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2 Morphology of the Venom Apparatus JAN VAN MARLE and TOM PIEK Farmacologisch Laboratorium Universiteit van Amsterdam Amsterdam, The Netherlands

I. Morphology of the Cuticular Parts 17 II. Morphology of the Glandular Parts 20

III. Structure and Histochemistry of Venom Glands 27 References 41

I. MORPHOLOGY OF THE CUTICULAR PARTS

The embedding technique, discovered by Swammerdam, made possible his extensive studies on the chitinous parts of the sting of bees and wasps (Swammerdam, 1672-1673). He described the sting of the honey-bee worker as consisting of three parts, two of them, the lancets, moving inside the sting sheath (see Chapter 1, Fig. 3 and Table I).

The stinging apparatus of Aculeata, except that of Sapygidae, Chrysididae and Drynidae, is a transformed ovipositor, and its parts are readily identified with those of the ovipositor of other insects (Bischoff, 1927; Snodgrass, 1956). Detailed descriptions of the chitinous parts and their muscles have been given by several authors (Table I). This table shows the paucity of information on solitary Aculeata.

In the honey-bee the entire stinging organ includes two sets of parts that are anatomically and functionally distinct. One is the large basal part, which is the principal motor apparatus, the other is the long shaft, which is the piercing instrument. The sting is protracted and retracted by muscles, described by Snodgrass (1956) (Fig. 1). The mechanism of ejecting venom from the venom reservoir into the sting has been described by Arnhart (1929) and by Maschwitz and Kloft (1971). During protraction of the sting the air present in the bulb of the sting is pressed out. During retraction the venom is sucked up from the reservoir, and renewed protraction drives out the venom. These pumping movements are visible at the extracted sting apparatus.

A comparable sting apparatus has been described for the carpenter bee Xylocopa virginica by Hermann and Mullen (1974). This bee is reluctant to

17 VENOMS OF THE HYMENOPTERA Copyright © 1986 by Academic Press Inc. (London) Ltd.

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Jan van Marie and Tom Piek

Table I References to Descriptions of Structure

and Functions of the Ovipositor and the Sting of Hymenoptera

Taxa References

Symphyta

Apocrita Terebrantia

Ichneumonoidea

Cynipodea Chalcidoidea

Aculeata (Bethyloidea) (Scolioidea) Pompiloidea Spheciodea Vespoidea

Apoidea

Formicoidea

Zirngiebl (1936, 1938) Tait (1962)

Soliman (1941) Venkatraman and Subba Rao (1954) Frühauf (1923) King (1962) King and Copland (1969) Copland and King (1971, 1972a-c, 1973)

Salman (1929) Rathmayer (1962) Schlusche (1936) Rietschel (1937) Flemming (1957) Crough and Smith (1958) Hunt and Hermann (1970) Zander (1899, 1951) Rietschel (1937) Snodgrass (1942, 1956) Hermann and Mullen (1974) Dewitz (1877) Hermann and Blum (1966, 1967a,b, 1968) Hermann (1968a-c, 1969a,b) Hermann et al (1970) Blum and Hermann (1978)

sting, yet it has a well-developed venom apparatus. Hermann and Chao (1983) described the furcula, a small but important piece between muscles and sting, to play a role in the maneuverability of the sting. The furcular remnant in Formicidae intermediates between muscles and venom canal, thus altering the direction of venom dispersal (Hermann, 1983).

The above described type of venom ejection was called by H. Bischoff (personal communication to Olberg, 1959, p. 2) the valve-pump type, in contrast to the injection type. The injection type ejection of venom is realized by contraction of the venom reservoir muscles, described in the subsequent

2. Morphology of the Venom Apparatus 19

Fig. 1 Mechanism of sting protraction and retraction in the honey-bee worker. Redrawn from Snodgrass (1956). The sting is held in the retracted position (between the sheath lobes) by the muscles 196 and 199. The sting is protracted by the muscles 197 and 198 (numbers according to Snodgrass, 1956).

section. Among the Aculeata the valve type is present in Sphecidae, Apoidea and Formicidae, the injection type is present in Vespidae and Pompilidae. Most of the Terebrantia possess an injection type of venom ejection. However, some species have a pumping type, although this type seems to be different in mechanism compared with that of the aculeate wasps (H. Bischoff, personal communication to Olberg, 1959, p. 2).

