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Morphology and ultrastructure of the dufour's and venom gland in the ant, Myrmica rubra (L.) (Hymenoptera : Formicidae)
Int. J. InsectMorphol. &EmbryoL, Vol. 15, No. 1/2, pp. 13to 25, 1986.
0020- 7322/86$3.00 + .00 © 1986PergamonPressLtd.
Printed in Great Britain.
MORPHOLOGY
AND ULTRASTRUCTURE
OF THE DUFOUR'S
AND VENOM GLAND IN THE ANT, MYRMICA (HYMENOPTERA
RUBRA
(L.)
• FORMICIDAE)
JOHAN B1LLEN* Limburgs Universitair Centrum, Department SBM, B-3610 Diepenbeek, Belgium and Laboratorium voor Systematiek en Ecologie, K.U.Leuven, Naamsestraat 59, B-3000 Leuven, Belgium
(Accepted 29 M a y 1985)
A b s t r a c t - - T h e morphology and fine structure of the D u f o u r ' s and venom gland, as well as their entrance into the sting, are described in the myrmicine ant, Myrmica rubra (Hymenoptera : Formicidae). The epithelial cells that constitute the D u f o u r ' s gland wall, contain a well-developed smooth endoplasmic reticulum. Older workers, compared with younger ones, show an increasing number of multilamellar inclusions. The venom gland secretory cells are arranged in 2 free filaments that carry the secretion to the reservoir. Their cytoplasm shows an intracellular collecting ductule with surrounding microvillar sheath, and an abundance of free ribosomes. However, a well-organized granular endoplasmic reticulum, which is typical in species with a more powerful sting, does not occur. Both the D u f o u r ' s and venom gland ducts are characterized by the insertion of extensive muscle fibres, which act as a precise and mutually independent control mechanism for the discharging activities of the 2 glands. Index descriptors (in addition to those in title) : Sting, colony defence, alarm pheromone, trail pheromone. INTRODUCTION
AMONG the ants of the North Temperate Zone, the red myrmicine species, Myrmica rubra (L.), has been one of the most favourite research subjects for myrmecologists. More than 100 papers have been devoted to this species. For example, it served as a model in M.V. Brian's extensive work on the reproductive biology of myrmicine ants, many behavioural analyses have been performed by M.-C. Cammaerts, while the pheromone gland chemistry was thoroughly studied by Morgan et al. (1972, 1977). Therefore, its basic biology is well understood. The Dufour's gland and venom gland secretions have been analysed very carefully, clear ethological responses being attributed to the chemical compounds from both glands. The Dufour's gland components mainly act in recruitment of nestmates and in territorial marking (Cammaerts et al., 1977), while the venom gland is the source of the trail pheromone (Evershed et al., 1982). In contrast with these fairly recent contributions, is the morphological information that goes back to Janet's descriptions of M. rubra at the turn of the century. For both the
*Senior Research assistant of the Belgian National Fund for Scientific Research. 13
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JOHAN BILLEN
Dufour's and venom glands, our actual knowledge is restricted to one of his papers (Janet, 1898). Notwithstanding the amazing accuracy in the description of the sting structure (Kugler, 1978), no special attention has been paid to the sting associated glands themselves. For this reason and in order to supply the recent ethological studies with a morphological dimension, we report, in this paper, the histology as well as the ultrastructure of both the D u f o u r ' s and venom glands in Myrmica rubra. Since no notable differences were found between the glands of workers and queens, our description refers to the workers only unless stated otherwise. At the same time, an ultrastructural report of these glands in a myrmicine species forms part of a comparative ultrastructural study of the pheromone-producing glands in ants from several subfamilies. MATERIAL
AND
METHODS
Workers and queens were collected from nests in Alken, Belgium, and near Roermond in the Netherlands. After killing by momentary submersion in liquid nitrogen, the ants were dissected and the glands fixed in 2070 cold glutaraldehyde buffered with sodium cacodylate (0.05 M) and saccharose (0.15 M) at pH 7.3. Tissues were postfixed in 2% osmiumtetroxide and prestained with a 2% uranyl acetate solution before dehydration in a graded acetone series. Thin sections were stained with uranyl acetate and lead citrate and examined in a Philips EM 400 electron microscope. Transverse sections at 5 or 10 ~.m intervals from complete abdomens were stained with methylene blue and thionin and were used for microreconstruction of the gland region considered. In 17 workers, gland measurements were achieved by microscopic projection and accurate drawing with a curvimeter. RESULTS
1. General anatomy In M. rubra, the venom and Dufour's glands are by far the most distinct abdominal glands in both the queens and workers. The Dufour's gland is a sac-like structure measuring approximately 600 x 250 g m (Fig. 1). The reservoir is lined with a thin monolayered glandular epithelium, which occupies nearly 15% of the total volume of 0.009 m m 3 (Fig. 2). The venom gland, on the other hand, is composed of 2 free 720 _+ 80 ~m long filaments with a constant diameter of 60 _+ 7 ~tm, except for their narrower proximal-fifth near the entrance into the reservoir. The filaments comprise the glandular cells from where the secretion reaches the thin-walled reservoir (Fig. 2). The worker reservoir has a diameter of 370 _ 55 Ixm and a volume of approximately 0.02 m m 3, while it is some 30% smaller in the queen. The reservoir finally continues in the narrow venom gland duct (length nearly 300 Ixm), which opens in the sting bulb. The D u f o u r ' s gland also terminates at the sting base ventrad of the venom gland duct (Figs. 1; 11). The sting itself is 580 _+ 50 ~tm long, and essentially comprises the unpaired dorsal sting shaft and 2 ventral lancet parts. 2. DuJour's gland ultrastructure The wall of Dufour's gland consists of a unicellular layer of epithelial cells with a constant thickness ranging between 1 and 5 ~tm, according to individual ants (Figs. 2; 3). The cell shape correspondingly varies from nearly squamous (Fig. 6) to a cuboidal form, the latter being more frequently observed. A cuticular layer with a thickness of approxi-
D u f o u r ' s a n d V e n o m G l a n d in M y r m i c a rubra
15
/
\ \ \
0.2 mm
\
~t
FIG. 1. T h r e e - d i m e n s i o n a l r e c o n s t r u c t i o n o f a b d o m e n o f a M . rubra w o r k e r . L e f t h i n d q u a r t e r h a s b e e n r e m o v e d to s h o w the v e n o m a n d D u f o u r ' s g l a n d s . D G = D u f o u r ' s g l a n d ; Lv = lancet valves; P G f = v e n o m g l a n d free f i l a m e n t s ; P G r = v e n o m g l a n d reservoir; St = sting.
mately 0.5 gm forms the lining near the lumen side. It is comprised of an electron-dense epicuticle (40 nm) and an electron-lucid endocuticle that has a fibrillar appearance in its upper part (Figs. 3 - 6). Each cell contains a large, centrally located and rounded nucleus (flattened in the very thin epithelia), which possesses large quantities of dispersed heterochromatin (Figs. 3; 5; 6). The cytoplasm shows a well-developed smooth endoplasmic reticulum, numerous, scattered ribosomes, and a few mitochondria (Figs. 3; 4). The endoplasmic reticulum, which is conspicuous in young workers (Fig. 4, one-day-old), decreases considerably in old individuals, which are characterized by the appearance of numerous multilamellar inclusions (Fig. 5, old foraging worker). These inclusions represent more or less spherical bodies with an array of concentric membranes surrounding a core that may have a heterogeneous configuration. They often contain 2 or more of such onion-like figures, and can reach a diameter of up to 2 ~tm (Fig. 5). The cells are connected by septate junctions near their apical surface. Here, the lateral cell borders may form some interdigitations (Figs. 3; 5). The basal cell membrane closely adheres to the thin basement membrane, which has a thickness of approximately 40 nm. The gland i:~ surrounded by a well-developed and continuous muscle layer (Fig. 3). Nerve fibres (Fig. 6) and tracheoles may sometimes be observed around the gland.
