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Structure and sensory equipment of the ovipositor of Habrobracon hebetor (Say) (Hymenoptera: Braconidae)
Micron 39 (2008) 1255–1261
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Structure and sensory equipment of the ovipositor of Habrobracon hebetor (Say) (Hymenoptera: Braconidae) Hany K.M. Dweck *, Neveen S. Gadallah, Essam Darwish Department of Entomology, Faculty of Science, Cairo University, Giza, Egypt
A R T I C L E I N F O
A B S T R A C T
Article history: Received 15 December 2007 Received in revised form 30 March 2008 Accepted 31 March 2008
The microsculpture of various structures of the ovipositor of Habrobracon hebetor (Say) (Hymenoptera: Braconidae) is described from scanning and transmission electron microscopy. These include: the ovipositor egg canal, valvillus, seal of the first valvulae, interlocking mechanism (olistheter) connecting the first and second valvulae, an olistheter-like interlocking mechanism connecting the two pieces of the first valvulae, annulation, microtrichia of the third valvulae, and the ovipositor sensory equipment. Better understanding of the microsculpture of these components may make their roles in stinging, oviposition, and the host selection process more clear. ß 2008 Elsevier Ltd. All rights reserved.
Keywords: Microsculpture Valvillus Seal Olistheter Annulation Microtrichia
1. Introduction The insect ovipositor is a complex structure consisting of up to seven interlocking sclerites associated with the 8th and 9th abdominal segments in insect females (termed the genital segments). Female insects use the ovipositor to deposit eggs in specific locations such as deep within the soil, in plant tissues, under the bark of trees, and even within the bodies of other insects. The basic organization of female genitalia shows a remarkable uniformity among Hymenoptera. It consists of two pairs of valvifers and three pairs of valvulae derived from the 8th and the 9th abdominal segments (LeRalec and Wajnberg, 1990). The first valvifers (gonocoxites VIII) are continuous with the rami of the first valvulae (gonapophyses VIII). The second valvifers (gonocoxites IX) extend as the third valvulae (gonostyli) and ventrally bear the fused second valvulae (gonapophyses IX). The interlocked first and second valvulae form the shaft of the ovipositor and enclose the egg-canal. The ovipositor of parasitic Hymenoptera is a specialized organ with which the female probes and drills the substrate where a host lives, pierces the integument of the host, injects the substances from the accessory glands, perceives stimuli involved in the host
* Corresponding author. Tel.: +20 25676826; fax: +20 25728843. E-mail address: [email protected] (Hany K.M. Dweck). 0968-4328/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2008.03.012
selection process, guides and deposits eggs (van Lenteren, 1981; Quicke et al., 1995). Hosts attacked by these parasitoids are diverse. Members of most pterygote orders, of all developmental stages, fixed or mobile, that are exposed or concealed in different substrates, can be parasitized. This wide range of situations produces a diversity of constraints to which the ovipositor must be adapted. Therefore, morphological and functional features of the ovipositor should vary with host diversity. Variation in ovipositor structure has been used for classification of parasitoids, for clarifying phylogenetic relationships (e.g., Austin and Field, 1997) and, recently, in several cases to relate form with function (Field and Austin, 1994; LeRalec et al., 1996; Quicke and Fitton, 1995; Quicke et al., 1995). Habrobracon hebetor is a gregarious, idiobiont (one that prevents any further development of the host after initial parasitization), larval ectoparasitoid of pyralid moths. The use of H. hebetor for biological control of stored product moths has been well documented in the literature (Keever et al., 1986; Press et al., 1982; Scho¨ller and Prozell, 2001). The advantage of H. hebetor as a biological control agent for stored product moths in retail stores and warehouses is that it attacks the wandering larval stages of the moths and may serve to reduce secondary infestation of packaged goods (Cline et al., 1984; Cline and Press, 1990). The purpose of the present work is to describe structure and sensory equipment of the ovipositor of H. hebetor (Say) (Hymenoptera: Braconidae) in order to better understand their roles in stinging, oviposition, and the host selection process.
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2. Materials and methods 2.1. Stock culture of parasitoids The Mediterranean flour moth, Ephestia kuehniella and the ectoparasitoid, H. hebetor used in the present study were collected from Cairo flour mills (Egypt). A laboratory culture of E. kuehniella was reared in glass jars on whole-wheat flour previously heated in order to ensure the absence of any infestation. The stock culture of the parasitoid H. hebetor was reared on 25–30-day-old host larvae in cylindrical glass jars (10 cm 17 cm) containing infested flour. The parasitoids and their hosts were maintained at 28 1 8C and 60 10 RH with a photoperiod of 14:10 (L:D). 2.2. Light microscopy Ovipositors were dissected from specimens using fine forceps, gradually dehydrated through a series of ethyl alcohol (20, 50, 70, 80, 90, 95, 100% ethanol), then held in 100% xylene for 48 h and mounted on glass slides in Canada balsam for examination using a Cambridge light microscope. Measurements of the different parts of the ovipositor were made using an ocular micrometer and were taken from 50 individuals. 2.3. Scanning electron microscopy The dissected parts were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3) for at least 4 h at 4 8C, washed in 0.1 M phosphate buffer (overnight), immersed in a postfixative of 1% buffered osmium tetroxide for 1 h at 4 8C and then gradually dehydrated in acetone, critical point dried in an Autosamdri1-815, and finally sputter coated with gold in a SPI-ModuleTM Sputter Coater. Examination and photography were done using Philips ALx 30 and Jeol JSM-5200 scanning electron microscopes at different accelerating voltages. Images were then recorded digitally on a computer. Measurements (in mm) were obtained from photographs of at least 50 individual sensilla of the same type and were used to calculate means. 2.4. Transmission electron microscopy Different parts under examination were fixed in 2.5 glutaraldehyde in 0.1 M phosphate buffer (pH 7.3) for at least 4 h at 4 8C, briefly washed in 0.1 M phosphate buffer (overnight), and postfixed in 1% osmium tetroxide in 0.1 M phosphate buffer for 1 h at 4 8C. After dehydration in a graded series of acetone, they were embedded in Epon-Araldite. Thin sections were cut with a glass knife using a Reichert-Jung Ultracut E, collected on colloid grids, contrasted with uranyl acetate and lead citrate, and finally observed in a Jeol JEM-1200 EX II. Semi-thin sections were stained with toluidine blue (1%) and observed under a Cambridge light microscope. Drawings were then made using a camera lucida.