The tip of the ovipositor of Terebrantia, as well as that of the sting of Aculeata, may possess a discriminating power. In Terebrantia this power may be used to discriminate between pupae that are suitable and those that are unsuitable for the development of the progeny, in Aculeata it may be used to locate the nerve centres of the prey (see Chapter 4). Within the group of the Terebrantia discrimination of hosts by the ovipositor has been described for Braconidae by Narayanan and Chaudhuri (1954), for Ichneumonidae by Wylie (1958), and for Chalcidoidea by Varley (1941).

The ovipositors of various Chalcidoidea have been examined and all have sense organs of some kind to the tip and along the ovipositor (Fulton, 1933; Varley, 1941). Salt (1937) found one of the first indications that contact chemoreceptors may exist on the ovipositor of a chalcid wasp (Trichogramma sp.). This wasp is able to distinguish between parasitized and unparasitized hosts. Dethier (1947), using techniques similar to those employed in testing tarsal chemoreceptors in insects, has demonstrated that the ichneumonid wasp Nemeritis canescens responds to chemical stimulation of the desheathed ovipositor. The similarities between his data and those obtained when several compounds were tested on gustatory receptors on the mouthparts and tarsi of other insect species suggest a common mechanism of action for all these contact chemoreceptors (Dethier, 1947).

20 Jan van Marie and Tom Piek

Pore structures were found near the tip of the sheath of the ovipositor of Pimpla sp. (Ichneumonidae), near the tip of the stylet of the ovipositor of Synergus sp. (Cynipoidea), on the ovipositors of Diastrophus sp. (Cynipoidea), and on the ovipositor of Nasonia vitripennis and Pteromalus puparum (Chalcidoidea), as well as on the stylets of the stings of Apis mellifera (Apoidea) and Vespa vulgaris (Vespoidea) (King and Fordy, 1970).

This short summary of the evidence for the presence of receptors at the tip of the ovipositor or sting shows that while data are available on the Terebrantia as well as on the social wasps, bees and ants, no data are present concerning the large group of solitary aculeate wasps, which often sting into nerve ganglia of the prey (see Chapter 4). Therefore, the interesting question of what type of receptors may be involved in finding the nerve centres remains unanswered.

II. MORPHOLOGY OF THE GLANDULAR PARTS

A review of the relationship of the venom apparatus of Hymenoptera to elements of the reproductive system in other insects has been given by Robertson (1968). Buds of the genitalia appear in the eighth and ninth abdominal sterna of third-instar larvae in the Symphyta, and of the earliest larval instar in the more highly evolved Apocrita. The two buds on the eighth sternum become the ventral valves of the ovipositor, or sting, the two outer buds on the ninth become the dorsal valves, and the remaining two the inner valves. The spermatheca is invaginated from the basis of the ventral valves of the eighth sternum, and the venom gland from the base of the inner valves of the ninth sternum. A more anterior, median invagination of the ninth sternum gives rise to the Dufour's gland. It is therefore clear that the lumen of these glands is covered with a chitin layer. Therefore, the distinction between glandular and chitinous parts is not completely correct, although generally accepted.

Figure 2 shows the reproductive organs and the venom gland of the sphecid wasp Sphex maxillosus as described by Fabre (1856). This venom gland is paired and extensively branched. The unpaired ductus venatus is partly swollen, forming the venom reservoir. Although this basic type of glandular part of the venom reservoir is found in nearly all Hymenoptera some diversity exists concerning the branching of the gland, and in some species an unpaired venom gland is found (Fig. 3). A problem may be the position of the venom reservoir in Braconidae (Fig. 3, number 3), which is not a swollen part of the unpaired ductus venatus but has the appearance of a venom gland, one among a large number of venom glands ending at a single point on the beginning of the ductus venatus (Fig. 4). A comparative morphology of the

2. Morphology of the Venom Apparatus

Fig. 2 Reproductive organs and venom gland of Fabre's 'Sphex ä ailes jaunes' (Sphex flavipennis). According to the table of concordance (Legros, 1924, p. 437), this is Sphex maxillosus F. Note the venom reservoir with the highly branched venom glands. From Fabre (1856).