3. Venom gland ultrastructure Secretory cells constitute the greater part of the wall of the 2 free filamentous venom glands. They appear as polygonal cells with a large, rounded nucleus that is located near the periphery of the filaments. The cytoplasm contains numerous small mitochondria and
16
JOHAN BILLEN
PGf
A
B
PGr
Nf
DG
C
c t ¸ :,
FIG. 2. General m o r p h o l o g y of 2 g l a n d s entering sting (A), and schematical r e p r e s e n t a t i o n o f cytological o r g a n i z a t i o n of secretory cells in v e n o m (B) and D u f o u r ' s g l a n d (C). ct = cuticle; D C = duct cell; D G = D u f o u r ' s gland; id = i n t r a c e l l u l a r ductule; M f = muscle fibre; N f = nerve fibre; PGf = v e n o m g l a n d free filaments; P G r = v e n o m g l a n d reservoir; SC = secretory cell.
an abundance of free ribosomes (Figs. 8; 9). However, a well-organized granular endoplasmic reticulum is rarely found. A few microtubules and small vacuoles of low electron density are scattered in the cytoplasm. Each cell is supplied with a straight or gently curved fine cuticular ductule that is surrounded by a sheath of tightly packed microvilli (Fig. 7). Its cuticular lining consists of a thin and fenestrated epicuticle and a more extensive, fibrillar endocuticle. In association with this intracellular formation, are sometimes multilamellar inclusions that may penetrate the microvillar sheath (Fig. 9). Corresponding with each intracellular ductule, is an extracellular duct that belongs to a separate duct cell, which carries the secretion of the glandular cell to the central lumen of the filament. The junction of the draining intracellular ductule and the efferent duct is characterized by a considerable change in the cuticular lining : the fibrillar endocuticle does not continue in the duct cell, whereas the narrow and interrupted epicuticle becomes much thickened and continuous prior to entering the duct cell, in which it proceeds as the only cuticular lining of the duct (Fig. 9).
FIG. 3. E l e c t r o n m i c r o g r a p h o f D u f o u r ' s g l a n d wall s h o w i n g epithelial structure and s u r r o u n d i n g muscle fibres. FIG. 4. A p i c a l c y t o p l a s m in a 1-day-old w o r k e r with well-developed, s m o o t h e n d o p l a s m i c reticulum. FIG. 5. A p i c a l c y t o p l a s m in a n old f o r a g i n g worker. Note occurrence of m u l t i l a m e l l a r inclusions and scattered free ribosomes. FIc;. 6. Very thin D u f o u r ' s gland e p i t h e l i u m with flattened nuclei, a n d s u r r o u n d i n g muscle and nerve fibres. ct = cuticle; M f = muscle fibre; M L B - m u l t i l a m e l l a r body; N = nucleus; N f = nerve fibre; SER s m o o t h e n d o p l a s m i c reticulum; sj = septate j u n c t i o n . Scale bar for Figs. 3 - 6 = 1 ~tm.
D u f o u r ' s and Venom Gland in Myrmica rubra
17
18
JOHAN BILLEN
Dufour's and Venom Gland in Myrmica rubra
19
The duct cells form a thin layer adjacent to the intima that lines the lumen of the filament. Narrow cytoplasmic strands including the cuticular ductules, which have a mean internal diameter between 0.1 and 0.15 Ixm, penetrate between the secretory cells and assure the ,;tructural link between these and the central collecting duct of the filament. The duct cells contain few organelles since they function to guide the secretion. Apart from some mitochondria and free ribosomes, they mainly contain numerous microtubules (Figs. 8; 9). The secretion collected by both free filaments finally reaches the reservoir, where it is stored until it will be released. The reservoir wall consists of a very thin epithelium of squamous cells with a thickness of nearly 0.5 ~m (Fig. 10). It is covered with a relatively thin cuticle (approx. 0.25 ~tm), in which the electron-dense epicuticle and the electronlucid endocuticle have approximately the same thickness. A distinct granular basement m e m b r a n e lines the epithelium at the hemocoel side. Muscle fibres were never observed in relationship with the venom gland reservoir or the filaments. A single nerve fibre was occasionally found near the periphery of the gland filaments.
4. The sting base and entrance of the Dufour's and venom glands Both the D u f o u r ' s and venom gland ducts curve downwards into the posterior region of the abdomen before entering the sting base. At the same time, the D u f o u r ' s gland duct assumes a narrow slit-like shape. It always occurs ventrally to the more rounded venom gland duct, in which the cuticle at the dorsal side has considerably thickened (Fig. 11). The 2 ducts open on top of a tissue bulb that nearly extends as far into the.hollow sting base as the region of the lancet valves (Fig. 1). The entrance of the ducts into the sting shows obvious changes in their epithelium and surrounding musculature (Figs. 11 - 14). On both the dorsal and ventral sides of the slitlike orifice of the Dufour's gland, are extensive muscle fibres. The ventral bundles extend to the medioventral side of the last abdominal sternite. The origin of the dorsal fibres could not be determined. The ventral part of the venom gland duct serves as the insertion area for 2 muscle bundles that have their origin on the left and right lateroventral wall of the 7th abdominal sternite, respectively (Fig. 11). A few micrometres more posteriorly, the 2 lancets appear and accomplish ventral closing of the sting by a tongue-and-groove articulation with the sting bulb and shaft. H e r m a n n (1984, Fig. 6.33, pp. 233) illustrates a similar relationship between the sting and the ducts from the venom reservoir and Dufour's gland in Paraponera clavata (Formicidae : Ponerinae). The epithelial duct cells and the muscle fibres display a distinct interdigitating aspect in their contact area. The most conspicuous characteristic of the duct cells of both the D u f o u r ' s (]Figs. 12; 13) and venom glands (Fig. 14) in the region of muscular attachment, is the large number of microtubules in the cytoplasm. These appear as well-oriented FJ(;. 7. Detail of intracellular collecting ductule of venom gland secretory cell, showing granular endocuticle, fenestrated epicuticle and surrounding sheath of micro-villi. FIG. 8. Free filament lumen and adjacent duct cells containing microtubules. FIc. 9. Junction of venom gland duct and secretory cell. Note thickening of cuticular lining when entering duct cell. Secretory cells contain numerous mitochondria and free ribosomes. Multilamellar bodies may penetrate between microvilli. FJ~. 10. Part of reservoir wall showing squamous epithelium and relatively thin cuticular lining. bm - basement membrane; ct = cuticle; DC - duct cell; ed = extracellular ductule; id = intrace]tlular ductule; M = mitochondria; MLB = multilamellar b o d y ; ' M T = microtubules; mv = microvilli; SC = secretory cell. Scale in Figs. 7 - 10 is 1 p.m.
