were obtained: aquatic firefly larvae of L. leii, L. lateralis and L. cruciata, together with terrestrial firefly larvae of Diaphanes sp. 1, Diaphanes sp. 2, P. pectoralis, P. analis, Pyrocoelia sp. and La. noctiluca. First, second, fourth and sixth instar larvae of L. leii were obtained from a laboratory colony located in Yuan An County, Yi Chang City, Hubei Province, China. Laboratory colonies were maintained under a natural photoperiod in aquaria containing freshwater of 25°C. Captive larvae were fed fresh aquatic snails Bellamya purificata Heude 1890 every 2 days (Fu et al. 2006b). Third to fourth instar larvae of L. lateralis were obtained from Higashi-Yamato, Tokyo, Japan in March, 2007. Fourth instar larvae of L. cruciata were obtained from Musashi-Murayama, Tokyo, Japan in March, 2007. Larvae of D. sp. 1 were collected from E Mei Mountain, Sichuan Province, China in September and November, 2007. Larvae of D. sp. 2 were collected from Guang Shui County, Sui Zhou City, Hubei Province, China in August, 2007. Larvae of P. pectoralis were collected from the campus of Huazhong Agricultural University (Wuhan City, Hubei Province, China) in July 2007. Larvae of P. analis were collected from Zhu Hai City, Guangzhou Province, China in April, 2007. Larvae of P. sp. were collected from Mt. Tai, Shandong Province, China in September, 2007. Larvae of L. noctiluca were collected from Wendover, Buckinghamshire, UK in October 2007. Organ morphology In order to study the number of larval organs and their locations, we placed the larvae of each species in individual Petri dishes. Each larva was gently touched with tweezers on their tergites, so that the areas from which the organs protruded could be photographed by digital camera (Nikon D70 s with a 60-mm macro lens) and their number could be determined. For detailed observations of the organ surfaces, we used SEM. Specimens were prepared by the following three methods: (1) some larvae were disturbed with forceps to elicit organ eversion. The larvae were then quickly killed by emersion in liquid nitrogen, but not subjected to further dissection. (2) Larvae were fixed by submergence in liquid nitrogen for 30 s prior to further processing. The two preparation methods had different purposes: the first was used to observe the outer surface of the organs; the second allowed us to observe the inner surface of the organs. For that purpose the sternites were cut longitudinally from the
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Fig. 1 Eversible organs in several genera of larval fireflies (arrows indicate eversible organs, scale bar = 5 mm). a Aquatic firefly L. leii, b aquatic firefly L. cruciata, c terrestrial firefly D. sp. 1, d terrestrial firefly D. sp. 2, e terrestrial firefly L. noctiluca, f terrestrial firefly P. pectoralis
prothorax towards the abdominal apex. Tracheae, fat bodies and intestine were carefully removed. Larvae treated in this way were then fixed for 24 h in 4°C cold, phosphatebuffered 2.5% glutaraldehyde with a pH of 7.4. The larvae were then rinsed twice in phosphate buffer at 10-min intervals and post-fixed for 3–4 h in 1% osmium tetroxide at room temperature, followed by two rinses in phosphate buffer and dehydration in a graded series of ethanol with 12 h stays at each concentration (30, 50, 70, 80, 90 and 100%). The specimens were then placed in acetone for two 12-h periods before finally being subjected to critical point drying. Each larva was attached with double-sided sticky tape to an aluminium stub and sputter-coated with gold to a thickness of about 200 nm. The specimens were observed under a JSM-6390 LV SEM at an accelerating voltage of 25 kV (Fu et al. 2006a, 2007; Mora et al. 2001). In the third treatment, for ultrastructural observations, the larvae were first disturbed to project their organs outward. The organs were then carefully dissected and fixed in 2% cold glutaraldehyde at pH 7.3 in a 50-M sodium cacodylate buffer (with 150 M sucrose added). After postfixation in 2% cold osmium tetroxide in the same buffer, the organs were dehydrated through a graded acetone series and embedded in araldite. Semi-thin sections for light