Fig. 3 A selection of glandular venom apparatuses of Hymenoptera. All representatives show a venom gland, mostly paired and highly branched, and a venom reservoir. The venom reservoir is part of the ductus venatus, except in Braconidae (3). Nearly all show a second gland, the Dufour's gland, which is smaller, unpaired and not branched, except in some Apoidea (15, 16). In the Sphecoidea a third gland is frequently present (7-10). In part of the groups the venom bladder is muscular 2, 3, 4, 12, 13, 14, see also Fig. 5).

1. Emphytus tibialis (B); 2. Ichneumon lineata (B); 3. Vipio terrefactor (P); 4. Megascolia flavifrons (P); 5. Diamma bicolor (R); 6. Priocnemis variegata (B); 7. Astata boops (B); 8. Philanthus coronatus (B); 9. Crabro cephalotus (B); 10. Ammophila sabulosa (B); 11. Cerceris arenaria (B); 12. Vespa crabro (B); 13. Vespula germanica (B); 14. Polistes gallica (B); 15. Halictus leucosius (B); 16. Mechachile sericans (P); 17. Andrena pilipes (B); 18. Xylocopa violacea (Β'); 19. Bombus muscorum (B); 20. Apis mellifera (Z); 21. Camponotus pennsylvanicus (H and B). B, Bordas (1894); B', Bordas (1908); H & B, Hermann and Blum (1968); P, Pawlowsky (1914); R, Robertson (1968); Z, Zander (1951).


Fig. 4 Venom apparatus of Microbmcon hebetor (Braconidae). The central venom reservoir is surrounded by eight venom gland tubes (see also Fig. 11). The ductus venatus ends at the beginning of the ovipositor or sting, together with the Dufour's gland, also called the alkaline gland. The venom reservoir is surrounded by muscles (see also Fig. 11). After Soliman (1941).

venom apparatuses of female braconids has been described by Edson and Vinson (1979).

In Aculeata sometimes no venom reservoir seems to be present. This has been found, for example, in the wasp Ampulex compressa (Sphecidae) (see the next section and also Chapter 5, Figs. 22-25).

In the preceding section Bischof fs distinction between the Hymenoptera with a valve-pump type of venom ejection and Hymenoptera with an injection type ejection of venom was described. In the first type the driving force was realized by movements of the chitinous parts of the sting apparatus. In the latter type the driving force comes from the action of muscles present around in the wall of the venom reservoir. Figure 5 shows a few examples of muscle fibres present either around the wall of the reservoir or attached to the ductus venatus of Aculeata. In Vespidae the wall of the reservoir is covered with a muscular sheath; in some Scoliidae only a part of the venom reservoir is muscular (Pawlowsky, 1914). Figure 5(c,d) shows that muscle fibres attached at the fused duct from both venom and Dufour's gland could open this duct during ejection of venom driven out by contraction of the reservoir muscles.

In Braconidae two types of venom reservoirs were found by Edson et al


Fig. 5 Muscle fibres present in the wall of the venom reservoir (a,b) and ductus venatus (c,d) of Hymenoptera. (a) Megascolia flavifrons (Pawlowsky, 1914); (b) Vespa sp. (Pawlowsky, 1914); (c) Vespa crabro (Bordas, 1908); (d) Xylocopa violacea (Bordas, 1908).

(1982). The reservoir of type 1 (Doryctes, Coeloides, Bracon, Rogas) has a relatively thick muscular sheath which is innervated, while the longitudinal and circular muscles of the type 2 (Chelonus, Phanerotoma, Cardiochiles, Meteorus) reservoir consist of scattered fibres which are not innervated.