Fig. 1. Ovipositor of Habrobracon hebetor (lateral aspect); 40. B = bulb; T9 = tergite 9; V1, V2, and V3 = first, second, and third valvulae; Vf1, Vf2 = first and second valvifers.
and second valvulae. When held together, these structures form internal lumina that serve as passageways for eggs and/or venom. The paired first valvulae (=lower valves of Quicke et al., 1999) are separated from each other along most of their length. The posterior tip of each first valvula has barbs or serrations on its lateral side (Fig. 2). The second valvulae (=upper valves of Quicke et al., 1999) are fused along their entire length and are expanded proximally to form an enlarged bulb. Near their apex, the second valvulae are somewhat enlarged and slightly decurved, bearing no barbs or serrations. The first and second valvulae are joined together by an interlocking mechanism (=olistheter of Quicke et al., 1999) (Figs. 3b–d and f and 4a and b). Each olistheter consists of a projected tongue (rhachis) (Fig. 5a) that is vertically positioned on the ventral wall of each second valvula in H. hebetor. Each rhachis fits into a groove (aulax) (Fig. 6b) in the dorsal wall of each first valvula, but this interlocking mechanism does not extend to the apex of the ovipositor (Fig. 3a). Close to the apex of the ovipositor shaft, the two first valvulae become interlocked dorsally by a mechanism similar to the olistheter (Figs. 3a–d and 4a). A single distinct valvillus (Quicke et al., 1992) is situated on the inner surface of the distal third of each first valvula (Fig. 6a). This
3. Results 3.1. Ovipositor morphology The female genitalia of H. hebetor exhibits the basic organization typically seen in Hymenoptera (Fig. 1) (Smith, 1970). The ovipositor shaft is on average 2.38 0.13 mm long (about half of adult body length) and 0.07 0.01 mm thick (n = 50). It is a rigid, yet flexible, structure that ends in a pointed tip and consists of the first
Fig. 2. Interlocked first (V1) and second (V2) valvulae of ovipositor of H. hebetor just proximal to ovipositor tip. Ca = campaniform sensilla; T = teeth.
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Fig. 3. Diagram of serial transverse sections from distal end of ovipositor of H. hebetor: (a–f) distal to proximal sections. A = aulax; EC = egg canal; L = lumen; R = rhachis; S = mid-longitudinal septum; V1, V2 = first and second valvulae.
valvillus is obviously concave and the position of the valvillar insertion in the egg canal was found to be approximately medial. There is a thin longitudinal cuticular flap that forms a seal (Quicke et al., 1994) by protruding into the egg or venom canal from the medioventral part of each first valvula (Fig. 4b). The surface of the egg canal within the ovipositor of H. hebetor is not smooth but possesses complex microsculpture consisting of ctenidia and subctenidia. The ventral wall in the proximal portion of the second valvulae has well-developed fine projections across most of the surface between the rhachises (Fig. 5b). Rows of these posteriorly-directed ctenidia are also located on the inner surface of each first valvula both anterior and posterior to the valvillus (Fig. 6b). Another distinct type of setiform structure (=subctenidial setae of Rahman et al., 1998) was found associated with those ctenidia posterior to the valvillus (Fig. 6c).
The third valvulae (=the ovipositor sheath of Quicke et al., 1999) are one segmented and are fleshy, ridged and annulated externally in the basal two thirds of their length (Fig. 7a), but smooth in the distal third (Fig. 7b). The third valvulae unsheath both the first and second valvulae when they are not in use. The inner surface of the third valvulae is almost entirely covered with very dense, flexible cuticular microtrichia (=cuticular spines of Ne´non et al., 1997) (Fig. 7c). The density of the microtrichia varies along the length of the third valvulae, the tip usually having the highest concentration. 3.2. Sensory equipment Three morphologically distinct types of sensilla have been identified on the ovipositor shaft, third valvulae, and second valvifers of the ovipositor of H. hebetor from observations utilizing
Fig. 4. Transverse sections of ovipositor of H. hebetor: (a) distal and (b) medial. A = aulax; C = ctenidia; EC = egg canal; L = lumen; R = rhachis; Sl = seal; V1, V2 = first and second valvulae.
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Fig. 5. Ventral wall of second valvula of ovipositor of H. hebetor: (a) distal end and (b) proximal end. C = ctenidia; R = rhachis; S = protruding margins of mid-longitudinal septum.
SEM and TEM. These types of sensilla will each be addressed individually. 3.2.1. Styloconic sensilla Styloconic sensilla are present in two distinct groups. The first group (5.97 1.19 mm in shaft length) is arranged linearly (12–18 sensilla in number) on each second valvifer at the point where it articulates with the first ramus (Fig. 8a). The second group of styloconic sensilla (1.4 0.14 mm in shaft length) is present in the form of a hair plate (12–18 sensilla in number) at the point of articulation between each first and second valvifer (Fig. 8b). 3.2.2. Trichoid sensilla Two types of trichoid sensilla are found covering the entire external surface of the third valvulae. The first type, long trichoid sensilla, is distributed along the entire length of the third valvulae (Fig. 7a and b). They are inserted in sockets and each external process (54.4 32.4 mm in length) has slight longitudinal grooves and gradually tapers to a sharp point. The cuticle of this sensillum exhibits a thick and non-porous wall,
which is not innervated by dendrites, as seen in the TEM reprint (Fig. 9b). The second type of trichoid sensilla, short trichoid sensilla, is similar to the first type except that they are located between those of the first type in the distal end of the third valvulae and are shorter in overall length than those of the first type (10.45 4.45 mm in length) (Fig. 7b and 9a). 3.2.3. Campaniform sensilla Campaniform sensilla, with an irregular and depressed cuticular structure (Fig. 2), are found along the ovipositor shaft. However, they are found more concentrated at the tip of the second valvulae (Fig. 10). This sensillar type is innervated by a single dendritic segment which forms a tubular body just before entering the valvula cuticle (Fig. 11). 4. Discussion Most of the ovipositor structures examined in the present work show a correlation between their morphological characteristics and their function. Several of these will now be discussed.
Fig. 6. Dorsal wall of first valvula of ovipositor of H. hebetor: (a) valvillus near distal end, (b) posterior to valvillus and (c) anterior to valvillus. A = aulax; Ct = ctenidia; STS = subctenidial setae; white arrow = valvillus.
Fig. 7. Third valvula of ovipositor of H. hebetor: (a) lateral wall, (b) tip and (c) median wall. Mi = microtrichia; LST = long trichoid sensilla; SST = short trichoid sensilla.
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Fig. 8. Second valvifer (Vf 2) of ovipositor of H. hebetor: (a) at articulation point with first ramus and (b) at articulation point with first valvifer. SS = styloconic sensilla.
Fig. 9. Transverse sections of the proximal portions of (a) short and (b) long trichoid sensilla. Note the thick and groove non-porous sensillum wall (SW) and the sensillum lymph (SL), which is not innervated by dendrites.
