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Sense organs on the ovipositor of Macrocentrus cingulum Brischke (Hymenoptera: Braconidae): their probable role in stinging, oviposition and host selection process
Journal of Asia-Pacific Entomology 16 (2013) 343–348
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Sense organs on the ovipositor of Macrocentrus cingulum Brischke (Hymenoptera: Braconidae): their probable role in stinging, oviposition and host selection process☆ Tofael Ahmed a, b, 1, Tian-tao Zhang a, 1, Kang-lai He a, Shu-xiong Bai a, Zhen-ying Wang a,⁎ a b
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China Bangladesh Sugarcane Research Institute, Ishurdi-6620, Pabna, Bangladesh
a r t i c l e
i n f o
Article history: Received 3 January 2013 Revised 25 March 2013 Accepted 23 April 2013 Keywords: Morphology Sense organ Macrocentrus cingulum Ovipositor Electron microscopy
a b s t r a c t Parasitoid wasps from the insect order Hymenoptera can be deployed successfully as biological control agents for a number of pests, and have previously been introduced for the control of corn pest insect species from the Lepidopteran genus Ostrinia. Organs on the ovipositor of parasitoid wasps have mechanical and tactile senses that coordinate the complex movements of egg laying, and the ovipositor of Hymenopteran insects have evolved associated venom glands as part of their stinging defense. The ovipositor of parasitic wasps has evolved an additional function as a piercing organ that is required for the deposition of eggs within suitable host larvae. The morphology and ultrastructure of sense organs on the ovipositor and sheath of Macrocentrus cingulum Brischke (Hymenoptera: Braconidae) are described using scanning and transmission electron microscopy. Three types of sensilla trichodea were shown to be abundant on the outer sheath of the ovipositor, with types II and III being most distal, and the inner surface of the ovipositor covered with microtrichia, more densely near the apex. Sensilla coeloconica are distributed on both ventral and dorsal valves, while campaniform sensilla and secretory pores are only located on the dorsal valve. The olistheter-like interlocking mechanism, as well as the morphology of the ventral and dorsal valve tips and the ventral valve seal may be important in stinging, oviposition and in the host selection process. © 2013 The Authors. Published by Elsevier B.V. on behalf of Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society. All rights reserved.
Introduction The parasitic wasp Macrocentrus cingulum Brischke (Hymenoptera: Braconidae) (syn. M. grandii Goidanich) is a polyembryonic endoparasitoid of the Asian corn borer, Ostrinia furnacalis (Lepidoptera: Pyralidae) and the European corn borer, O. nubilalis (Edwards and Hopper, 1999; Hu et al., 2003). The native range of M. cingulum includes most of Europe as well as Asia, where it is distributed in Japan, Korea and China (Watanabe, 1967). Polyembryonic development is advantageous for rapid propagation of a biological control agent, whereby multiple offspring can be produced from a single zygote (Xu et al., 2010). Furthermore, oviposition by M. cingulum has been shown to be highly specific for species of the genus Ostrinia (Losey et al., 2001), which
☆ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ⁎ Corresponding author at: Institute of Plant Protection, Chinese Academy of Agricultural Sciences, No.2 West Yuanmingyuan Road, Beijing 100193, China. Tel.: +86 10 62815945; Fax: +86 10 62896114. E-mail address: [email protected] (Z. Wang). 1 Tofael AHMED and Tian-tao ZHANG contributed equally to this work.
identified this parasitoid as an ideal candidate for introduction into North America for the control of O. nubilalis populations in corn producing areas (Parker, 1931). The efficacy of parasitoid infection of host insects in corn fields has since been determined to range from 4 to 23% in the region (Siegel et al. 1986). The insect ovipositor is a complex structure associated with the 8th and 9th abdominal segments in females (Dweck et al., 2008). Among the endopterygote insects, members of the order Hymenoptera (bees and wasps) have evolved a well-developed ovipositor apparatus that performs multiple functions. The ancestral function of insect ovipositors is solely for the deposition of eggs onto suitable substrates, which involves the coordination of multiple sensory inputs. The ovipositors of Hymenoptera also have associated venom glands, and ducts for the injection of venom into prey insects, or for use as a defense mechanism, and this additional development required the evolution of unique ovipositor structures. Additionally, parasitic Hymenopteran wasps have specialized sensory organs (Le Ralec et al., 1996) which are required for the location, recognition and acceptance of suitable host insects for oviposition (Papp, 1974). These functions are coordinated by complex physical structures that originate from gonad tissues (Snodgrass, 1931, 1933). Previous studies of Hymenopteran ovipositor sense organs stylets and sheaths, by scanning and transmission electron microscopy (SEM
1226-8615/$ – see front matter © 2013 The Authors. Published by Elsevier B.V. on behalf of Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society. All rights reserved. http://dx.doi.org/10.1016/j.aspen.2013.04.015