The venom reservoir type in Braconidae mentioned above seems to function in a manner schematized in Fig. 6. Beard (1971) described that as the muscles (not visible in the figure, see next section) contract, the reservoir shortens, the spiral structure is compressed and venom is ejected. No valves are evident to prevent backflow into the glands, but according to Beard (1971) the configuration of the gland ducts and the base of the contractible reservoir are such as to suggest that the ducts may be wholly or partially compressed at the moment of contraction. A comparable mechanism has been suggested earlier for the venom reservoir of the braconid wasps Microbracon hebetor (= Habrobracon juglandis) (Bender, 1943), and Lysiphlebus fabarum (Tremblay, 1964). However, in Stenobracon deesae, Venkatraman and Subba Rao (1954) have described a similar structure of the venom glands with a spiracular reservoir in the middle, but here the bulb at the base of the reservoir was provided with a circular valve below the openings of the venom glands. If in M. brevicornis no backflow occurs, the volume of the discharge should be equal to the relaxed volume minus the contracted volume of the reservoir. Beard (1971) measured in the venom reservoir of M brevicornis the helical lining of the cavity and estimated the volume at 1 pi. As extirpated reservoirs were very refractory to stimuli that might induce contraction, Beard (1971) had to speculate from insufficient evidence that the compressed volume could be in the range of 0.6 pi, giving a tidal volume of about 0.4 pi, if contraction


Fig. 6 Schematic representation of contraction and relaxation of the venom reservoir of Microbracon brevicornis working as an automatic syringe. When the bulb muscles (not shown in the figure) contract, the cavity shortens, the spiral is compressed and venom is ejected. No valves are evident to prevent backflow into the glands, but the configuration of the gland ducts and the base of the contractile reservoir are such as to suggest that the ducts may be wholly or partially compressed at the moment of contraction, vg, Venom gland; vr, venom reservoir. After Beard (1971). See also Fig. 4.

is maximal. Hase (1924) reported that the volume of single droplets on the sting of M hebetor was 0.3 pi. According to Beard, M brevicornis can sting and paralyse as many as 100 larvae of the moth Plodia interpunetella per day with a mean of 40, and as many as 1700 per lifetime with a mean of ~ 900. This is accomplished by sustained synthesis of venom at a turnover rate of about eight times the residual volume per day. The total venom produced by the wasp in its lifetime amounts to only 0.10-0.25 μΐ.

In some species of Andrenidae (Apoidea) the venom reservoir seems to have also a muscular wall, but in the majority of groups of Apoidea it is nonmuscular (Robertson, 1968). However, Bridges (1979) reported that secretory cells present around the proximal third of the venom reservoir of the honey-bee worker, Apis mellifera (Fig. 7), are surrounded by a fine muscle reticulum.

In the worker of honey-bees the venom gland is a long, thin, distally bifurcated, integumentary gland with a cuticular lining. Secretory cells, duct-forming and squamous epithelial cells are found along the length of the gland, and additional secretory cells are also present in the reservoir (Fig. 7). A comparable ultrastructure of the venom gland of the chalcidid wasp Nasonia vitripennis has been described by Ratcliffe and King (1967). The presence of secretory cells in part of the venom reservoir is not restricted to the above-described Hymenoptera. Figure 8 shows the distribution of secretory elements


Fig. 7 Diagrammatic representation of the venom gland of a worker of the honey-bee, Apis mellifera. From Owen and Bridges (1976).

among the parts of the venom apparatus of a number of Hymenoptera. Secretory cells seem to be present frequently in those parts of venom reservoirs which are adjacent to the true glands. In the braconid wasp Vipio terref actor even the ductus venatus contains secretory cells (Pawlowsky, 1914).