4.1. Stinging function As a female H. hebetor mounted its host by using its antennal sensory receptors (Dweck and Gadallah, 2008), her abdomen was bent forward, the ovipositor was unsheathed and stinging of the host took place. Stinging by this species is a mechanism used to puncture the host’s integument in order to acquire a meal of hemolymph and to inject venom into the host larva. Two to three
Fig. 10. Tip of second valvulae of ovipositor of H. hebetor showing campaniform sensilla (Ca).
stings were observed by this female. Duration of a sting usually lasts from 2 to 3 min (Darwish et al., 2003). Stinging results in paralysis of motor function which facilitates oviposition by the wasp. The barbs or serrations present at the distal end of each first valvula on the ovipositor of H. hebetor are used for successful puncturing of the host integument. Their proportions and relative densities vary widely between taxa (van Achterberg and Quicke, 1991). In Oryilus lepiodus (Hawke et al., 1973), Biosteres (Opius) longicaudatus (Greany et al., 1977), Trybliographa rapae (Brown and Anderson, 1998), Trichogramma galloi and T. pretiosum (Coˆnsoli et al., 1999), these serrations may act to pierce the host cuticle and hold the ovipositor in position in the cuticle as oviposition takes place. In addition to passing eggs, the ovipositor of H. hebetor also has to channel liquid venoms into the host. A number of adaptations have evolved for the purpose of preventing leakage of these fluids from the ovipositor in H. hebetor. The olistheters connecting both first and second valvulae are orientated vertically, thus allowing the first valvulae to separate relatively easily. This change in orientation helps to maintain a tighter contact between the medioventral walls of the first valvulae thus reducing loss of venom before it reaches the tip. Also, H. hebetor has a fine longitudinal flap or seal along the ventromedial part of each piece of the first valvulae. The presence of venom within the egg canal of an H. hebetor ovipositor will force these two flexible little flaps together in order to form a fluid-tight seal. This seal in an H. hebetor ovipositor has also been observed in almost all other examined ovipositors of braconid species (Quicke and van Achterberg, 1990;
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(Soliman, 1940). The ovipositor is then extended to its full length, being held between and guided by the third valvulae. Apparently, great effort and exertion are needed in order to extrude the comparatively large egg out of the ovipositor. The egg becomes apparent at the middle part of the ovipositor and slowly begins its descent within by sliding the first and second valvulae back and forth upon one another. Traction is provided by the internal microsculpture (ctenidia and subctenidia) until the egg is gently laid on the substratum. This microsculpture also appears to prevent the egg from withdrawing back into the wasp (Quicke et al., 1999). The single distinct valvillus situated on the inner surface of the distal third of each first valvula may help to hold the eggs in place within the ovipositor prior to oviposition, as was suggested by Rogers (1972) for the ichneumonid Venturia canescens. Extensive study of their distribution in many parasitoids as well as in the Aculeata, Vespoidea, and Apoidea has been done by Quicke et al. (1992). 4.3. Host selection process
Fig. 11. Medial transverse section of second valvula lumen of ovipositor of H. hebetor showing tubular body (white arrow) of a campaniform sensillum.
Quicke et al., 1994). The second valvulae of H. hebetor are very blunt and rounded at the apex and are totally unsuited for penetrating through host cuticle. However, serial sections through the ovipositor reveal an olistheter-like interlocking of the mediodorsal part of the first valvulae just prior to the apex, thereby forming a slender evenomation tube. The same type of olistheter-like interlocking has been observed near the ovipositor tip in several other braconids, such as in Zagyptogastra (Quicke, 1991), Aleiodes, Ligulibracon, and Odontobracon (Quicke et al., 1994). This may be an adaptation to injecting venom into the host while laying the egg externally (Quicke, 1997). The annulation of the third valvulae in H. hebetor makes them flexible. Flexion might facilitate handling during stinging, preventing the third valvulae from interfering with the ovipositor shaft. As the ovipositor shaft begins to penetrate the host integument, the third valvulae slide basally along the former, forming gradually expanding loops where they are separated from the ovipositor shaft. These observations indicate that the third valvulae might serve as supports. The microtrichia lining the inner surfaces of the third valvulae are probably involved in cleaning the ovipositor sensilla between stingings. It is important that the sensilla of the ovipositor shaft are kept clean for the ovipositor to remain functional. The sensilla are concentrated at the tip, which might explain why the microtrichia are most densely concentrated at the tip of the third valvulae. LeRalec et al. (1996) and Ne´non et al. (1997) described similar dense cuticular microtrichia along the inner surface of the third valvulae in some aphidiine braconids and the ichneumonid Megarhyssa atrata, respectively. 4.2. Ovipositional function After paralysis, a female mounts and externally examines the host. If the host is found to be acceptable, she acquires a good hold on the host and curves her much-dilated abdomen under her
Three morphologically distinct types of sensilla have been identified on the first, second, and third valvulae and on the second valvifers of the H. hebetor ovipositor from SEM and TEM observations. These types of sensilla have previously been described in other parasitic Hymenoptera (Brown and Anderson, 1998; Chiappini and Solinas, 2002; Coˆnsoli et al., 1999; Ganesalingam, 1972; Greany et al., 1977; Gutierrez, 1970; Hawke et al., 1973; LeRalec, 1991; LeRalec et al., 1996; LeRalec and Wajnberg, 1990; Ne´non et al., 1997). Styloconic sensilla are present in two distinct groups. The first group occurs in a linear arrangement on the second valvifer at the point of articulation with the first ramus and is thought to function as a mechanoreceptor. It is probably involved in detecting movement of the ramus against the second valvifer as occurs when the first valvulae are moved alternately (Quicke et al., 1999). This type of sensillum has also been described in some species belonging to the Chalcidoidea, Cynipoidea, Ceraphronoidea and Proctotrupoidea (LeRalec, 1991; LeRalec et al., 1996; Mason, 1984), but are absent in aphidiine braconids (LeRalec et al., 1996). The second group of styloconic sensilla occurs in the form of a hair plate at the point of