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and TEM), have proven useful for defining species-specific morphologies (Le Ralec and Wajnberg, 1990; Brown and Anderson, 1998; Rahman et al., 1998; Vilhhelmsen, 2000; Roux et al., 2005; Dweck et al., 2008; Nacro and Nenon, 2009; Wang et al., 2010; Shah, 2012). Additionally, variation in ovipositor structures has been useful for the classification of parasitoids, as well as for determining phylogenetic relationships (Austin and Field, 1997) and relating ovipositor structure with possible function (Quicke and Fitton, 1995; Quicke et al., 1995; Dweck et al., 2008). The present study aims to determine the morphology and ultrastructure of sense organs on the ovipositor stylets and sheaths of M. cingulum Brischke (Hymenoptera: Braconidae), and to confirm the classification of the different types of sensilla using SEM and TEM. The goal of this research was to better understand the morphology of the sense organs on this structure, which is used in parasitoid host location and oviposition. Materials and methods Experimental Insects The parasitoid wasp, M. cingulum Brischke (Hymenoptera: Braconidae) was reared in the laboratory on the host insect, O. furnacalis, at a constant 25 °C temperature and a 16:8 light:dark photoperiod as described by Hu et al. (2003). All laboratory procedures were conducted at the Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China. Scanning electron microscopy (SEM) The ovipositor with sheaths was dissected from newly emerged M. cingulum parasitoids using a fine forceps, and was placed into a pre-fixation solution (2.5% glutaraldehyde in 0.1 M phosphate buffer solution (PBS), pH 7.4) and incubated at 4 °C for 24 h. Tissues were then changed to a post-fixation solution (1% buffered osmium tetraoxide) for overnight incubation at 4 °C. The specimens were then dehydrated by passage through a series of 30, 50, 70, 80, 90, 95 and 99.9% ethyl alcohol for 1 h each, and were then placed in absolute (100%) alcohol for 15–20 min. The ovipositor stylets were then mounted on stubs with carbon coated double sticky tape and were sputter coated with gold particles in a SPI-Module TM Sputter Coater. Examination and photography were performed using a SEM (JEOL JSM-35) with accelerating voltage set at 6.0 kV. Images were recorded digitally and stored electronically. The ovipositor and sensilla length were measured from digital images using Adobe Photoshop version CS3. At least six ovipositors were investigated, and 18 sensilla of each type were examined to calculate mean values.
Results Ovipositor morphology The female genitalia of M. cingulum showed that the basic organization is remarkably uniform among the Hymenopteran insects (Rahman et al., 1998). The ovipositors averaged 4.42 ± 0.41 mm in length, and consisted of ventral and dorsal valves and an ovipositor sheath (Fig. 1). When fused together, the internal lumen functions as a passageway for eggs and/or venom. The paired ventral valves are independent along most of their length, while dorsal valves were fused along their entire length. The tip of the dorsal valve was observed to be enlarged and slightly curved to form a characteristic notch like structure located approximately 1 mm from the distal tip (Fig. 2). The dorsal and ventral valves are interlocked and form a tongue-like structure called the “rachis” (Figs. 2 and 3). The dorsal and ventral valves are parallel with respect to one another, and the former fits into a grove of the latter called the “aulax” (Fig. 3). This interlocking system does not extend to the apex of the ovipositor. Longitudinal thin chitinous flaps form a seal-like structure, which protrudes into the egg canal from the medial ventral portion of each of the lower valves (Fig. 3), and may function as a seal to prevent the loss of egg/venom from the canal. The single segmented ovipositor sheath was flexible and well sclerotized, with transverse annulations from the base along the entire length, with the exception of the most distal part (Fig. 4). The sensilla trichodea covered the external surface from base to apex with an evenly spaced distribution. Three types of sensilla trichodea were distinctly observed at the apex of the ovipositor sheath. The apex of the ovipositor sheath was pointed and heavily sclerotized, and the inner surface was densely covered with flexible cutaneous microtrichia (Fig. 5). These microtrichia on the ovipositor sheath were unevenly distributed with the highest density at the apex (Fig. 5).
Sensory structures on the M. cingulum ovipositor Three types of sensilla were identified on the ventral and dorsal valves, as well as on the ovipositor sheath of the M. cingulum ovipositor
Transmission electron microscopy (TEM) The ovipositor stylets were dissected as described previously and was then placed into a pre-fixation solution (2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2) for 4 h at 4 °C. Fixation took place overnight in fixation buffer 25% glutaraldehyde and 10% acrolein diluted in a 0.1 M cacodylate buffer (pH 7.2). Samples were briefly washed with fixation buffer, placed into post-fixation buffer (1% osmium tetroxide in 0.1 M cacodylate buffer) for 1.5 h and then dehydrated as described previously. After dehydration, the fixed specimens were treated with propylene oxide, and were then transferred to embedding molds with Spur's resin and polymerized in an oven set at 60 °C for 24 h. Ultrathin sections were cut with an ultramicrotome (Reichert, OMU-3) using a diamond blade and were mounted on 50 mesh pioloform coated grids, stained with uranyl acetate and lead citrate, and finally observed in a TEM (JEOL-1200 EX) with accelerating voltage set at 6.0 kV, and images were then recorded digitally and stored on a computer.