Fig. 8 Distribution of secretory elements (dots) of venom apparatuses of (a) Mechachilae sericans (Apoidea: Mechachilidae); (b) Megascoliaflavifrons (Scolioidea); (c) Vespa sp. (Vespidea); (d) Vipio terref actor (Ichneumonoidea: Braconidae); (e) Camponotus pennsylvanicus (For-micoidea). (a-d) From Pawlowsky (1914); (e) from Hermann and Blum (1968).


III. STRUCTURE AND HISTOCHEMISTRY OF VENOM GLANDS

The venom apparatus of the Hymenoptera develops from an invagination of the ninth abdominal sternum (Robertson, 1968). Consequently, from an anatomical point of view the lumen of both the venom glands and the venom reservoir are considered exterior; moreover the whole lumen of the venom apparatus is lined with a cuticular intima. Since secretion of the venom by the secretory cells of the glands and by the secretory cells of the venom reservoir, if present, takes place through this cuticular lining, it necessitates an adaptation of the ultrastructure of the secretory cells. In a cross section of the venom gland three layers can be distinguished: around the lumen of the gland a cuticular lining is present; next to this lining the ectodermal cells are situated, by which the cuticle is secreted; and on these ectodermal cells

Fig. 9 Diagram of the wall of a venom gland in cross section. BM, basement membrane; C, cuticula; ect, ectodermal cell; G, Golgi apparatus; Lu, lumen; mit, mitochondrion; S, secretory cell; V, vacuole; Vo, vesicular organeile; RER, rough endoplasmic reticulum. Arrow, cuticular funnel.

the secretory cells are situated (Fig. 9). In most cases the ectodermal cells are small and atrophied.

From the lumen of the venom gland or the venom reservoir small chitinous funnels run to each secretory cell, ending in a vesicular organelle. This vesicular organelle forms a space inside the secretory cell lined with microvilli, in which the venom is secreted (Fig. 9). Shape and size of this vesicular organelle are dependent on the nutritional state of the animal (Ratclif fe and King, 1970) and on the age of the secretory cells (van Marie, 1977). After secretion of the venom in the vesicular organelle the venom will reach the lumen of the gland or reservoir via the funnel. This morphological


specialization enabling secretion through the cuticle is not unique for secretory cells belonging to the venom apparatus but is found wherever secretion has to take place through the cuticula (Smith, 1968). Ultrastructurally, the secretory cells of the venom glands and venom reservoir are similar to class 3 gland cells described by Noiret and Quennedey (1974).

Notwithstanding the extensive information available as regards the anatomy of the venom apparatus of Hymenoptera, little information can be found concerning its histology and ultrastructure. Consequently any form of generalization must be treated with prudence.

It appears that in Hymenoptera two types of venom gland are present. Type A contains secretory cells which do not change during the life span of the wasps. This type seems to be present only in the venom apparatus of some Ichneumonoidea (King and Copland, 1969; Ratcliffe and King, 1969; van Marie, 1977; Edson et al., 1982). Type B consists of secretory cells with an ultrastructure changing with the age of the cells. In these venom glands every stage of development may be found, ranging from young undeveloped cells via actively secreting to old pycnotic cells. One example is the venom gland of a wasp belonging to the Chalcidoidea, Pteromalus puparwn, in which all cells progress synchronously through the various stages of development (Fig. 10). This type of secretory cell is also found in the venom glands of other groups of the Hymenoptera investigated (Kanwar and Sethi, 1971; Owen and Bridges, 1976; Bar-Nea et al., 1976; van Marie, 1977).

In type A venom glands as present in Microbracon hebetor (van Marie, 1977), in the Braconidae investigated by Edson et al. (1982), in Nasonia vitripennis (Ratcliffe and King, 1969) and perhaps in Mymaridae (King and Copland 1969), the secretory cells are large and cuboidal, containing a well-developed secretory organelle. Light microscopically, no structures can be observed in the strongly basophilic cytoplasm except the nucleus and the secretory organelle (Fig. 11). All cells present in the gland have the same ultrastructural appearance (Fig. 12). The ultrastructure of the cells is dominated by an extensive rough endoplasmic reticulum (RER) either arranged in stacks or in swollen vesicles. The cells contain numerous Golgi complexes and small mitochondria. In the vicinity of the Golgi complexes large numbers of small vesicles are present. In some species (N. vitripennis, Ratcliffe and King, 1969) the vesicles are filled with electron-dense secretory material, in others the secretory material is not electron-dense (van Marie, 1977; Edson et al., 1982). Sometimes vacuoles may be present (Edson et al., 1982). The small vesicles or vacuoles filled with secretory material coalesce with the lumen of the secretory organelle which becomes filled with secretory material.