microscopy were stained with Delafield’s haematoxylin and eosin; ultra-thin sections for TEM, double-stained with uranyl acetate and lead citrate for a few minutes each, were examined in a Hitachi H-7650 TEM microscope at 75 kV accelerating voltage following Fu et al. (2007) and MeyerRochow and Liddle (1988). Behaviour To observe the behavioural responses elicited by threats on larvae of L. lateralis and L. cruciata, we divided individuals into two treatment groups: (1) 20 fourth instar larvae were kept in water; (2) 20 fourth instar larvae were placed in Petri dishes and carefully dried with filter paper. Each group of the larvae was tested at night (25–27°C air temperature, 70–85% RH or 23°C water temperature for the group tested in water). A 3-mm diameter wooden dowel was used to stimulate the larvae. Four stimulus treatments were used to simulate increasing levels of molestation: (1) moving the dowel’s tip at a rate of approximately 2 cm/s several times to within 0.5 cm of the larva; (2) gently touching the tergites of a larva with the dowel; (3) quickly turning the larva over with the dowel and (4) squeezing the larva between dowel and substrate. The controlled group
10 Meso- and meta-thoracic segments, and abdominal segment 1–8
Position of organs
Forked
Organs shape
Weak
Intensity of glow
Information of Both have wings adults
Easy
Easy
Organs eversion
Both have wings
Weak
Discernable ‘pine-oil’ smell
Smell Discernable ‘pineassociated oil’ smell with organs eversion
Forked
0.2
Organ length/ 0.2 larval body length
Meso- and metathoracic segments, and abdominal segment 1–8
Pairs of organs 10
Both have wings
Weak
Easy
Discernable ‘pine-oil’ smell
Forked
0.3
Meso- and meta-thoracic segments, and abdominal segment 1–8
10
Female adult wingless
Strong
Seldom
Weak ‘mint’ smell
Thoracic organs forked; abdominal organs columnar
0.03
Meso- and meta-thoracic segments, and abdominal segment 1–7
9
Diaphanes sp. 1
Luciola cruciata
Luciola leii
Luciola lateralis
Terrestrial firefly
Aquatic firefly
Table 1 Description of eversible organs and the other information in larval fireflies
7
Lampyris noctiluca 9
Pyrocoelia analis, P pectoralis, P. sp.
Female adult wingless
Strong
Seldom
Thoracic organs forked; abdominal organs columnar Weak ‘mint’ smell
0.03
Female adult wingless
Very strong
Hardly
None
Columnar
0.03
Female have elytra buds
Very strong
Hardly
None
Columnar
0.02
Meso- and Abdominal Meso- and meta-thoracic segment 1–7 meta-thoracic segments, and segments, abdominal segment and abdominal 1–7 segment 1–7
9
Diaphanes sp. 2
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Table 2 The morphology of eversible organs by SEM Aquatic firefly
Terrestrial firefly
Luciola leii
Luciola lateralis
Luciola cruciata
Diaphanes sp. 1
Diaphanes sp. 2
Lampyris noctiluca
Pyrocoelia analis, P. pectoralis, P. sp. 9–19
Disc diameter (lm)
7
7
7
24–36
22–26
10–15
Disc location
Surface
Surface
Surface
Surface
Surface
Pit
Pit
Globular body diameter (lm)
6
6
6
26
26
20
20
Protuberance diameter (lm)
7
7
7
10–13
8–10
8–10
8–13
Protuberance stem
Present
Present
Present
Present
Present
Present
Present
Protuberance spines numbers (lm)
2–6
4–7
7–10
None
None
None
None
Protuberance character
Solid
Solid
Solid
Solid
Solid
Hollow
Hollow
consisted of 20 fourth instar larvae kept in Petri dish with water and without any form of stimulation. The elicited behaviours were recorded on video under red light (Krall et al. 1999; Fu et al. 2007). To observe the responses elicited by threats on terrestrial larvae of Pyrocoelia, Diaphanes and L. noctiluca, we placed 20 larvae in Petri dishes and tested them using the same methods described above. For L. noctiluca, defensive behaviour was also observed in the field, by placing 20 larvae on the nests of the wood ant Formica rufa L. 1761. One skirmish of D. sp. 2 with ants Camponotus albosparsus Bingham 1903 was also observed in August, 2006 by accident.