articulation of the first and second valvifers. These may also function as mechanoreceptors and are probably involved in detecting the position and movements of the first valvifer relative to the second valvifer (Quicke et al., 1999). This group of sensilla has been found to be reduced to a few sensilla, or just one sensillum, in some small species of Chalcidoidea (Copland and King, 1971a,b, 1972a,b; LeRalec, 1991). Trichoid sensilla as found to entirely cover the third valvulae externally have also been seen in the ichneumonid M. atrata (Ne´non et al., 1997), the trichogrammatid Trichogramma maidis (LeRalec and Wajnberg, 1990). No olfactory or gustatory function can be attributed to this sensillar type as it does not posses any pore system and sensory neuron as revealed by TEM. However, trichoid sensilla may serve as mechanoreceptors due to their socket-like insertion into the third valvulae cuticle and their spatial arrangement. They may probably respond to air currents and vibration during host searching. In the laboratory, Darwish et al. (2003) found that the third valvulae never touch the host during any step of the host selection process. Campaniform sensilla found on the ovipositor shaft of H. hebetor appear to be strictly tactile as it is innervated by only one neuron, each of which possesses a tubular body, and because it has no opening to the exterior. LeRalec and Wajnberg (1990), Coˆnsoli et al. (1999), and Chiappini and Solinas (2002) described similar
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sensilla on the ovipositors of Trichogramma maidis, T. galloi and T. pretiosum, and Anagrus breviphragma, respectively. These sensilla were found along the length of the ovipositor shaft of H. hebetor, as well as at the tip, and most likely are involved in perceiving tactile cues by which H. hebetor discriminates between different stages of the same host. In the laboratory, Soliman (1940) found that before oviposition in the host takes place, H. hebetor females take quite a long time (sometimes as long as 3 h) inspecting the paralyzed caterpillar and its surroundings, using the tip of the ovipositor as a feeler. Doutt (1959) reported that before H. hebetor females initiate oviposition, they try to paralyze all hosts (even in different stages) in the vicinity. Darwish et al. (2003) found that H. hebetor females prefer late-instar hosts (30-day-old larvae) for oviposition. Wang (1991) observed the same preference for H. hebetor parasitizing the larvae of Plodia interpunctella. The use of age-dependant cues has also been cited as a mechanism by which Cotesia glomerata discriminates between different stages of the same host species (Mattiacci and Dicke, 1995a,b). Acknowledgements Sincere thanks to Dr. Roy Vogtsberger (Department of Biology, Midwestern State University, USA) and Dr. Delphine Bourdais (Unite´ d’Ecologie et de Bioge´ographie, Universite´ catholique de Louvain, Belgium.) for reviewing the manuscript and supplying us with many helpful suggestions and advice. References van Achterberg, C., Quicke, D.L.J., 1991. A new genus of Braconinae with depressed ovipositor-tip from the Oriental region (Hymenoptera: Braconidae). Zool. Med. Leiden. 64, 199–202. Austin, A.D., Field, T.O., 1997. The ovipositor system of the scelionid and platygastrid wasps (Hymenoptera: Platygastroidea): comparative morphology and phylogenetic implications. Invert. Taxon. 11, 1–87. Brown, P.E., Anderson, M., 1998. Morphology and ultrastructure of sense organs on the ovipositor of Trybliographa rapae, a parasitoid of the cabbage root fly. J. Insect. Physiol. 44, 1017–1025. Chiappini, E., Solinas, C., 2002. Ovipositor sensory structures of Anagrus breviphragma Soyka and their possible significance. In: Melika, G., Thuro´czy, C. (Eds.), Parasitic Wasps: Evolution, Systematics, Biodiversity and Biological Control. Agroinform Kiado´ & Nyomda, Budapest, Hungary, pp. 267–271. Cline, L.D., Press, J.W., 1990. Reduction in almond moth (Lepidoptera: Pyralidae) infestation using commercial packaging of foods in combination with the parasitic wasp, Bracon hebetor (Hymenoptera: Braconidae). J. Econ. Entomol. 83, 1110–1113. Cline, L.D., Press, J.W., Flaherty, B.R., 1984. Preventing the spread of the almond moth (Lepidoptera: Pyralidae) from infested food debris to adjacent uninfested packages, using the parasite Bracon hebetor (Hymenoptera: Braconidae). J. Econ. Entomol. 77 (2), 331–333. Coˆnsoli, F.L., Kitajima, E.W., Postali Parra, J.R., 1999. Sensilla on the antenna and ovipositor of the parasitic wasps Trichogramma galloi Zucchi and T. pretiosum Riley (Hymenoptera: Trichogrammatidae). Microsc. Res. Tech. 45, 313–324. Copland, M.J.W., King, P.E., 1971a. The structure and possible function of the reproductive system in some Eulophidae and Tetracampidae. Entomologist 104, 4–28. Copland, M.J.W., King, P.E., 1971b. The structure of the reproductive system in the Chalcididae. Entomol. Mon. Mag. 107, 230–239. Copland, M.J.W., King, P.E., 1972a. The structure of the female reproductive system in the Pteromalidae (Hymenoptera: Chalcidoidea). Entomologist 105, 77–96. Copland, M.J.W., King, P.E., 1972b. The structure of the female reproductive system in the Tormidae (Hymenoptera: Chalcidoidea). Trans. R. Ent. Soc. Lond. 124, 191–212. Darwish, E., El-shazly, M., El-Sherif, H., 2003. The choice of probing sites by Bracon hebetor Say (Hymenoptera: Braconidae) foraging for Ephestia kuehniella Zeller (Lepidoptera: Pyralidae). J. Stored. Prod. Res. 39, 265–276. Doutt, R.L., 1959. Distribution of eggs by Microbracon (Hymenoptera: Bracoidae). Ecology 40, 302–303. Dweck, H.K.M., Gadallah, N.S., 2008. Description of the antennal sensilla of Habrobracon hebetor. Bio Control doi:10.1007/s10526-007-9145-6.
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Comparative morphology of the ovipositor of some parasitic Hymenoptera in relation to characteristics of their hosts. Can. Entomol. 128, 413–433. Mason, W.R.M., 1984. Structure and movement of the abdomen of female Pelecinus polyturator (Hymenoptera: Pelecinidae). Can. Entomol. 116, 419–426. Mattiacci, L., Dicke, M., 1995a. The parasitoid Cotesia glomerata (Hymenoptera: Braconidae) discriminates between first and fifth larval instars of its host Pieris brassicae, on the basis of contact cues from frass, silk, and herbivore-damaged leaf tissue. J. Insect. Behav. 8, 485–498. Mattiacci, L., Dicke, M., 1995b. Host age discrimination during host location by Cotesia glomerata, a larval parasitoid of Pieris brassicae. Entomol. Exp. Appl. 76, 37–48. Ne´non, J.P., Kacem, J., Lelannic, J., 1997. Structure, sensory equipment, and secretions of the ovipositor in a giant species of Hymenoptera. Megarhyssa atrata F. (Ichneumonidae: Pimplinae). Can. Entomol. 