Electron microscopically, two cell types can be distinguished in Microbracon

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hebetor, reflecting the state of metabolic activity (van Marie, 1977), cells with a dark cytoplasmic matrix (the metabolically active cells) and cells with a light cytoplasmic matrix (the metabolically inactive cells). The light or dark appearance of the cells is the only difference present between the secretory gland cells inM hebetor. In the whole lifespan of M hebetorthe ultrastructure of the venom glands remains unchanged. No generative changes are observed. Neither is the ultrastructure of the venom glands affected by a prolonged stinging behaviour (van Marie, 1977). However, starvation seriously affects the ultrastructure of the secretory cells, resulting in degenerative changes. The amount of organelles is significantly reduced. Especially in the vesicular organelle the size and number of microvilli is markedly decreased (Ratcliffe and King, 1970). In M. hebetor some indication exists that not all the venom glands produce the same secretion (Fig. 1 lb). Within two of the eight venom glands a component is present which is extractable with chloroform; however, the secretory cells of these venom glands do not differ ultrastructurally from the cells of the six remaining glands (van Marie, 1977). In the venom apparatus of two of nine braconid wasps investigated electron microscopically (Edson et al., 1982) distinct particles were observed. Rod-shaped particles, 40 nm in diameter and 120 nm in length and irregularly-shaped particles -200 nm in diameter were observed in Biosteres longicaudatus Ash. The venom apparatus of Meteorus leviventris Wesmael contains hexagonal particles ~ 50 nm in diameter and ovoid particles measuring 110 x 170 nm. The viral origin of these particles has not been established with certainty.

Type B venom glands, as far as can be generalized from a preliminary and consequently limited survey, were found to be present in all groups of the Hymenoptera except some Ichneumonoidea. They are observed in Pteromalus (Chalcidoidea), Campsomeris and Scolia (Scolioidea) and Ampulex (personal observations), as well as in Philanthus (Sphecoidea), in Vespa (Vespoidea) and Apis (Apoidea) (van Marie, 1977; Bar-Nea, 1976; Kanwar and Sethi, 1971; Owen and Bridges, 1976).

Compared to the uniform appearance of the secretory cells of the venom glands of the braconid type, this type presents a varied light microscopical and ultrastructural aspect. Under the light microscope the cells appear filled with vacuoles, sometimes containing eosinophilic or basophilic material (Fig. 13). In the glands one secretory cell type develops during a relatively short

Fig. 11 Venom apparatus of Microbracon hebetor Say. (a) Cross-section showing the muscular reservoir and its eight surrounding venom glands; (b) details of both types of venom glands. Gl, gland with secretory material of normal appearance; G2, gland with secretory material containing a chloroform-extractable component, (c) Detail of the venom reservoir with gland cells between the muscles (arrows). G, venom glands; Lu, lumen; R, reservoir. Small arrows, cuticular lining of the reservoir; arrow heads, vesicular organelle. Scale: 25 μτη.


period from an inactive undeveloped cell to a functionally active secretory cell. After an active period this cell degenerates and what is left are small dark remains without recognizable ultrastructure. In Philanthus triangulum these degenerated cells remain present in the venom glands (van Marie, 1977). In Apis mellifera they are perhaps actively phagocytized (Autrum and Kneitz, 1959; Cruz Landim et aL, 1967).