20
JOHAN BILLEN
Fl(;. 1I. Cross-section drawing through sting base (SB) showing muscle fibres inserting on ducts of both venom (PG) and Dufour's gland (DG). bundles that extend across the cell in a direction that is parallel to the underlying muscle fibres. The considerable interdigitating contact area between the duct epithelium and muscle fibres is studded on both sides with hemidesmosomes to which the microtubules and myofilaments attach. The attachment of the epithelial microtubules to the apical cell membrane goes along with dense thickenings of the latter, that probably also correspond with hemidesmosomes. In this way, a strong contact is achieved with the cuticular lining of the duct.
DISCUSSION The use of the sting as a weapon in colony defence or prey conquest is a commonly known aspect of ant behaviour (Hermann and Blum, 1981). During the act of stinging, a venomous secretion is injected into the victim through the hollow sting shaft. The venom originates from the venom gland which, together with Dufour's gland, opens into the sting base. Also, trail substances are often emitted through the sting by periodical touching of the substrate and are equally produced by either the venom or Dufour's gland. In the c o m m o n temperate red ant Myrmica rubra, Dufour's gland appears as a thinwalled and pear-shaped sac. The gland is entirely composed of simple epithelial cells that correspond with the class 1 glandular cells according to Noirot and Quennedey (1974). The cytoplasm mainly contains a well-developed smooth endoplasmic reticulum, which turns out to be a common feature of the ant Dufour's gland (Billen, 1985). The occurrence of an elaborate smooth endoplasmic reticulum in insect pheromone-secreting glands in general, should be related to the production of small non-proteinaceous molecules, such as hydrocarbons (Noirot and Quennedey, 1974). When growing older, the M. rubra
Dufour's and Venom Gland in Myrmica rubra
21
FJ(is. 12; 13. Cross-section through Dufour's gland duct near to its opening in sting bulb. Opening (Fig. 12) and closing (Fig. 13) are result of muscular forces and cuticular elasticity, respectively, ct cuticle; hd - hemidesmosomes; Mf muscle fibres; MT microtubules. Scale - I gm.
D u f o u r ' s g l a n d is c h a r a c t e r i z e d by t h e a p p e a r a n c e o f several m u l t i l a m e l l a r i n c l u s i o n s . T h e s e , to s o m e e x t e n t , m a y by c o n s i d e r e d as c y t o l y s o s o m e s , i n d i c a t i n g c e l l u l a r d e g e n e r a t i o n ( O w e n a n d B r i d g e s , 1976; G a m a a n d D a C r u z L a n d i m , 1977; D e l f i n o et al., 1983), t h o u g h t h e y are a c t u a l l y o f u n c e r t a i n s i g n i f i c a n c e ( N o i r o t a n d Q u e n n e d e y , 1974).
22
JOHAN BILLEN
F~G. 14. Detail o f ventral part of the v e n o m g l a n d duct in region of m u s c u l a r a t t a c h m e n t . E p i d e r m a l m i c r o t u b u l e s (MT) serve in t r a n s m i t t i n g m u s c u l a r p u l l i n g force to cuticle (ct). lad h e m i d e s m o s o m e s ; M f = muscle fibres, scale - 1 ~tm.
On the other hand, their conspicuous occurrence in Dufour's gland of some Israeli formicine ants was interpreted by micellar figures containing the hydrocarbon secretion of the gland (Hefetz and Orion, 1982). Also in M. rubra, the principal Dufour's gland constituents are hydrocarbons in the C,3 to C~9 range (Morgan and Wadhams, 1972), in addition to a very volatile component comprising ethanal, acetone, and butanone (Morgan et al., 1977). Moreover, the behavioural activity of the Dufour's gland in colony defence and recruitment of nestmates, appears to be age-dependent (Cammaerts-Tricot, 1975). Young workers do not participate actively in the alarm and defence reactions of the society and possess a Dufour's gland secretion that has an attractive power for foragers of lower magnitude than the glands of older workers (Cammaerts-Tricot, 1974). Whether this behavioural evolution according to age in M. rubra is related to an increase in multilamellar bodies in the Dufour's gland cells in aging workers, and hence the interpretation of them as secretion figures, remains unanswered. The answer may require a cytochemical approach. In contrast to the very simple epithelial composition of the Dufour's gland, the venom gland has 2 free filaments containing secretory cells that open into a big, thin-walled reservoir. According to Noirot and Quennedey (1974), the secretory cells correspond to class 3. They form part of functional units, each comprising a secretory cell, an intracellular collecting ductule and a short duct cell that carries the secretion to the filament lumen. Ultrastructural information dealing with the venom gland in social Hymenoptera so far refers to descriptions in the honey bee (Owen and Bridges, 1976), the wasps Polistes gallicus (Delfino et al., 1983) and Vespa orientalis (Kanwar and Kanwar, 1975), the formicine ant Camponotus rufipes (Gama and Da Cruz Landim, 1977) and in
Dufour's and Venom Gland in Myrmica rubra
23
the Old and New World army ants (Billen, 1985). Although differences may exist in the relative proportions and general shape of the 2 filaments and reservoir, the cellular organization is quite similar following these descriptions. The occurrence of an intracellular ductule facilitates the collection of secretion that will easily cross the fibrillar endocuticle and fenestrated epicuticle. Within the duct cell, the much thickened and continuous epicuticular lining ensures a merely guiding function and prevents any loss of secretion. The cytoplasm of the secretory cells in M. rubra is characterized by an abundance of free ribosomes, but lacks a well-organized granular endoplasmic reticulum. Chemical analysis of the venom in Myrmica ants reveals a rather small proteinaceous fraction representing less than 12°70 of the total, which contains hyaluronidase with venom spreading properties. The major, non-proteinaceous fraction mainly corresponds with histamine and a large number of naturally occurring amino acids (Jentsch, 1969). The rather weak proteinaceous composition of Myrmica venom corresponds with the absence of a well-developed granular endoplasmic reticulum. The Myrmica sting, though still causing an unpleasant feeling, is much less powerful when compared with the extremely painful sti~aging from ants in the subfamilies Myrmeciinae, Ponerinae and Ecitoninae. Representatives of these groups all possess a strong proteinaceous venom (Blum and Hermann, 1978), and were all found to contain a venom gland with a very elaborate granular endoplasmic reticulum (Billen, 1985, and unpublished observations). Contrary to these more fearful species, the Myrmica venom gland also lacks any hemolytic constituenls (Jentsch, 1969). Its moderate toxicity perhaps may be related to the relatively thin cuticular lining (0.25 Ixm) of the venom gland reservoir in M. rubra. This may be compared with the more toxic species where this protective layer can reach a thickness of several micrometres (Billen, 1985). In addition to its function in secreting defensive substances, the venom gland in Myrmica ants is the source of the trail pheromone, which was identified as 3-ethyl-2, 5-dimethylpyrazine (Evershed et al., 1982). As was observed for the behavioural activity of the Dufour's gland components, the trail following behaviour in M. rubra attains its most obvious responses in old foraging workers (Cammaerts-Tricot and Verhaeghe, 1974). As !it is also these foragers that will be the first to encounter and eventually sting enemies, their venom glands are subjected to a dual function. However, the method by which the workers are able to discriminate between either trail or venom substances to be ,released from the same gland, remains an intriguing subject. It, in fact, would be most wasteful to add toxic components to odour trails or even more meaningless to attack invaders by injecting a small fraction of trail substances along with the real venom. A similar question concerns the discriminating possibility between the Dufour's and venom gland constituents, as they are both emitted through the sting. The extensive muscular supply affecting the duct region of the 2 glands clearly illustrates their separate discharginj~ abilities. It is suggested that the slit-like orifices of each gland can be opened independently by contraction of the inserting muscles, and that closure is effected passively through cuticle elasticity. In this way, the Dufour's gland duct is opened by the combined action of dorsally and ventrally occurring muscle fibres. On the other hand, the venom gland duct is only affected by ventrally inserting muscles. The lack of dorsal muscles, due to the obstruction by the sting shaft, is functionally compensated for by the extremely sclerotized and hence rigid dorsal cuticle. An additional closing muscle overlaying the venom gland duct, as described by Janet (1898), was not observed.