129, 789–799. Press, J.W., Cline, L.D., Flaherty, B.R., 1982. A comparison of two parasitoids, Bracon hebetor (Hymenoptera: Braconidae) and Venturia canescens (Hymenoptera: Ichneumonidae), and a predator Xylocoris flavipes (Hemiptera: Anthocoridae) in suppressing residual populations of the almond moth, Ephestia cautella (Lepidoptera: Pyralidae). J. Kansas. Entomol. Soc. 55 (4), 125–128. Quicke, D.L.J., 1991. Ovipositor mechanics of the braconine wasp genus Zagyptogastra and the ichneumonid genus Pritomerus. J. Nat. Hist. 25, 971–977. Quicke, D.L.J., 1997. Parasitic Wasps. Chapman and Hall, London, pp. 1–470. Quicke, D.L.J., van Achterberg, C., 1990. Phylogeny of the subfamilies of the family Braconidae (Hymenoptera: Ichneumonoidea). ZoologischesVerhandelingen. Leiden 258, 1–95. Quicke, D.L.J., Fitton, M.G., 1995. Ovipositor steering mechanism in parasitic wasps of the families Gasteruptiidae and Aulacidae (Hymenoptera). Proc. R. Soc. Lond. B 261, 99–103. Quicke, D.L.J., Fitton, M.G., Harris, J., 1995. Ovipositor steering mechanisms in braconid wasps. J. Hymen Res. 4, 110–120. Quicke, D.L.J., Fitton, M.G., Ingram, S.N., 1992. Phylogenetic implications of the structure and distribution of ovipositor valvilli in the Hymenoptera (Insecta). J. Nat. Hist. 26, 587–608. Quicke, D.L.J., Fitton, M.G., Tunstead, J.R., Ingram, S.N., Gaitens, P.V., 1994. Ovipositor structure and relationships within the Hymenoptera, with special reference to the Ichneumonoidea. J. Nat. Hist. 28, 635–682. Quicke, D.L.J., Leralec, A., Vilhelmsen, L., 1999. Ovipositor structure and function in the parasitic Hymenoptera with an exploration of new hypothesis. Rendiconti 47, 197–239. Rahman, M.H., Fitton, M.G., Quicke, D.L.J., 1998. Ovipositor internal microsculpture in the Braconidae (Insecta Hymenoptera). Zool. Scripta 27, 319–331. Rogers, D., 1972. The ichneumon wasp Venturia canescens: ovipositor and avoidance of superparasitism. Entomol. Exp. Appl. 15, 190–194. Scho¨ller, M., Prozell, S., 2001. Die Mehlmottenschlupfwespe Habrobracon hebetor (Hymenoptera: Braconidae) als Antagonist vorratsscha¨dlicher Motten Gesunde Pflanz 53, 82–89. Smith, E.L., 1970. Evolutionary morphology of the external insect genitalia. II. Hymenoptera. Ann. Entomol. Soc. Am. 63, 1–27. Soliman, H.S., 1940. Studies on the biology of Microbracon hebetor Say (Hymenoptera: Braconidae). Bull. Soc. Fouad. Le Caire 24, 215–224. Wang, X.W., 1991. Studies on the relationship between host size and sex ratio of Bracon hebetor (Hymenoptera: Braconidae). Chin. J. Biol. Cont. 71, 16–18.
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Structure and sensory equipment of the ovipositor of Habrobracon hebetor (Say) (Hymenoptera: Braconidae) Hany K.M. Dweck *, Neveen S. Gadallah, Essam Darwish Department of Entomology, Faculty of Science, Cairo University, Giza, Egypt
A R T I C L E I N F O
A B S T R A C T
Article history: Received 15 December 2007 Received in revised form 30 March 2008 Accepted 31 March 2008
The microsculpture of various structures of the ovipositor of Habrobracon hebetor (Say) (Hymenoptera: Braconidae) is described from scanning and transmission electron microscopy. These include: the ovipositor egg canal, valvillus, seal of the first valvulae, interlocking mechanism (olistheter) connecting the first and second valvulae, an olistheter-like interlocking mechanism connecting the two pieces of the first valvulae, annulation, microtrichia of the third valvulae, and the ovipositor sensory equipment. Better understanding of the microsculpture of these components may make their roles in stinging, oviposition, and the host selection process more clear. ß 2008 Elsevier Ltd. All rights reserved.
Keywords: Microsculpture Valvillus Seal Olistheter Annulation Microtrichia
1. Introduction The insect ovipositor is a complex structure consisting of up to seven interlocking sclerites associated with the 8th and 9th abdominal segments in insect females (termed the genital segments). Female insects use the ovipositor to deposit eggs in specific locations such as deep within the soil, in plant tissues, under the bark of trees, and even within the bodies of other insects. The basic organization of female genitalia shows a remarkable uniformity among Hymenoptera. It consists of two pairs of valvifers and three pairs of valvulae derived from the 8th and the 9th abdominal segments (LeRalec and Wajnberg, 1990). The first valvifers (gonocoxites VIII) are continuous with the rami of the first valvulae (gonapophyses VIII). The second valvifers (gonocoxites IX) extend as the third valvulae (gonostyli) and ventrally bear the fused second valvulae (gonapophyses IX). The interlocked first and second valvulae form the shaft of the ovipositor and enclose the egg-canal. The ovipositor of parasitic Hymenoptera is a specialized organ with which the female probes and drills the substrate where a host lives, pierces the integument of the host, injects the substances from the accessory glands, perceives stimuli involved in the host
* Corresponding author. Tel.: +20 25676826; fax: +20 25728843. E-mail address: [email protected] (Hany K.M. Dweck). 0968-4328/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2008.03.012
selection process, guides and deposits eggs (van Lenteren, 1981; Quicke et al., 1995). Hosts attacked by these parasitoids are diverse. Members of most pterygote orders, of all developmental stages, fixed or mobile, that are exposed or concealed in different substrates, can be parasitized. This wide range of situations produces a diversity of constraints to which the ovipositor must be adapted. Therefore, morphological and functional features of the ovipositor should vary with host diversity. Variation in ovipositor structure has been used for classification of parasitoids, for clarifying phylogenetic relationships (e.g., Austin and Field, 1997) and, recently, in several cases to relate form with function (Field and Austin, 1994; LeRalec et al., 1996; Quicke and Fitton, 1995; Quicke et al., 1995). Habrobracon hebetor is a gregarious, idiobiont (one that prevents any further development of the host after initial parasitization), larval ectoparasitoid of pyralid moths. The use of H. hebetor for biological control of stored product moths has been well documented in the literature (Keever et al., 1986; Press et al., 1982; Scho¨ller and Prozell, 2001). The advantage of H. hebetor as a biological control agent for stored product moths in retail stores and warehouses is that it attacks the wandering larval stages of the moths and may serve to reduce secondary infestation of packaged goods (Cline et al., 1984; Cline and Press, 1990). The purpose of the present work is to describe structure and sensory equipment of the ovipositor of H. hebetor (Say) (Hymenoptera: Braconidae) in order to better understand their roles in stinging, oviposition, and the host selection process.