Aside from the nucleus and a prominent vesicular organelle, the young undeveloped cells contain only a few mitochondria and a sparse fragmentary RER. In the course of development the cells become filled with RER and the cytoplasmatic matrix turns increasingly darker. Vacuoles appear to be filled with flocculent secretory material of varied electron density. These vacuoles fuse with the vesicular organelle. This fusion is accompanied by a progressive disappearance of the large numbers of microvilli that are present in the young undeveloped cells. Subsequently, a large space filled with secretory material develops around the chitinous funnel which connects the vesicular organelle with the lumen of the venom gland. This way of secretion is also described in the silk glands of Bombyx mori (Matsuuri and Tashiro, 1976).

With increasing age the cells become progressively darker and diminish in size. They become filled with whorled figures and finally they no longer contain any recognisable ultrastructure. The degenerated remains either stay in the glands or are phagocytized (Owen and Bridges, 1976; van Marie, 1977). This development on the secretory cells of the venom glands may be asynchronous (i.e. in Philanthus and in Apis) or synchronous (i.e. in Pteromalus). In the first case cells in various stages of development can be observed (Fig. 14); in the second case all cells have the same structural appearance (Fig. 10).

A venom reservoir is not present in all Hymenoptera. In Ampulex compressa, for instance, after fusion of the two lateral venom glands the ductus venatus maintains the aspect of a venom gland. The secretory cells disappear distally from the point where the duct of the alkaline gland meets the ductus venatus. From this point to the orifice of the ductus venatus close to the sting the wall of the venom duct consists of a cuticular intima and a squamous ectodermal epithelium. No muscles are present around or attached to the venom glands or the ductus venatus.

In only a few instances in the literature has the fine structure of the venom gland and venom reservoir been studied in some detail. The species studied

Fig. 12 Cross-section through a venom gland of Microbracon hebetor Say with light (L) and dark (D) cells. In both cell types a vesicular organelle (Vo) is present. Lu, lumen of the gland; N, nucleus. Arrow, ectodermal cells and cuticular lining of the lumen of the gland. Scale: 0.5 /im.


Fig. 13 Cross-section through the venom glands of the sphecid wasps Philanthus triangulum L. (a) and Ampulex compressa (b). The secretory cells of both venom glands are vacuolated. Around the lumen the cuticular lining is visible (arrows). In A. compressa the small nuclei of the ectodermal cells surrounding this lining are easily distinguishable. Scale: 25 μτη.


demonstrate such variety in structure and composition of the venom reservoir that any form of generalization is impossible. If present, the venom reservoir can be considered as a specialization of the wall of the venom duct (except in Braconidae with a type 1 venom apparatus). In the Braconidae two types of venom apparatus can be distinguished (Edson et al., 1982). They do not differ in ultrastructure of the venom glands, which are all of the same braconid type; however, they do differ in the structure of the venom reservoir. Type 1 reservoirs are built essentially like a hollow muscle (Fig. 11). On a cuticular lining longitudinally oriented muscles are attached. Between these muscle fibres secretory cells are present which are similar in ultrastructure to the cells of the venom gland (Fig. 15). In Microbracon hebetor these cells seem to be metabolically inactive (van Marie, 1977); in other braconid species they present a metabolically active ultrastructural appearance (Edson et al., 1982).

Unlike the muscles of reservoir type 2, the muscles of the reservoir type 1 are innervated. The synapses contain small vesicles (Edson et al., 1982) as well as large neurosecretory granules (personal observation). The cuticular intima of the type 1 reservoir shows a prominent lining. This thickening may be circular or spiral (Beard, 1971; van Marie, 1977; Edson et al., 1982). It has been suggested that the lined intima acts antagonistically towards the muscles of the reservoir. The walls of type 2 reservoirs consist only of squamous ectodermal cells and cuticular lining. The lining has no thickening; throughout the reservoir it is evenly shaped (Edson et al., 1982). Between the squamous cells secretory cells can be observed. Muscle fibres are present around type 2 reservoirs, but nowhere are they attached to the wall. The muscles together with tracheoles and trachea end cells form a sheath around the reservoir. The muscles do not seem to be innervated.