Results The aquatic larvae of L. leii (Fig. 1a), L. cruciata (Fig. 1b) and L. lateralis possess ten pairs of similar pale white, forked eversible organs. The organs are located laterally on the meso- and metathorax and above the tracheal gills on each of the eight abdominal segments (Table 1). The organs of L. cruciata are thinner and longer than those of L. leii and L. lateralis (Table 1). Larval instars 1–6 of L. leii all possess eversible organs. The terrestrial larvae of D. sp. 1 (Fig. 1c) and D. sp. 2 (Fig. 1d) possess nine pairs of pale white eversible organs of either forked or columnar shape. The two pairs of forked organs are located laterally on the mesoand metathorax and the seven pairs of unforked, columnshaped organs are situated above the spiracles on abdominal segments 1–7. The eversible organs of L. noctiluca larvae were very similar to those of Pyrocoelia, except that they have just seven pairs of small pale white, mammiform eversible organs on the abdominal segments. In their retracted state the organs of L. noctiluca are concealed within slit-like pouches of unsclerotized cuticle adjacent to the dorsal margin of abdominal pleurites 1–7, lying dorsal and somewhat anterior to the spiracle (Fig. 1e). The terrestrial larvae of Pyrocoelia possess nine pairs of small pale white, mammiform eversible organs. The organs are located
laterally on the meso- and metathorax, and above the spiracles on each of the abdominal segments 1–7 (Fig. 1f). Organ morphology External Luciola species The morphological characteristics of the eversible organs in the aquatic fireflies L. lateralis, L. cruciata and L. leii are similar (Table 2; Fig. 1a, b). The organs themselves are transparent, fork-shaped sacs, immersed in haemolymph. When the organs are everted, their white or yellow colour depends on the amount of haemolymph in them. On the external surfaces of the larval organs of all three Luciola species, numerous flower-shaped protuberances were discovered. The protuberances are slightly different among the three species. The protuberances of L. leii are equipped with two to six long apical spines (Table 1). From the observation of first, second, fourth and sixth instar L. leii larvae, each instar possesses the eversible organs (Fig. 2a–d). In the first and second instar larval organs, the protuberances are equipped with three, four and five apical spines but are less densely packed than in the other instars (Fig. 2a, b). The protuberances of L. lateralis are similar to L. leii but with one more apical spine in the middle than those of L. leii. In fourth instar larvae of L. lateralis, the protuberances are equipped with six or seven apical spines (Fig. 3a–e). The protuberances of L. cruciata, were equipped with more than six irregular apical spines, arranged like a crown (Fig. 4a–c). The protuberances extend from round ‘discs’, the diameter of which (7 lm) was the same as that of the protuberances. SEM observations revealed the presence of numerous ‘globular bodies’ on the wrinkled inner surfaces of the organs in all three species (Figs. 2e, f, 3f, 4d–f). In the middle of the globular bodies of each species there are round foveae (Figs. 2f, 3f, 4f). Light- and TEM observations revealed that each protuberance is connected to the globular body via a narrow
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Fig. 2 External morphology of eversible organs of aquatic firefly L. leii (arrows indicate everted eversible organs). a Pairs of fork-shaped eversible organs in first instar of larva, b single fork-shaped organ in first instar of larva, c two-spined protuberance on the surface of fourth instar of larval eversible organ, d six-spined protuberance on the surface of sixth instar of larval eversible organ, e the inner surface of organ, f globular structure with central fovea
stalk in the aquatic fireflies, L. lateralis and L. cruciata. The protuberances occur in high density and the 0.1-lm thick and 0.2-lm long stalk that connects each protuberance with its underlying globular body leads to a fovea in the middle of the globular body (Figs. 2, 3, 4, 9). Diaphanes species In D. sp. 1, on the external surface of the organ, numerous spherical protuberances measuring 10– 13 lm in diameter were discovered (Fig. 5a–c). Although there are no apical spines on the protuberances, there are irregular ‘veins’ (Fig. 5c). Each spherical protuberance extends from a large, round disc the diameter of which varies from 24 to 36 lm (Fig. 5c). SEM revealed some protuberances that had broken at the base, leaving only the disc (Fig. 5b). The organs of D. sp. 2 are very similar to those of D. sp. 1 in terms of the numbers of organs and their position (Fig. 6a). The only difference between the larval organs of the two Diaphanes species is in the shape of the protuberances (Figs. 5c, 6b). The protuberances of D. sp. 2
are spear-shaped with ridges around them (Fig. 6c). SEM observations revealed numerous round, triangular or rectangular ‘globular bodies’ on the wrinkled internal surfaces of the organ (Figs. 5d, e, 6d, e). There are foveae on the centre of the globular bodies, similar to those of the Luciola species (Figs. 5f, 6f). La. noctiluca The microscopic features of the organs are well preserved in the larval exuviae, where they are often easier to observe than in the larva itself. The external surface of the organ carries an array of circular pits approximately 10–15 lm in diameter (dimensions varying according to the size of the larva; Fig. 7a). The base of each pit is occupied by a flat disc, again approximately 10– 15 lm in diameter, 5-lm thick and perforated by a central canal (Fig. 7b). Viewed with optical microscopy the disc appears pale brown, in contrast to the surrounding colourless cuticle. This colouration, together with the disc’s resistance to distortion during the preparation of