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2. Materials and methods 2.1. Stock culture of parasitoids The Mediterranean flour moth, Ephestia kuehniella and the ectoparasitoid, H. hebetor used in the present study were collected from Cairo flour mills (Egypt). A laboratory culture of E. kuehniella was reared in glass jars on whole-wheat flour previously heated in order to ensure the absence of any infestation. The stock culture of the parasitoid H. hebetor was reared on 25–30-day-old host larvae in cylindrical glass jars (10 cm 17 cm) containing infested flour. The parasitoids and their hosts were maintained at 28 1 8C and 60 10 RH with a photoperiod of 14:10 (L:D). 2.2. Light microscopy Ovipositors were dissected from specimens using fine forceps, gradually dehydrated through a series of ethyl alcohol (20, 50, 70, 80, 90, 95, 100% ethanol), then held in 100% xylene for 48 h and mounted on glass slides in Canada balsam for examination using a Cambridge light microscope. Measurements of the different parts of the ovipositor were made using an ocular micrometer and were taken from 50 individuals. 2.3. Scanning electron microscopy The dissected parts were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3) for at least 4 h at 4 8C, washed in 0.1 M phosphate buffer (overnight), immersed in a postfixative of 1% buffered osmium tetroxide for 1 h at 4 8C and then gradually dehydrated in acetone, critical point dried in an Autosamdri1-815, and finally sputter coated with gold in a SPI-ModuleTM Sputter Coater. Examination and photography were done using Philips ALx 30 and Jeol JSM-5200 scanning electron microscopes at different accelerating voltages. Images were then recorded digitally on a computer. Measurements (in mm) were obtained from photographs of at least 50 individual sensilla of the same type and were used to calculate means. 2.4. Transmission electron microscopy Different parts under examination were fixed in 2.5 glutaraldehyde in 0.1 M phosphate buffer (pH 7.3) for at least 4 h at 4 8C, briefly washed in 0.1 M phosphate buffer (overnight), and postfixed in 1% osmium tetroxide in 0.1 M phosphate buffer for 1 h at 4 8C. After dehydration in a graded series of acetone, they were embedded in Epon-Araldite. Thin sections were cut with a glass knife using a Reichert-Jung Ultracut E, collected on colloid grids, contrasted with uranyl acetate and lead citrate, and finally observed in a Jeol JEM-1200 EX II. Semi-thin sections were stained with toluidine blue (1%) and observed under a Cambridge light microscope. Drawings were then made using a camera lucida.
Fig. 1. Ovipositor of Habrobracon hebetor (lateral aspect); 40. B = bulb; T9 = tergite 9; V1, V2, and V3 = first, second, and third valvulae; Vf1, Vf2 = first and second valvifers.
and second valvulae. When held together, these structures form internal lumina that serve as passageways for eggs and/or venom. The paired first valvulae (=lower valves of Quicke et al., 1999) are separated from each other along most of their length. The posterior tip of each first valvula has barbs or serrations on its lateral side (Fig. 2). The second valvulae (=upper valves of Quicke et al., 1999) are fused along their entire length and are expanded proximally to form an enlarged bulb. Near their apex, the second valvulae are somewhat enlarged and slightly decurved, bearing no barbs or serrations. The first and second valvulae are joined together by an interlocking mechanism (=olistheter of Quicke et al., 1999) (Figs. 3b–d and f and 4a and b). Each olistheter consists of a projected tongue (rhachis) (Fig. 5a) that is vertically positioned on the ventral wall of each second valvula in H. hebetor. Each rhachis fits into a groove (aulax) (Fig. 6b) in the dorsal wall of each first valvula, but this interlocking mechanism does not extend to the apex of the ovipositor (Fig. 3a). Close to the apex of the ovipositor shaft, the two first valvulae become interlocked dorsally by a mechanism similar to the olistheter (Figs. 3a–d and 4a). A single distinct valvillus (Quicke et al., 1992) is situated on the inner surface of the distal third of each first valvula (Fig. 6a). This
3. Results 3.1. Ovipositor morphology The female genitalia of H. hebetor exhibits the basic organization typically seen in Hymenoptera (Fig. 1) (Smith, 1970). The ovipositor shaft is on average 2.38 0.13 mm long (about half of adult body length) and 0.07 0.01 mm thick (n = 50). It is a rigid, yet flexible, structure that ends in a pointed tip and consists of the first
Fig. 2. Interlocked first (V1) and second (V2) valvulae of ovipositor of H. hebetor just proximal to ovipositor tip. Ca = campaniform sensilla; T = teeth.
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Fig. 3. Diagram of serial transverse sections from distal end of ovipositor of H. hebetor: (a–f) distal to proximal sections. A = aulax; EC = egg canal; L = lumen; R = rhachis; S = mid-longitudinal septum; V1, V2 = first and second valvulae.
valvillus is obviously concave and the position of the valvillar insertion in the egg canal was found to be approximately medial. There is a thin longitudinal cuticular flap that forms a seal (Quicke et al., 1994) by protruding into the egg or venom canal from the medioventral part of each first valvula (Fig. 4b). The surface of the egg canal within the ovipositor of H. hebetor is not smooth but possesses complex microsculpture consisting of ctenidia and subctenidia. The ventral wall in the proximal portion of the second valvulae has well-developed fine projections across most of the surface between the rhachises (Fig. 5b). Rows of these posteriorly-directed ctenidia are also located on the inner surface of each first valvula both anterior and posterior to the valvillus (Fig. 6b). Another distinct type of setiform structure (=subctenidial setae of Rahman et al., 1998) was found associated with those ctenidia posterior to the valvillus (Fig. 6c).
The third valvulae (=the ovipositor sheath of Quicke et al., 1999) are one segmented and are fleshy, ridged and annulated externally in the basal two thirds of their length (Fig. 7a), but smooth in the distal third (Fig. 7b). The third valvulae unsheath both the first and second valvulae when they are not in use. The inner surface of the third valvulae is almost entirely covered with very dense, flexible cuticular microtrichia (=cuticular spines of Ne´non et al., 1997) (Fig. 7c). The density of the microtrichia varies along the length of the third valvulae, the tip usually having the highest concentration. 3.2. Sensory equipment Three morphologically distinct types of sensilla have been identified on the ovipositor shaft, third valvulae, and second valvifers of the ovipositor of H. hebetor from observations utilizing
Fig. 4. Transverse sections of ovipositor of H. hebetor: (a) distal and (b) medial. A = aulax; C = ctenidia; EC = egg canal; L = lumen; R = rhachis; Sl = seal; V1, V2 = first and second valvulae.
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Fig. 5. Ventral wall of second valvula of ovipositor of H. hebetor: (a) distal end and (b) proximal end. C = ctenidia; R = rhachis; S = protruding margins of mid-longitudinal septum.