24
JOHAN BILLEN
The epidermal microtubules extending from the myoepidermal junction to the cuticle provide great strength and serve in transmitting the muscular pulling force to the cuticle (Lai-Fook, 1967; Caveney, 1969; Billen, 1982). However, the muscle fibres inserting on the Dufour's and venom gland ducts must be considered as a control rather than an ejection mechanism. In this respect, the discharging activity of the Dufour's gland is regulated by the combined effect of the duct musculature and the muscle fibres surrounding the reservoir sac. In the venom gland, the situation is different, because of the absence, in M. rubra, of muscle fibres around the reservoir or free filaments. According to Janet (1898), the venom should be sucked into the sting base by the movements o f the piston-like lancet valves. Although these valves have a function in pumping venom into the wound, while the lancets are simultaneously cutting it deeper (Hermann and Blum, 1966), it remains doubtful whether they alone can cause a powerful venom injection. An indirect pressure effect from surrounding tissues onto the v e n o m gland reservoir of Myrmica may occur in order to force venom from the reservoir region into the sting. Many other ant groups do possess reservoir muscles to perform this function (unpublished data). Acknowledgements--1 am very grateful for the skillful assistance of Els Plaum in preparing the microscope sections. 1 sincerely thank Dr E. J. M. Evesham for reading the original manuscript. REFERENCES BILLEN, J. P. J. 1982. The Dufour gland closing apparatus in Formica sanguinea Latreille (Hymenoptera, Formicidae). Zoomorphology 99" 235 44. BILLEN, J. 1985. Comparative ultrastructure of the poison and Dufour glands in Old and New World army ants. Actes Coll. Insectes Soc. 2: 1 7 - 26. BI uM, M. S. and H. R. HERMANN. 1978. Venom and venom apparatuses of the Formicidae : Myrmeciinae, Ponerinae, Dorylinae, Pseudomyrmecinae, Myrmicinae, and Formicinae, pp. 801 - 894. In S. BETTIN1 (ed.) A r t h r o p o d Venoms, Handb. Exp. Pharm. 48, Springer-Verlag, Berlin, Heidelberg, New York. CAMMAERTS, M.-C., E. D. MORGAN and R. TYLER. 1977. Territorial marking in the ant Myrmica rubra L. (Formicidae). Biol. Behav. 2:263 72. CAMMAERTS-TRiCOT,M.-C. 1974. Production and perception of attractive pheromones by differently aged workers of Myrmica rubra (Hymenoptera Formicidae). lnsectes Soc. 21: 2 3 5 - 48. CAMMAERTS-TRICOT, M.-C. 1975. Ontogenesis of the defence reactions in the workers of Myrmica rubra L. (Hymenoptera : Formicidae). Anita. Behav. 23: 124-30. C~,MMAERTS-TRIcCrr, M.-C. and J.-C. VERHAEGHE. 1974. Ontogenesis of trail pheromone production and trail following behaviour in the workers of Myrmica rubra L. (Formicidae). Insectes Soc. 21:275 - 8 2 . CAVENEY, S. 1969. Muscle attachment related to cuticle architecture in Apterygota. J. Cell Sci. 4:541 - 5 9 . DELHNO, G., M. T. MAmNO PICCIOLI and C. CALLONI. 1983. Ultrastructure of the venom glands in Polistes gallicus (L.) (Hymenoptera Vespidae). Monit. ZooL ltal. (N.S.) 17: 2 6 3 - 77. EVERSHED, R. P., E. D. MORGAN and M.-C. CAMMAERTS. 1982. 3-Ethyl-2, 5-dimethylpyrazine, the trail pheromone from the venom gland in eight species of Myrmica ants. Insect Biochem. 12:383 - 91. GAMA, V. and C. DA CRUZ LANDIM. 1977. Ultra-estrutura das gl~.ndulas anexas ao ferr~o de Camponotus rufipes (Fabricius) ( H y m e n o p t e r a - Formicidae). Bolm. ZooL Univ. S. Paulo 2:135 - 57. HEFETZ, A. and T. OR~ON. 1982. Pheromones of ants of Israel : I. The a l a r m - defence system of some larger Formicinae. Israel J. EntomoL 16: 8 7 - 97. HERMANN, H. R. 1984. Elaboration and reduction of the venom apparatus in aculeate Hymenoptera, pp. 201 -249. In H. R. HERMANN (ed.) Defensive Mechanisms in Social Insects. Praeger, New York. HERMANN, H. R. and M. S. BLUM. 1966. The morphology and histology of the hymenopterous poison apparatus. 1. Paraponera clavata (Formicidae). Ann. Entomol. Soc. Amer. 59: 3 9 7 - 409. HERMANN, H. R. and M. S. BLUM. 1981. Defensive mechanisms in the social Hymenoptera, pp. 7 7 - 197. In H. R. HERMANN (ed.) Social Insects, Vol. 2. Academic Press, New York. JANET, C. 1898. Sur un organe non decrit servant a la fermeture du reservoir du venin, et sur le mode de fonctionnement de l'aiguillon chez les Fourmis. C. R. Acad. Sci. 127: 6 3 8 - 41. JENTSCH, J. 1969. A procedure for purification of Myrmica venom : the isolation of the convulsive component. Proc. 6th Int. Congress 1. U.S.S.1., Bern 1969. pp. 6 9 - 7 5 .
Dufour's and Venom Gland in Myrmica rubra
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KANWAR, K. C. and U. KANWAR. 1975. Fine structure of the venom gland of Vespa orientalis. Toxicon 13: 102 -- 03. KUGLER, C. 1978. A comparative study of the myrmicine sting apparatus (Hymenoptera, Formicidae). Stud. Entomol. 20:413 - 548. Lm-FooK, 1967. The structure of developing muscle insertions in insects. J. MorphoL 123:503 - 2 8 . MORGAN, E. D. and L. J. WADHAMS. 1972. Chemical constituents of Dufour's gland in the ant, Myrmica rubra. J. Insect Physiol. 18:1125 - 35. MORGAN, E. D., R. C. TYLER and M. C. CAMMAERTS. 1977. Identification of the components of Dufour gland secretion of the ant Myrmica rubra and responses to them. J. Insect Physiol. 23:511 - 15. NomoT, C. aad A. QUENNEDEY. 1974. Fine stucture of insect epidermal glands. Annu. Rev. Entomol. 19: 61 - 80. OWEN, M. D. and A. R. BRIDGES. 1976. Aging in the venom glands of queen and worker honey bees (Apis mell~fera L.) : some morphological and chemical observations. Toxicon 14:1 - 5.