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Fig. 3 External morphology of eversible organs in aquatic firefly L. lateralis. a Numerous protuberances on the eversible organs, b four-spined protuberance, c five-spined protuberance, d six-spined protuberance, e seven-spined protuberance, f globular structure with fovea in the middle
specimens, suggests that it is partially sclerotized, forming a relatively rigid base to the pit. Arising from the centre of the disc is an unsclerotized protuberance, varying in shape from spherical to elongate and with a diameter of approximately 4–7 lm (Fig. 7b, c). In fresh specimens the apex of the protuberance lies more or less level with, or extends slightly beyond, the rim of the pit (Fig. 7c). Using optical microscopy it appears as a vesicle with walls about 1 lm in thickness. The base of the protuberance is strongly constricted where it joins the disc and on the inner face of the disc a globular body about 3–4 lm in diameter is often visible (Fig. 7d–f). Pyrocoelia species In overall appearance, the eversible organs of Pyrocoelia fireflies were all similar and most closely resembled those of L. noctiluca. Numerous spherical or elongate protuberances protrude from the centre of pits on the surface of the eversible organs (Fig. 8a–d).
There were also numerous globular bodies on the internal surface of the organs (Fig. 8e, f). Ultrastructure of eversible organs Sections of the globular bodies showed that they consist of well-developed secretory cells, while TEM revealed that the ultrastructure of the secretory cells is similar in all of the firefly species studied (Fig. 9). The secretory cells themselves display hemispherical outlines and are surrounded by haemolymph. The walls of the organs are 0.2 lm thick and considerably folded. The nuclei of the secretory cells occupy positions in the basal part and are more or less spherical in shape. The cytoplasm of the secretory cells is characterized by numerous mitochondria of the crista type and an extensive system of cisternae and tubular endoplasmic reticulum. TEM revealed that the protuberances of Pyrocoelia larval eversible organs are hollow at the base
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Fig. 4 External morphology of eversible organs in aquatic firefly L. cruciata (arrows indicate eversible organs either everted retracted within the body). a Fork-shaped eversible organs, b numerous protuberances on the eversible organs, c crown-shaped protuberance, d pairs of forkshaped eversible organs, retracted, e single organ, retracted, f globular structures
(Fig. 9e). Similarly, optical microscopy has shown the protuberances of L. noctiluca to be hollow (Fig. 9f). Behaviour In the three aquatic Luciola fireflies, when tested in the laboratory, most of the larvae showed the same defence reactions. When the dowel was moved towards, 18 of 20 larvae ceased to crawl and withdrew their heads under the pronotum; when gently touching the tergites of larvae, 17 of 20 larvae glowed but there was no organ eversion or smell; when quickly turning the larva over with the dowel, 16 of 20 larvae glowed strongly but there was still no organ eversion or smell; when squeezing the larvae between dowel and substrate, 17 of 20 larvae glowed and everted their organs as well as releasing a pine-oil smell, but the organs were soon retracted. The newly hatched larvae of L. leii also possess the eversible organs and could release
the pine oil-like smell when forceps were used to press the tergites of the larvae. So it appears that all larval instars of L. leii possess the eversible organs. But all first instar larvae preferred to escape in the first instance instead of everting their organs. However, after subsequent stimulation they too everted their organs and released the characteristic smell. The control larvae crawled freely in the Petri dish and glowed irregularly. No organs were everted and no ‘pine-oil’ smell was detected. In tests with terrestrial larvae, most Diaphanes larvae (Fig. 10) showed similar defensive behaviour to the aquatic Luciola larvae when subjected to the same stimulation, but they emitted a weak smell of mint, rather than pine oil. Observations in the field show that all La. noctiluca larvae placed on wood ant (Formica rufa) nests usually make no attempt to curl their bodies into a defensive posture, even when submerged beneath an almost unbroken carpet of ants. Instead they crawl in a normal manner, at a
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Fig. 5 External morphology of eversible organs in terrestrial firefly D. sp. 1 (arrows indicate everted eversible organs). a Pairs of organs everted from body, b numerous protuberances on the external surface of organs, c round protuberances on the internal surface of organs, d single column-shaped organ rest in body, e single fork-shaped organ, retracted, f globular structure with central fovea