SEM and TEM. These types of sensilla will each be addressed individually. 3.2.1. Styloconic sensilla Styloconic sensilla are present in two distinct groups. The first group (5.97 1.19 mm in shaft length) is arranged linearly (12–18 sensilla in number) on each second valvifer at the point where it articulates with the first ramus (Fig. 8a). The second group of styloconic sensilla (1.4 0.14 mm in shaft length) is present in the form of a hair plate (12–18 sensilla in number) at the point of articulation between each first and second valvifer (Fig. 8b). 3.2.2. Trichoid sensilla Two types of trichoid sensilla are found covering the entire external surface of the third valvulae. The first type, long trichoid sensilla, is distributed along the entire length of the third valvulae (Fig. 7a and b). They are inserted in sockets and each external process (54.4 32.4 mm in length) has slight longitudinal grooves and gradually tapers to a sharp point. The cuticle of this sensillum exhibits a thick and non-porous wall,
which is not innervated by dendrites, as seen in the TEM reprint (Fig. 9b). The second type of trichoid sensilla, short trichoid sensilla, is similar to the first type except that they are located between those of the first type in the distal end of the third valvulae and are shorter in overall length than those of the first type (10.45 4.45 mm in length) (Fig. 7b and 9a). 3.2.3. Campaniform sensilla Campaniform sensilla, with an irregular and depressed cuticular structure (Fig. 2), are found along the ovipositor shaft. However, they are found more concentrated at the tip of the second valvulae (Fig. 10). This sensillar type is innervated by a single dendritic segment which forms a tubular body just before entering the valvula cuticle (Fig. 11). 4. Discussion Most of the ovipositor structures examined in the present work show a correlation between their morphological characteristics and their function. Several of these will now be discussed.
Fig. 6. Dorsal wall of first valvula of ovipositor of H. hebetor: (a) valvillus near distal end, (b) posterior to valvillus and (c) anterior to valvillus. A = aulax; Ct = ctenidia; STS = subctenidial setae; white arrow = valvillus.
Fig. 7. Third valvula of ovipositor of H. hebetor: (a) lateral wall, (b) tip and (c) median wall. Mi = microtrichia; LST = long trichoid sensilla; SST = short trichoid sensilla.
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Fig. 8. Second valvifer (Vf 2) of ovipositor of H. hebetor: (a) at articulation point with first ramus and (b) at articulation point with first valvifer. SS = styloconic sensilla.
Fig. 9. Transverse sections of the proximal portions of (a) short and (b) long trichoid sensilla. Note the thick and groove non-porous sensillum wall (SW) and the sensillum lymph (SL), which is not innervated by dendrites.
4.1. Stinging function As a female H. hebetor mounted its host by using its antennal sensory receptors (Dweck and Gadallah, 2008), her abdomen was bent forward, the ovipositor was unsheathed and stinging of the host took place. Stinging by this species is a mechanism used to puncture the host’s integument in order to acquire a meal of hemolymph and to inject venom into the host larva. Two to three
Fig. 10. Tip of second valvulae of ovipositor of H. hebetor showing campaniform sensilla (Ca).
stings were observed by this female. Duration of a sting usually lasts from 2 to 3 min (Darwish et al., 2003). Stinging results in paralysis of motor function which facilitates oviposition by the wasp. The barbs or serrations present at the distal end of each first valvula on the ovipositor of H. hebetor are used for successful puncturing of the host integument. Their proportions and relative densities vary widely between taxa (van Achterberg and Quicke, 1991). In Oryilus lepiodus (Hawke et al., 1973), Biosteres (Opius) longicaudatus (Greany et al., 1977), Trybliographa rapae (Brown and Anderson, 1998), Trichogramma galloi and T. pretiosum (Coˆnsoli et al., 1999), these serrations may act to pierce the host cuticle and hold the ovipositor in position in the cuticle as oviposition takes place. In addition to passing eggs, the ovipositor of H. hebetor also has to channel liquid venoms into the host. A number of adaptations have evolved for the purpose of preventing leakage of these fluids from the ovipositor in H. hebetor. The olistheters connecting both first and second valvulae are orientated vertically, thus allowing the first valvulae to separate relatively easily. This change in orientation helps to maintain a tighter contact between the medioventral walls of the first valvulae thus reducing loss of venom before it reaches the tip. Also, H. hebetor has a fine longitudinal flap or seal along the ventromedial part of each piece of the first valvulae. The presence of venom within the egg canal of an H. hebetor ovipositor will force these two flexible little flaps together in order to form a fluid-tight seal. This seal in an H. hebetor ovipositor has also been observed in almost all other examined ovipositors of braconid species (Quicke and van Achterberg, 1990;
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(Soliman, 1940). The ovipositor is then extended to its full length, being held between and guided by the third valvulae. Apparently, great effort and exertion are needed in order to extrude the comparatively large egg out of the ovipositor. The egg becomes apparent at the middle part of the ovipositor and slowly begins its descent within by sliding the first and second valvulae back and forth upon one another. Traction is provided by the internal microsculpture (ctenidia and subctenidia) until the egg is gently laid on the substratum. This microsculpture also appears to prevent the egg from withdrawing back into the wasp (Quicke et al., 1999). The single distinct valvillus situated on the inner surface of the distal third of each first valvula may help to hold the eggs in place within the ovipositor prior to oviposition, as was suggested by Rogers (1972) for the ichneumonid Venturia canescens. Extensive study of their distribution in many parasitoids as well as in the Aculeata, Vespoidea, and Apoidea has been done by Quicke et al. (1992). 4.3. Host selection process
Fig. 11. Medial transverse section of second valvula lumen of ovipositor of H. hebetor showing tubular body (white arrow) of a campaniform sensillum.