0020- 7322/86$3.00 + .00 © 1986PergamonPressLtd.
Printed in Great Britain.
MORPHOLOGY
AND ULTRASTRUCTURE
OF THE DUFOUR'S
AND VENOM GLAND IN THE ANT, MYRMICA (HYMENOPTERA
RUBRA
(L.)
• FORMICIDAE)
JOHAN B1LLEN* Limburgs Universitair Centrum, Department SBM, B-3610 Diepenbeek, Belgium and Laboratorium voor Systematiek en Ecologie, K.U.Leuven, Naamsestraat 59, B-3000 Leuven, Belgium
(Accepted 29 M a y 1985)
A b s t r a c t - - T h e morphology and fine structure of the D u f o u r ' s and venom gland, as well as their entrance into the sting, are described in the myrmicine ant, Myrmica rubra (Hymenoptera : Formicidae). The epithelial cells that constitute the D u f o u r ' s gland wall, contain a well-developed smooth endoplasmic reticulum. Older workers, compared with younger ones, show an increasing number of multilamellar inclusions. The venom gland secretory cells are arranged in 2 free filaments that carry the secretion to the reservoir. Their cytoplasm shows an intracellular collecting ductule with surrounding microvillar sheath, and an abundance of free ribosomes. However, a well-organized granular endoplasmic reticulum, which is typical in species with a more powerful sting, does not occur. Both the D u f o u r ' s and venom gland ducts are characterized by the insertion of extensive muscle fibres, which act as a precise and mutually independent control mechanism for the discharging activities of the 2 glands. Index descriptors (in addition to those in title) : Sting, colony defence, alarm pheromone, trail pheromone. INTRODUCTION
AMONG the ants of the North Temperate Zone, the red myrmicine species, Myrmica rubra (L.), has been one of the most favourite research subjects for myrmecologists. More than 100 papers have been devoted to this species. For example, it served as a model in M.V. Brian's extensive work on the reproductive biology of myrmicine ants, many behavioural analyses have been performed by M.-C. Cammaerts, while the pheromone gland chemistry was thoroughly studied by Morgan et al. (1972, 1977). Therefore, its basic biology is well understood. The Dufour's gland and venom gland secretions have been analysed very carefully, clear ethological responses being attributed to the chemical compounds from both glands. The Dufour's gland components mainly act in recruitment of nestmates and in territorial marking (Cammaerts et al., 1977), while the venom gland is the source of the trail pheromone (Evershed et al., 1982). In contrast with these fairly recent contributions, is the morphological information that goes back to Janet's descriptions of M. rubra at the turn of the century. For both the
*Senior Research assistant of the Belgian National Fund for Scientific Research. 13
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Dufour's and venom glands, our actual knowledge is restricted to one of his papers (Janet, 1898). Notwithstanding the amazing accuracy in the description of the sting structure (Kugler, 1978), no special attention has been paid to the sting associated glands themselves. For this reason and in order to supply the recent ethological studies with a morphological dimension, we report, in this paper, the histology as well as the ultrastructure of both the D u f o u r ' s and venom glands in Myrmica rubra. Since no notable differences were found between the glands of workers and queens, our description refers to the workers only unless stated otherwise. At the same time, an ultrastructural report of these glands in a myrmicine species forms part of a comparative ultrastructural study of the pheromone-producing glands in ants from several subfamilies. MATERIAL
AND
METHODS
Workers and queens were collected from nests in Alken, Belgium, and near Roermond in the Netherlands. After killing by momentary submersion in liquid nitrogen, the ants were dissected and the glands fixed in 2070 cold glutaraldehyde buffered with sodium cacodylate (0.05 M) and saccharose (0.15 M) at pH 7.3. Tissues were postfixed in 2% osmiumtetroxide and prestained with a 2% uranyl acetate solution before dehydration in a graded acetone series. Thin sections were stained with uranyl acetate and lead citrate and examined in a Philips EM 400 electron microscope. Transverse sections at 5 or 10 ~.m intervals from complete abdomens were stained with methylene blue and thionin and were used for microreconstruction of the gland region considered. In 17 workers, gland measurements were achieved by microscopic projection and accurate drawing with a curvimeter. RESULTS
1. General anatomy In M. rubra, the venom and Dufour's glands are by far the most distinct abdominal glands in both the queens and workers. The Dufour's gland is a sac-like structure measuring approximately 600 x 250 g m (Fig. 1). The reservoir is lined with a thin monolayered glandular epithelium, which occupies nearly 15% of the total volume of 0.009 m m 3 (Fig. 2). The venom gland, on the other hand, is composed of 2 free 720 _+ 80 ~m long filaments with a constant diameter of 60 _+ 7 ~tm, except for their narrower proximal-fifth near the entrance into the reservoir. The filaments comprise the glandular cells from where the secretion reaches the thin-walled reservoir (Fig. 2). The worker reservoir has a diameter of 370 _ 55 Ixm and a volume of approximately 0.02 m m 3, while it is some 30% smaller in the queen. The reservoir finally continues in the narrow venom gland duct (length nearly 300 Ixm), which opens in the sting bulb. The D u f o u r ' s gland also terminates at the sting base ventrad of the venom gland duct (Figs. 1; 11). The sting itself is 580 _+ 50 ~tm long, and essentially comprises the unpaired dorsal sting shaft and 2 ventral lancet parts. 2. DuJour's gland ultrastructure The wall of Dufour's gland consists of a unicellular layer of epithelial cells with a constant thickness ranging between 1 and 5 ~tm, according to individual ants (Figs. 2; 3). The cell shape correspondingly varies from nearly squamous (Fig. 6) to a cuboidal form, the latter being more frequently observed. A cuticular layer with a thickness of approxi-
D u f o u r ' s a n d V e n o m G l a n d in M y r m i c a rubra
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/
\ \ \
0.2 mm
\
~t
FIG. 1. T h r e e - d i m e n s i o n a l r e c o n s t r u c t i o n o f a b d o m e n o f a M . rubra w o r k e r . L e f t h i n d q u a r t e r h a s b e e n r e m o v e d to s h o w the v e n o m a n d D u f o u r ' s g l a n d s . D G = D u f o u r ' s g l a n d ; Lv = lancet valves; P G f = v e n o m g l a n d free f i l a m e n t s ; P G r = v e n o m g l a n d reservoir; St = sting.
mately 0.5 gm forms the lining near the lumen side. It is comprised of an electron-dense epicuticle (40 nm) and an electron-lucid endocuticle that has a fibrillar appearance in its upper part (Figs. 3 - 6). Each cell contains a large, centrally located and rounded nucleus (flattened in the very thin epithelia), which possesses large quantities of dispersed heterochromatin (Figs. 3; 5; 6). The cytoplasm shows a well-developed smooth endoplasmic reticulum, numerous, scattered ribosomes, and a few mitochondria (Figs. 3; 4). The endoplasmic reticulum, which is conspicuous in young workers (Fig. 4, one-day-old), decreases considerably in old individuals, which are characterized by the appearance of numerous multilamellar inclusions (Fig. 5, old foraging worker). These inclusions represent more or less spherical bodies with an array of concentric membranes surrounding a core that may have a heterogeneous configuration. They often contain 2 or more of such onion-like figures, and can reach a diameter of up to 2 ~tm (Fig. 5). The cells are connected by septate junctions near their apical surface. Here, the lateral cell borders may form some interdigitations (Figs. 3; 5). The basal cell membrane closely adheres to the thin basement membrane, which has a thickness of approximately 40 nm. The gland i:~ surrounded by a well-developed and continuous muscle layer (Fig. 3). Nerve fibres (Fig. 6) and tracheoles may sometimes be observed around the gland.