normal speed, briefly stopping and retracting their heads into the pronotum when the head is touched by an ant. Eversion of the defensive organs appears to be stimulated by direct contact with the ants and the organs are retracted within a few seconds when contact ceases (attempts to stimulate eversion by positioning a larva over a 32% aqueous solution of formic acid were unsuccessful, again suggesting that eversion is in response to tactile rather than olfactory cues). In rare cases the ants were not repelled and succeeded in biting the larva, causing large droplets of haemolymph to form on various parts of the body. Numerous times on such occasions an ant was observed to touch one of these droplets with its antennal flagellum and in each case the ant recoiled immediately, moving away briskly and attempting to clean its antennae. Similarly an ant touching a droplet of haemolymph with its mouthparts would instantly retreat to a distance of a few centimetres
before wiping its mouthparts energetically on the surface of the nest, as if to clean them. These observations suggest that distasteful substances occur throughout the haemolymph and are not confined entirely to the defensive organs. It is sometimes also possible to stimulate eversion by holding a larva by the thorax and using a probe to prod the abdomen in the region of the organs. Again, eversion is brief and the organs are retracted as soon as the stimulation stops. When testing Pyrocoelia firefly larvae, 19 of 20 large larvae glowed after disturbance (especially when they were well fed and fat) and were less mobile, retracting their heads into the pronotum and glowing strongly, whereas when exposed to the same stimulation 20 small larvae ran away, instead of remaining immobile, and glowed. Throughout the whole test, no organ eversion or release of repellant haemolymph was observed.
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Fig. 6 External morphology of eversible organs in terrestrial firefly D. sp. 2 (arrows indicate everted eversible organs). a Pairs of organs everted from body, b numerous spear-shaped protuberances on single column-shaped organ, c spearshaped protuberance, d single column-shaped organ, retracted, e single fork-shaped organ, retracted, f globular structure with central fovea
Discussion The fact that in each of the species examined the eversible organs were deployed in response to a real or simulated attack, together with the avoidance response of predators upon contact with the organs, leads us to believe that the organs serve a significant defensive function, allowing the lampyrid larva to release controlled doses of toxic and/or repellent substances in order to deter or repel predators. This is consistent with earlier studies (Lloyd 1973; Blum and Sannasi 1974; Sivinski 1981; Ohba and Hidaka 2002), in which predators including raccoon dog (Nyctereutes procyonoides), bat (Myotis macrodactylus), dragonfly (Anotogaster sieboldii) and goby (Rhinogobius brunneus) exhibited avoidance responses to a range of Luciola and Pyrocoelia larvae. This defence mechanism is likely to be particularly effective against predators which hunt using predominantly olfactory cues, such as ants and ground
beetles (Carabidae) (Fu et al. 2007) lizards and some amphibians (De Cock 2004) and Insectivora (e.g. mice, see Underwood et al. 1997) and would complement the colouration and larval flashing that, at least in L. noctiluca, have been shown to act as aposematic cues to visually guided diurnal and nocturnal predators (De Cock and Matthysen 1999, 2001, 2003). The feature, which most clearly unites all of the eversible organs described here and distinguishes them from every other part of the lampyrid cuticle, is the approximately spherical protuberance. In La. noctiluca and Pyrocoelia the protuberances are hollow and may act as storage receptacles for the repellent entering from the globular body via the central pore in the disc. From here we conjecture that the repellent may be released either by gradual diffusion through the extremely thin walls of the protuberance, or more abruptly when the protuberance is ruptured by external pressure, for example by an attacking
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Fig. 7 External morphology of eversible organs in terrestrial firefly P. pectoralis (arrows indicate the irregular shapes of protuberances). a Numerous protuberances on single column-shaped organ, b irregularly shaped protuberances sit in pits, c irregularly shaped protuberance, d the pit with broken stalk inside, e single fork-shaped organ, retracted, f globular structure with central fovea
predator. In the other species, in which the protuberance is solid, it presumably serves some other function. The function of the apical spines, where present, is unclear but it is conceivable that they are hollow and operate in a way comparable to the urticating hairs of some plants, breaking along inherent lines of weakness to release the repellent. We might further surmise that the disc from which the protuberance arises serves to provide both a relatively solid base to support the protuberance and a collar to prevent any tearing of the delicate cuticle from spreading beyond the protuberance itself (Fig. 11). In L. noctiluca the protuberance is further protected by being set into a circular pit, the floor of which is reinforced by the disc, leaving just the distal face of the protuberance extending beyond the external surface of the organ. Although considerable progress has been made in characterizing the eversible organs of the ten species described here, at least two fundamental questions remain