The venom reservoir of Nasonia resembles the reservoir type 2 of the Braconidae insofar as that a large part of it consists of a cuticular intima without any structure and with a squamous ectodermal epithelium. However, it has a specialized area around the entrance of the duct of the venom gland (Ratcliffe and King, 1969). The venom duct divides radialy in fine ductules. After some distance these ductuli divide again and form a complex system of branched and interconnected ductules. The ductuli end in the venom reservoir. There are secretory cells situated in between the ductules. The basal part of these cells is invaginated, indicated an active uptake from the haemolymph. A continuous muscle sheath surrounds the venom reservoir.

The venom reservoir of Philanthus triangulum consists for the greatest part of a smooth cuticular intima with a squamous ectodermal epithelium. Only around the entrance of the venom duct are secretory cells present (Fig. 16). These cells are of the same type as the cells of the glands, probably at various stages of development and metabolic activity. Like the secretory cells of the


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Fig. 14 A cross-section through the venom glands of Philanthus triangulum L. showing young undeveloped cells (a) and cells at various stages of development and degeneration (b). The darker the cells the more degenerated and older they proved to be. Lu, lumen; N, nucleus; Vo, vesicular organeile; T, trachea. Arrow, cuticular lining and ectodermal cells. Scale: 0.5 /mi.


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40 Jan van Marie and Tom PieK

venom reservoir of Nasonia vitripennis, the secretory cells of the venom reservoir of P. triangulum are invaginated at their basal part (van Marie, 1977). The venom reservoir of P. triangulum is surrounded by a thin muscular sheath. However, this sheath is nowhere attached to the wall of the reservoir. It fuses with the muscles around the ductus venatus. Considering its fragility, it cannot be assumed that this muscle sheath is responsible for the extrusion of venom after stinging by the wasp (van Marie, 1977).

The venom reservoir of the honey-bee (Apis mellifera) is partly enveloped in a similar muscular sheath (Bridges, 1979; Bridges and Owen, 1984). A better developed musculature is found attached to the venom reservoir of Scolioidea and Vespoidea. These muscles evidently are involved in the extrusion of the venom (Bar-Neaef fl/., 1976; Bordas, 1894; Pawlowsky, 1914; Robertson, 1968).

In some species the venom gland and venom reservoir have been investigated histochemically to detect the presence of fat, proteins, (muco)poly-saccharides, RNA, etc., including Nasonia vitripennis (Ratcliffe and King, 1969); Microbracon hebetor and Philanthus triangulum (van Marie, 1977); Apis mellifera (Owen, 1971); and Vespa orientalis (Bar-Nea etal., 1976; Kanwar and Sethi, 1971). In the few species investigated a strong positive reaction was observed in the secretory cells if stained for RNA. This is in agreement with the electron microscopic findings of an abundant RER in the secretory cells. In M. hebetor no differences were observed between the secretory material in the venom gland and in the venom reservoir. Although it appeared that two out of eight venom glands contain a chloroform-extractable fat component (Fig. 11) it was not possible to make this component visible in any other way (van Marie, 1977). The secretory material in the venom reservoirs of N. vitripennis reacts much more with reagents specific for protein than does the secretory material in the venom glands. It has been suggested that the secretory cells of the venom reservoir are involved in this protein secretion (Ratcliffe and King, 1969). In the venom reservoirs of the honey-bee and some wasps (Vespula arenaria and V. maculata), dopamine and nor adrenalin have been demonstrated using the formalin-induced fluorescence technique (Owen, 1971; Bridges, 1980). No venom apparatuses of other species have been investigated using this technique.

More or less extensive histochemical investigations have been made on the venom apparatus of Philanthus triangulum, Nasonia vitripennis, Microbracon hebetor and Vespa orientalis. However, it seems impossible to characterize histochemically the secretory material present in the venom reservoir or in the venom gland of the species investigated. The histochemical results closely resemble the results obtained with other tissues of the animals investigated.


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