3. Venom gland ultrastructure Secretory cells constitute the greater part of the wall of the 2 free filamentous venom glands. They appear as polygonal cells with a large, rounded nucleus that is located near the periphery of the filaments. The cytoplasm contains numerous small mitochondria and
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JOHAN BILLEN
PGf
A
B
PGr
Nf
DG
C
c t ¸ :,
FIG. 2. General m o r p h o l o g y of 2 g l a n d s entering sting (A), and schematical r e p r e s e n t a t i o n o f cytological o r g a n i z a t i o n of secretory cells in v e n o m (B) and D u f o u r ' s g l a n d (C). ct = cuticle; D C = duct cell; D G = D u f o u r ' s gland; id = i n t r a c e l l u l a r ductule; M f = muscle fibre; N f = nerve fibre; PGf = v e n o m g l a n d free filaments; P G r = v e n o m g l a n d reservoir; SC = secretory cell.
an abundance of free ribosomes (Figs. 8; 9). However, a well-organized granular endoplasmic reticulum is rarely found. A few microtubules and small vacuoles of low electron density are scattered in the cytoplasm. Each cell is supplied with a straight or gently curved fine cuticular ductule that is surrounded by a sheath of tightly packed microvilli (Fig. 7). Its cuticular lining consists of a thin and fenestrated epicuticle and a more extensive, fibrillar endocuticle. In association with this intracellular formation, are sometimes multilamellar inclusions that may penetrate the microvillar sheath (Fig. 9). Corresponding with each intracellular ductule, is an extracellular duct that belongs to a separate duct cell, which carries the secretion of the glandular cell to the central lumen of the filament. The junction of the draining intracellular ductule and the efferent duct is characterized by a considerable change in the cuticular lining : the fibrillar endocuticle does not continue in the duct cell, whereas the narrow and interrupted epicuticle becomes much thickened and continuous prior to entering the duct cell, in which it proceeds as the only cuticular lining of the duct (Fig. 9).
FIG. 3. E l e c t r o n m i c r o g r a p h o f D u f o u r ' s g l a n d wall s h o w i n g epithelial structure and s u r r o u n d i n g muscle fibres. FIG. 4. A p i c a l c y t o p l a s m in a 1-day-old w o r k e r with well-developed, s m o o t h e n d o p l a s m i c reticulum. FIG. 5. A p i c a l c y t o p l a s m in a n old f o r a g i n g worker. Note occurrence of m u l t i l a m e l l a r inclusions and scattered free ribosomes. FIc;. 6. Very thin D u f o u r ' s gland e p i t h e l i u m with flattened nuclei, a n d s u r r o u n d i n g muscle and nerve fibres. ct = cuticle; M f = muscle fibre; M L B - m u l t i l a m e l l a r body; N = nucleus; N f = nerve fibre; SER s m o o t h e n d o p l a s m i c reticulum; sj = septate j u n c t i o n . Scale bar for Figs. 3 - 6 = 1 ~tm.
D u f o u r ' s and Venom Gland in Myrmica rubra
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JOHAN BILLEN
Dufour's and Venom Gland in Myrmica rubra
19
The duct cells form a thin layer adjacent to the intima that lines the lumen of the filament. Narrow cytoplasmic strands including the cuticular ductules, which have a mean internal diameter between 0.1 and 0.15 Ixm, penetrate between the secretory cells and assure the ,;tructural link between these and the central collecting duct of the filament. The duct cells contain few organelles since they function to guide the secretion. Apart from some mitochondria and free ribosomes, they mainly contain numerous microtubules (Figs. 8; 9). The secretion collected by both free filaments finally reaches the reservoir, where it is stored until it will be released. The reservoir wall consists of a very thin epithelium of squamous cells with a thickness of nearly 0.5 ~m (Fig. 10). It is covered with a relatively thin cuticle (approx. 0.25 ~tm), in which the electron-dense epicuticle and the electronlucid endocuticle have approximately the same thickness. A distinct granular basement m e m b r a n e lines the epithelium at the hemocoel side. Muscle fibres were never observed in relationship with the venom gland reservoir or the filaments. A single nerve fibre was occasionally found near the periphery of the gland filaments.
4. The sting base and entrance of the Dufour's and venom glands Both the D u f o u r ' s and venom gland ducts curve downwards into the posterior region of the abdomen before entering the sting base. At the same time, the D u f o u r ' s gland duct assumes a narrow slit-like shape. It always occurs ventrally to the more rounded venom gland duct, in which the cuticle at the dorsal side has considerably thickened (Fig. 11). The 2 ducts open on top of a tissue bulb that nearly extends as far into the.hollow sting base as the region of the lancet valves (Fig. 1). The entrance of the ducts into the sting shows obvious changes in their epithelium and surrounding musculature (Figs. 11 - 14). On both the dorsal and ventral sides of the slitlike orifice of the Dufour's gland, are extensive muscle fibres. The ventral bundles extend to the medioventral side of the last abdominal sternite. The origin of the dorsal fibres could not be determined. The ventral part of the venom gland duct serves as the insertion area for 2 muscle bundles that have their origin on the left and right lateroventral wall of the 7th abdominal sternite, respectively (Fig. 11). A few micrometres more posteriorly, the 2 lancets appear and accomplish ventral closing of the sting by a tongue-and-groove articulation with the sting bulb and shaft. H e r m a n n (1984, Fig. 6.33, pp. 233) illustrates a similar relationship between the sting and the ducts from the venom reservoir and Dufour's gland in Paraponera clavata (Formicidae : Ponerinae). The epithelial duct cells and the muscle fibres display a distinct interdigitating aspect in their contact area. The most conspicuous characteristic of the duct cells of both the D u f o u r ' s (]Figs. 12; 13) and venom glands (Fig. 14) in the region of muscular attachment, is the large number of microtubules in the cytoplasm. These appear as well-oriented FJ(;. 7. Detail of intracellular collecting ductule of venom gland secretory cell, showing granular endocuticle, fenestrated epicuticle and surrounding sheath of micro-villi. FIG. 8. Free filament lumen and adjacent duct cells containing microtubules. FIc. 9. Junction of venom gland duct and secretory cell. Note thickening of cuticular lining when entering duct cell. Secretory cells contain numerous mitochondria and free ribosomes. Multilamellar bodies may penetrate between microvilli. FJ~. 10. Part of reservoir wall showing squamous epithelium and relatively thin cuticular lining. bm - basement membrane; ct = cuticle; DC - duct cell; ed = extracellular ductule; id = intrace]tlular ductule; M = mitochondria; MLB = multilamellar b o d y ; ' M T = microtubules; mv = microvilli; SC = secretory cell. Scale in Figs. 7 - 10 is 1 p.m.