unanswered. Firstly, any substances released by the organs have yet to be identified. Toxic LBG have been detected in Photinus and Photuris species (Eisner et al. 1997; Gonza´lez et al. 1999a,b; Knight et al. 1999) and there is evidence for the presence of similar substances in L. noctiluca (Tyler et al. 2008), but so far there is no indication that these are the substances emitted by the eversible organs. In any case, the fact that the distinctive aromas of the secretions vary considerably between genera suggests that their chemical composition may well also vary. Secondly, the mechanism by which the organ is everted and retracted remains unclear. It seems possible that eversion is achieved by means of an increase in the hydrostatic pressure of the haemolymph, but having found no trace of muscle attachments on the internal surfaces of the organ, the method of retraction has yet to be explained. Turning now from functional to evolutionary considerations, the close similarities between the eversible organs of
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Fig. 8 External morphology of eversible organs in terrestrial firefly L. noctiluca. a Numerous protuberances on single column-shaped organ, b irregularly shaped protuberances sit in pits, c irregularly shaped protuberance, d single forkshaped organ rest in body close to a tracheal connected with gill (EO eversible organ, T tracheal), e single fork-shaped organ, f globular structure with central fovea
the species studied, in terms of their position, general morphology and microstructure provide strong evidence that these organs are homologous in all ten species. The discovery of similar organs in other, phylogenetically distant lampyrid genera coupled with the almost ubiquitous occurrence of bioluminescence amongst larval lampyrids and its absence in many adults, would lend support to the hypothesis that bioluminescence within the Lampyridae originally served as an aposematic rather than a courtship display and that the, either mono- or polyphyletic, appearance of organs to release repellent or toxic substances appeared earlier, in some (pre-)lampyrid ancestor(s). Yet, to date there is no proof that these organs have evolved from a single ancestor or appeared multiple times within the Lampyridae or are even pre-lampyrid in origin. The observed differences in the size, shape and scent of the organs in different genera may be the result of adaptation to different ecological environments. In particular it may be significant that the organs are appreciably larger,
more strongly scented (at least to humans) and more readily deployed in the three aquatic Luciola species than in the remaining, terrestrial ones. One possible explanation for this distinction is that larval aquatic fireflies received more predatory pressure than terrestrial ones (the larval aquatic fireflies need to find and construct pupal cells on land). The evolutionary precursors of the protuberances described here may well have been the hollow bristles and hairs that cover much of the surface of a typical lampyrid larva, from which it is possible to envisage protuberances being derived by a reduction in size, length-to-diameter ratio and degree of sclerotization. Indeed in L. noctiluca larvae a morphological series can be seen, from the elongated, heavily sclerotized bristles on the sclerites, through the smaller, more squat and more lightly sclerotized pyriform structures to be found around the margins of the eversible organ, to the virtually unsclerotized protuberances themselves (Trice et al. 2004).
Eversible organs in larval firefly
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Fig. 9 Ultrastructures of secretory cell connected with protuberances, secretory cell containing extensive numerous mitochondria and an extensive system of cisternae and tubular endoplasmic reticulum. ER endoplasmic reticulum. Scale bar: a 6 lm, b 5 lm, c 10 lm, d 1 lm, e 5 lm, f 5 lm. a L. lateralis, b L. cruciata, c D. sp. 1, d D. sp. 2, e P. pectoralis, f L. noctiluca
Fig. 10 Larva of D. sp. 2 attacked by a mass of Camponotus albosparsus ants showing everted defensive organs (indicated by arrows). This larva remained motionless and unharmed for 3 h, before being taken into the ants ‘nest’
In conclusion it would appear that, together with toxins, reflex bleeding, aposematic colouration and bioluminescence, the larval eversible organs form an important
Fig. 11 Illustration of external morphology of a typical protuberance and connected secretory cell. as Apical spines, pr protuberance, pw pit wall, st stalk, d disk, sc secretory cell, gb globular structure, f fovea
element in the fireflies’ defensive armoury, effective against a range of predators both on land and in water. Acknowledgments We thank Lihong Qing for the taking the SEM and TEM pictures, Lesley Ballantyne gave useful comments and
168 suggestions. This is contribution of Foundation of New Teacher from Chinese Educational Ministry 20070504083 and Hubei Provincial Foundation of Natural Science No. 4006-086064.
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