Quicke et al., 1994). The second valvulae of H. hebetor are very blunt and rounded at the apex and are totally unsuited for penetrating through host cuticle. However, serial sections through the ovipositor reveal an olistheter-like interlocking of the mediodorsal part of the first valvulae just prior to the apex, thereby forming a slender evenomation tube. The same type of olistheter-like interlocking has been observed near the ovipositor tip in several other braconids, such as in Zagyptogastra (Quicke, 1991), Aleiodes, Ligulibracon, and Odontobracon (Quicke et al., 1994). This may be an adaptation to injecting venom into the host while laying the egg externally (Quicke, 1997). The annulation of the third valvulae in H. hebetor makes them flexible. Flexion might facilitate handling during stinging, preventing the third valvulae from interfering with the ovipositor shaft. As the ovipositor shaft begins to penetrate the host integument, the third valvulae slide basally along the former, forming gradually expanding loops where they are separated from the ovipositor shaft. These observations indicate that the third valvulae might serve as supports. The microtrichia lining the inner surfaces of the third valvulae are probably involved in cleaning the ovipositor sensilla between stingings. It is important that the sensilla of the ovipositor shaft are kept clean for the ovipositor to remain functional. The sensilla are concentrated at the tip, which might explain why the microtrichia are most densely concentrated at the tip of the third valvulae. LeRalec et al. (1996) and Ne´non et al. (1997) described similar dense cuticular microtrichia along the inner surface of the third valvulae in some aphidiine braconids and the ichneumonid Megarhyssa atrata, respectively. 4.2. Ovipositional function After paralysis, a female mounts and externally examines the host. If the host is found to be acceptable, she acquires a good hold on the host and curves her much-dilated abdomen under her
Three morphologically distinct types of sensilla have been identified on the first, second, and third valvulae and on the second valvifers of the H. hebetor ovipositor from SEM and TEM observations. These types of sensilla have previously been described in other parasitic Hymenoptera (Brown and Anderson, 1998; Chiappini and Solinas, 2002; Coˆnsoli et al., 1999; Ganesalingam, 1972; Greany et al., 1977; Gutierrez, 1970; Hawke et al., 1973; LeRalec, 1991; LeRalec et al., 1996; LeRalec and Wajnberg, 1990; Ne´non et al., 1997). Styloconic sensilla are present in two distinct groups. The first group occurs in a linear arrangement on the second valvifer at the point of articulation with the first ramus and is thought to function as a mechanoreceptor. It is probably involved in detecting movement of the ramus against the second valvifer as occurs when the first valvulae are moved alternately (Quicke et al., 1999). This type of sensillum has also been described in some species belonging to the Chalcidoidea, Cynipoidea, Ceraphronoidea and Proctotrupoidea (LeRalec, 1991; LeRalec et al., 1996; Mason, 1984), but are absent in aphidiine braconids (LeRalec et al., 1996). The second group of styloconic sensilla occurs in the form of a hair plate at the point of articulation of the first and second valvifers. These may also function as mechanoreceptors and are probably involved in detecting the position and movements of the first valvifer relative to the second valvifer (Quicke et al., 1999). This group of sensilla has been found to be reduced to a few sensilla, or just one sensillum, in some small species of Chalcidoidea (Copland and King, 1971a,b, 1972a,b; LeRalec, 1991). Trichoid sensilla as found to entirely cover the third valvulae externally have also been seen in the ichneumonid M. atrata (Ne´non et al., 1997), the trichogrammatid Trichogramma maidis (LeRalec and Wajnberg, 1990). No olfactory or gustatory function can be attributed to this sensillar type as it does not posses any pore system and sensory neuron as revealed by TEM. However, trichoid sensilla may serve as mechanoreceptors due to their socket-like insertion into the third valvulae cuticle and their spatial arrangement. They may probably respond to air currents and vibration during host searching. In the laboratory, Darwish et al. (2003) found that the third valvulae never touch the host during any step of the host selection process. Campaniform sensilla found on the ovipositor shaft of H. hebetor appear to be strictly tactile as it is innervated by only one neuron, each of which possesses a tubular body, and because it has no opening to the exterior. LeRalec and Wajnberg (1990), Coˆnsoli et al. (1999), and Chiappini and Solinas (2002) described similar
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sensilla on the ovipositors of Trichogramma maidis, T. galloi and T. pretiosum, and Anagrus breviphragma, respectively. These sensilla were found along the length of the ovipositor shaft of H. hebetor, as well as at the tip, and most likely are involved in perceiving tactile cues by which H. hebetor discriminates between different stages of the same host. In the laboratory, Soliman (1940) found that before oviposition in the host takes place, H. hebetor females take quite a long time (sometimes as long as 3 h) inspecting the paralyzed caterpillar and its surroundings, using the tip of the ovipositor as a feeler. Doutt (1959) reported that before H. hebetor females initiate oviposition, they try to paralyze all hosts (even in different stages) in the vicinity. Darwish et al. (2003) found that H. hebetor females prefer late-instar hosts (30-day-old larvae) for oviposition. Wang (1991) observed the same preference for H. hebetor parasitizing the larvae of Plodia interpunctella. The use of age-dependant cues has also been cited as a mechanism by which Cotesia glomerata discriminates between different stages of the same host species (Mattiacci and Dicke, 1995a,b). Acknowledgements Sincere thanks to Dr. Roy Vogtsberger (Department of Biology, Midwestern State University, USA) and Dr. Delphine Bourdais (Unite´ d’Ecologie et de Bioge´ographie, Universite´ catholique de Louvain, Belgium.) for reviewing the manuscript and supplying us with many helpful suggestions and advice. References van Achterberg, C., Quicke, D.L.J., 1991. A new genus of Braconinae with depressed ovipositor-tip from the Oriental region (Hymenoptera: Braconidae). Zool. Med. Leiden. 64, 199–202. Austin, A.D., Field, T.O., 1997. The ovipositor system of the scelionid and platygastrid wasps (Hymenoptera: Platygastroidea): comparative morphology and phylogenetic implications. Invert. Taxon. 11, 1–87. Brown, P.E., Anderson, M., 1998. Morphology and ultrastructure of sense organs on the ovipositor of Trybliographa rapae, a parasitoid of the cabbage root fly. J. Insect. Physiol. 44, 1017–1025. Chiappini, E., Solinas, C., 2002. Ovipositor sensory structures of Anagrus breviphragma Soyka and their possible significance. In: Melika, G., Thuro´czy, C. (Eds.), Parasitic Wasps: Evolution, Systematics, Biodiversity and Biological Control. Agroinform Kiado´ & Nyomda, Budapest, Hungary, pp. 267–271. Cline, L.D., Press, J.W., 1990. Reduction in almond moth (Lepidoptera: Pyralidae) infestation using commercial packaging of foods in combination with the parasitic wasp, Bracon hebetor (Hymenoptera: Braconidae). J. Econ. Entomol. 83, 1110–1113. Cline, L.D., Press, J.W., Flaherty, B.R., 1984. Preventing the spread of the almond moth (Lepidoptera: Pyralidae) from infested food debris to adjacent uninfested packages, using the parasite Bracon hebetor (Hymenoptera: Braconidae). J. Econ. Entomol. 77 (2), 331–333. Coˆnsoli, F.L., Kitajima, E.W., Postali Parra, J.R., 1999. Sensilla on the antenna and ovipositor of the parasitic wasps Trichogramma galloi Zucchi and T. pretiosum Riley (Hymenoptera: Trichogrammatidae). Microsc. Res. Tech. 45, 313–324. Copland, M.J.W., King, P.E., 1971a. The structure and possible function of the reproductive system in some Eulophidae and Tetracampidae. Entomologist 104, 4–28. Copland, M.J.W., King, P.E., 1971b. The structure of the reproductive system in the Chalcididae. Entomol. Mon. Mag. 107, 230–239. Copland, M.J.W., King, P.E., 1972a. The structure of the female reproductive system in the Pteromalidae (Hymenoptera: Chalcidoidea). Entomologist 105, 77–96. Copland, M.J.W., King, P.E., 1972b. The structure of the female reproductive system in the Tormidae (Hymenoptera: Chalcidoidea). Trans. R. Ent. Soc. Lond. 124, 191–212. Darwish, E., El-shazly, M., El-Sherif, H., 2003. The choice of probing sites by Bracon hebetor Say (Hymenoptera: Braconidae) foraging for Ephestia kuehniella Zeller (Lepidoptera: Pyralidae). J. Stored. Prod. Res. 39, 265–276. Doutt, R.L., 1959. Distribution of eggs by Microbracon (Hymenoptera: Bracoidae). Ecology 40, 302–303. Dweck, H.K.M., Gadallah, N.S., 2008. Description of the antennal sensilla of Habrobracon hebetor. Bio Control doi:10.1007/s10526-007-9145-6.
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