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JOHAN BILLEN
Fl(;. 1I. Cross-section drawing through sting base (SB) showing muscle fibres inserting on ducts of both venom (PG) and Dufour's gland (DG). bundles that extend across the cell in a direction that is parallel to the underlying muscle fibres. The considerable interdigitating contact area between the duct epithelium and muscle fibres is studded on both sides with hemidesmosomes to which the microtubules and myofilaments attach. The attachment of the epithelial microtubules to the apical cell membrane goes along with dense thickenings of the latter, that probably also correspond with hemidesmosomes. In this way, a strong contact is achieved with the cuticular lining of the duct.
DISCUSSION The use of the sting as a weapon in colony defence or prey conquest is a commonly known aspect of ant behaviour (Hermann and Blum, 1981). During the act of stinging, a venomous secretion is injected into the victim through the hollow sting shaft. The venom originates from the venom gland which, together with Dufour's gland, opens into the sting base. Also, trail substances are often emitted through the sting by periodical touching of the substrate and are equally produced by either the venom or Dufour's gland. In the c o m m o n temperate red ant Myrmica rubra, Dufour's gland appears as a thinwalled and pear-shaped sac. The gland is entirely composed of simple epithelial cells that correspond with the class 1 glandular cells according to Noirot and Quennedey (1974). The cytoplasm mainly contains a well-developed smooth endoplasmic reticulum, which turns out to be a common feature of the ant Dufour's gland (Billen, 1985). The occurrence of an elaborate smooth endoplasmic reticulum in insect pheromone-secreting glands in general, should be related to the production of small non-proteinaceous molecules, such as hydrocarbons (Noirot and Quennedey, 1974). When growing older, the M. rubra
Dufour's and Venom Gland in Myrmica rubra
21
FJ(is. 12; 13. Cross-section through Dufour's gland duct near to its opening in sting bulb. Opening (Fig. 12) and closing (Fig. 13) are result of muscular forces and cuticular elasticity, respectively, ct cuticle; hd - hemidesmosomes; Mf muscle fibres; MT microtubules. Scale - I gm.
D u f o u r ' s g l a n d is c h a r a c t e r i z e d by t h e a p p e a r a n c e o f several m u l t i l a m e l l a r i n c l u s i o n s . T h e s e , to s o m e e x t e n t , m a y by c o n s i d e r e d as c y t o l y s o s o m e s , i n d i c a t i n g c e l l u l a r d e g e n e r a t i o n ( O w e n a n d B r i d g e s , 1976; G a m a a n d D a C r u z L a n d i m , 1977; D e l f i n o et al., 1983), t h o u g h t h e y are a c t u a l l y o f u n c e r t a i n s i g n i f i c a n c e ( N o i r o t a n d Q u e n n e d e y , 1974).
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JOHAN BILLEN
F~G. 14. Detail o f ventral part of the v e n o m g l a n d duct in region of m u s c u l a r a t t a c h m e n t . E p i d e r m a l m i c r o t u b u l e s (MT) serve in t r a n s m i t t i n g m u s c u l a r p u l l i n g force to cuticle (ct). lad h e m i d e s m o s o m e s ; M f = muscle fibres, scale - 1 ~tm.
On the other hand, their conspicuous occurrence in Dufour's gland of some Israeli formicine ants was interpreted by micellar figures containing the hydrocarbon secretion of the gland (Hefetz and Orion, 1982). Also in M. rubra, the principal Dufour's gland constituents are hydrocarbons in the C,3 to C~9 range (Morgan and Wadhams, 1972), in addition to a very volatile component comprising ethanal, acetone, and butanone (Morgan et al., 1977). Moreover, the behavioural activity of the Dufour's gland in colony defence and recruitment of nestmates, appears to be age-dependent (Cammaerts-Tricot, 1975). Young workers do not participate actively in the alarm and defence reactions of the society and possess a Dufour's gland secretion that has an attractive power for foragers of lower magnitude than the glands of older workers (Cammaerts-Tricot, 1974). Whether this behavioural evolution according to age in M. rubra is related to an increase in multilamellar bodies in the Dufour's gland cells in aging workers, and hence the interpretation of them as secretion figures, remains unanswered. The answer may require a cytochemical approach. In contrast to the very simple epithelial composition of the Dufour's gland, the venom gland has 2 free filaments containing secretory cells that open into a big, thin-walled reservoir. According to Noirot and Quennedey (1974), the secretory cells correspond to class 3. They form part of functional units, each comprising a secretory cell, an intracellular collecting ductule and a short duct cell that carries the secretion to the filament lumen. Ultrastructural information dealing with the venom gland in social Hymenoptera so far refers to descriptions in the honey bee (Owen and Bridges, 1976), the wasps Polistes gallicus (Delfino et al., 1983) and Vespa orientalis (Kanwar and Kanwar, 1975), the formicine ant Camponotus rufipes (Gama and Da Cruz Landim, 1977) and in
Dufour's and Venom Gland in Myrmica rubra
23
the Old and New World army ants (Billen, 1985). Although differences may exist in the relative proportions and general shape of the 2 filaments and reservoir, the cellular organization is quite similar following these descriptions. The occurrence of an intracellular ductule facilitates the collection of secretion that will easily cross the fibrillar endocuticle and fenestrated epicuticle. Within the duct cell, the much thickened and continuous epicuticular lining ensures a merely guiding function and prevents any loss of secretion. The cytoplasm of the secretory cells in M. rubra is characterized by an abundance of free ribosomes, but lacks a well-organized granular endoplasmic reticulum. Chemical analysis of the venom in Myrmica ants reveals a rather small proteinaceous fraction representing less than 12°70 of the total, which contains hyaluronidase with venom spreading properties. The major, non-proteinaceous fraction mainly corresponds with histamine and a large number of naturally occurring amino acids (Jentsch, 1969). The rather weak proteinaceous composition of Myrmica venom corresponds with the absence of a well-developed granular endoplasmic reticulum. The Myrmica sting, though still causing an unpleasant feeling, is much less powerful when compared with the extremely painful sti~aging from ants in the subfamilies Myrmeciinae, Ponerinae and Ecitoninae. Representatives of these groups all possess a strong proteinaceous venom (Blum and Hermann, 1978), and were all found to contain a venom gland with a very elaborate granular endoplasmic reticulum (Billen, 1985, and unpublished observations). Contrary to these more fearful species, the Myrmica venom gland also lacks any hemolytic constituenls (Jentsch, 1969). Its moderate toxicity perhaps may be related to the relatively thin cuticular lining (0.25 Ixm) of the venom gland reservoir in M. rubra. This may be compared with the more toxic species where this protective layer can reach a thickness of several micrometres (Billen, 1985). In addition to its function in secreting defensive substances, the venom gland in Myrmica ants is the source of the trail pheromone, which was identified as 3-ethyl-2, 5-dimethylpyrazine (Evershed et al., 1982). As was observed for the behavioural activity of the Dufour's gland components, the trail following behaviour in M. rubra attains its most obvious responses in old foraging workers (Cammaerts-Tricot and Verhaeghe, 1974). As !it is also these foragers that will be the first to encounter and eventually sting enemies, their venom glands are subjected to a dual function. However, the method by which the workers are able to discriminate between either trail or venom substances to be ,released from the same gland, remains an intriguing subject. It, in fact, would be most wasteful to add toxic components to odour trails or even more meaningless to attack invaders by injecting a small fraction of trail substances along with the real venom. A similar question concerns the discriminating possibility between the Dufour's and venom gland constituents, as they are both emitted through the sting. The extensive muscular supply affecting the duct region of the 2 glands clearly illustrates their separate discharginj~ abilities. It is suggested that the slit-like orifices of each gland can be opened independently by contraction of the inserting muscles, and that closure is effected passively through cuticle elasticity. In this way, the Dufour's gland duct is opened by the combined action of dorsally and ventrally occurring muscle fibres. On the other hand, the venom gland duct is only affected by ventrally inserting muscles. The lack of dorsal muscles, due to the obstruction by the sting shaft, is functionally compensated for by the extremely sclerotized and hence rigid dorsal cuticle. An additional closing muscle overlaying the venom gland duct, as described by Janet (1898), was not observed.
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JOHAN BILLEN
The epidermal microtubules extending from the myoepidermal junction to the cuticle provide great strength and serve in transmitting the muscular pulling force to the cuticle (Lai-Fook, 1967; Caveney, 1969; Billen, 1982). However, the muscle fibres inserting on the Dufour's and venom gland ducts must be considered as a control rather than an ejection mechanism. In this respect, the discharging activity of the Dufour's gland is regulated by the combined effect of the duct musculature and the muscle fibres surrounding the reservoir sac. In the venom gland, the situation is different, because of the absence, in M. rubra, of muscle fibres around the reservoir or free filaments. According to Janet (1898), the venom should be sucked into the sting base by the movements o f the piston-like lancet valves. Although these valves have a function in pumping venom into the wound, while the lancets are simultaneously cutting it deeper (Hermann and Blum, 1966), it remains doubtful whether they alone can cause a powerful venom injection. An indirect pressure effect from surrounding tissues onto the v e n o m gland reservoir of Myrmica may occur in order to force venom from the reservoir region into the sting. Many other ant groups do possess reservoir muscles to perform this function (unpublished data). Acknowledgements--1 am very grateful for the skillful assistance of Els Plaum in preparing the microscope sections. 1 sincerely thank Dr E. J. M. Evesham for reading the original manuscript. REFERENCES BILLEN, J. P. J. 1982. The Dufour gland closing apparatus in Formica sanguinea Latreille (Hymenoptera, Formicidae). Zoomorphology 99" 235 44. BILLEN, J. 1985. Comparative ultrastructure of the poison and Dufour glands in Old and New World army ants. Actes Coll. Insectes Soc. 2: 1 7 - 26. BI uM, M. S. and H. R. HERMANN. 1978. Venom and venom apparatuses of the Formicidae : Myrmeciinae, Ponerinae, Dorylinae, Pseudomyrmecinae, Myrmicinae, and Formicinae, pp. 801 - 894. In S. BETTIN1 (ed.) A r t h r o p o d Venoms, Handb. Exp. Pharm. 48, Springer-Verlag, Berlin, Heidelberg, New York. CAMMAERTS, M.-C., E. D. MORGAN and R. TYLER. 1977. Territorial marking in the ant Myrmica rubra L. (Formicidae). Biol. Behav. 2:263 72. CAMMAERTS-TRiCOT,M.-C. 1974. Production and perception of attractive pheromones by differently aged workers of Myrmica rubra (Hymenoptera Formicidae). lnsectes Soc. 21: 2 3 5 - 48. CAMMAERTS-TRICOT, M.-C. 1975. Ontogenesis of the defence reactions in the workers of Myrmica rubra L. (Hymenoptera : Formicidae). Anita. Behav. 23: 124-30. C~,MMAERTS-TRIcCrr, M.-C. and J.-C. VERHAEGHE. 1974. Ontogenesis of trail pheromone production and trail following behaviour in the workers of Myrmica rubra L. (Formicidae). Insectes Soc. 21:275 - 8 2 . CAVENEY, S. 1969. Muscle attachment related to cuticle architecture in Apterygota. J. Cell Sci. 4:541 - 5 9 . DELHNO, G., M. T. MAmNO PICCIOLI and C. CALLONI. 1983. Ultrastructure of the venom glands in Polistes gallicus (L.) (Hymenoptera Vespidae). Monit. ZooL ltal. (N.S.) 17: 2 6 3 - 77. EVERSHED, R. P., E. D. MORGAN and M.-C. CAMMAERTS. 1982. 3-Ethyl-2, 5-dimethylpyrazine, the trail pheromone from the venom gland in eight species of Myrmica ants. Insect Biochem. 12:383 - 91. GAMA, V. and C. DA CRUZ LANDIM. 1977. Ultra-estrutura das gl~.ndulas anexas ao ferr~o de Camponotus rufipes (Fabricius) ( H y m e n o p t e r a - Formicidae). Bolm. ZooL Univ. S. Paulo 2:135 - 57. HEFETZ, A. and T. OR~ON. 1982. Pheromones of ants of Israel : I. The a l a r m - defence system of some larger Formicinae. Israel J. EntomoL 16: 8 7 - 97. HERMANN, H. R. 1984. Elaboration and reduction of the venom apparatus in aculeate Hymenoptera, pp. 201 -249. In H. R. HERMANN (ed.) Defensive Mechanisms in Social Insects. Praeger, New York. HERMANN, H. R. and M. S. BLUM. 1966. The morphology and histology of the hymenopterous poison apparatus. 1. Paraponera clavata (Formicidae). Ann. Entomol. Soc. Amer. 59: 3 9 7 - 409. HERMANN, H. R. and M. S. BLUM. 1981. Defensive mechanisms in the social Hymenoptera, pp. 7 7 - 197. In H. R. HERMANN (ed.) Social Insects, Vol. 2. Academic Press, New York. JANET, C. 1898. Sur un organe non decrit servant a la fermeture du reservoir du venin, et sur le mode de fonctionnement de l'aiguillon chez les Fourmis. C. R. Acad. Sci. 127: 6 3 8 - 41. JENTSCH, J. 1969. A procedure for purification of Myrmica venom : the isolation of the convulsive component. Proc. 6th Int. Congress 1. U.S.S.1., Bern 1969. pp. 6 9 - 7 5 .
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KANWAR, K. C. and U. KANWAR. 1975. Fine structure of the venom gland of Vespa orientalis. Toxicon 13: 102 -- 03. KUGLER, C. 1978. A comparative study of the myrmicine sting apparatus (Hymenoptera, Formicidae). Stud. Entomol. 20:413 - 548. Lm-FooK, 1967. The structure of developing muscle insertions in insects. J. MorphoL 123:503 - 2 8 . MORGAN, E. D. and L. J. WADHAMS. 1972. Chemical constituents of Dufour's gland in the ant, Myrmica rubra. J. Insect Physiol. 18:1125 - 35. MORGAN, E. D., R. C. TYLER and M. C. CAMMAERTS. 1977. Identification of the components of Dufour gland secretion of the ant Myrmica rubra and responses to them. J. Insect Physiol. 23:511 - 15. NomoT, C. aad A. QUENNEDEY. 1974. Fine stucture of insect epidermal glands. Annu. Rev. Entomol. 19: 61 - 80. OWEN, M. D. and A. R. BRIDGES. 1976. Aging in the venom glands of queen and worker honey bees (Apis mell~fera L.) : some morphological and chemical observations. Toxicon 14:1 - 5.