Fig. 1. Ovipositor of M. cingulum (total view) 40×; “V” for ventral valve; and “D” for dorsal valve.
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Fig. 2. SEM micrograph of the tip of the ovipositor M. cingulum showing ventral (V) and dorsal (D) valves. Sensilla campaniform (Ca) on dorsal valve and sensilla coeloconica (COE) distributed over both valves. Barb-like apophyses (A) on ventral valve and rachis (R) on dorsal valve are also apparent.
shaft by SEM and TEM imaging. The structure and distribution of these sensilla types will be described individually in the following text.
Sensilla coeloconica Sensilla coeloconica (COE) were present along the distal two-thirds of the ventral valve and along most of the length of the dorsal valve. However, COE were found to be more densely distributed at the tip of the dorsal valve (Figs. 2 and 6), and occurred in a donut-shaped structure with a small formation protruding beyond the surface of the stylets. The external protuberance of these sensilla possessed a smooth surface, rounded body, and blunt tip that possessed a central pore-like opening. These sensilla could not be detected in ultrathin sections from TEM imaging.
Sensilla campaniform SEM results from M. cingulum showed that sensilla campaniform (Ca) were present and observed as irregular and depressed cuticle like structures, from which somewhat smooth raised domes protruded from the center (Fig. 6). These Ca possessed a tubular body just before entering the cuticle, which was innervated by a dendritic membrane (Fig. 7). Ca were shown to be located along the ventral and dorsal valves of the ovipositor, and were most densely distributed on the lateral side of the distal dorsal valve tip. Furthermore, each Ca was shown to be associated with one coeloconica sensillum (Fig. 2). Sensilla trichodea Sensilla trichodea (ST) were identified along the entire external surface of the ovipositor sheath through SEM imaging. ST type I were inserted into a socket which was elevated above the cuticle. Each ST rose from a conspicuous base and gradually tapered to a pointed tip. These sensilla were observed to have shallow longitudinal grooves that formed a spiral pattern around its surface. This sensillum also exhibits a thick cuticle and non-porous wall which was not innervated by dendrites, as seen in the TEM reprint (Fig. 8). ST type III were the shortest trichodea sensilla observed by SEM, whereas ST type II were longer than type III sensilla, but shorter than type I (Fig. 4). Both types II and III were similar to type I but were distinctly located at the distal end of the ovipositor sheath. Overall lengths of these three types I, II, and III were 65.65 ± 0.56, 38.82 ± 0.42, and 20.43 ± 0.48 μm, respectively. Secretory pores on the M. cingulum ovipositor Secretory pores (SP) were elongated and oval shaped with an eye-like appearance, having a central longitudinal slit, surrounded by a raised portion of cuticle (Figs. 2 and 9). These types of pores were observed only near the tip of the dorsal valve and below the rachis (R; Fig. 9), where they occurred in pairs on the dorsal valve. Each SP occurred longitudinally, and ran approximately parallel to the rachis on the tip of the dorsal valve. Discussion
Fig. 3. Transverse sections of ovipositor stylet, approximately one third from the distal end. The egg canal (EC) surrounded with paired ventral valve (V) and the two fused dorsal valves (D). The ventral and dorsal valves interlocking with rachis (R) which fit into aulax (A), and “L” for valve lumen and thin longitudinal chitinous flap that makes a seal (SI) like structure.
Morphological evidence can be used to infer the function of physical structures involved in parasitoid host location and oviposition (Quicke et al., 1994; Quicke and Fitton, 1995; Austin and Field, 1997). From the findings of our SEM and TEM studies of the ovipositor structure, we were able to summarize a possible correlation between the sensory equipment which was present, and their functions, during
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Fig. 4. SEM micrographs of third valve of the ovipositor of M. cingulum. A: micrograph of the pointed tip with three types of sensilla trichodea (ST I, ST II and ST III), B: sclerotized median wall with trichodea, and C: lateral wall with spring like annulation structure.
host selection and egg deposition in parasitoid actions. These aspects of M. cingulum ovipositor morphology are discussed in the sections below. Host selection process Three types of sensory organs and secretory pores were identified on the ventral and dorsal valves, and on the ovipositor sheath of the M. cingulum ovipositor, based on SEM and TEM observations. These types of sense organs have been described in other parasitic Hymenoptera (Le Ralec and Wajnberg, 1990; Brown and Anderson, 1998; van Lenteren et al., 2007; Dweck et al., 2008; Nacro and Nenon, 2009; Shah, 2012). Furthermore, the observation that these structures are conserved among species suggests they may have a common function and that they may be necessary for parasitism. Sensilla coeloconica were found on the distal two-thirds of the ventral valve, and on almost the entire length of the dorsal valve of M. cingulum. Such type of sensilla has also been described on the analogous structures of the parasitoid wasp species Trybliographa rapae (Brown and Anderson, 1998), Leptopilina heterotoma Nordlander (van Lenteren et al., 2007), Aprostocetus procerae (Nacro and Nenon, 2009) and Venturia canescens (Shah, 2012). Sensilla are known to function in the detection of external stimuli, and are likely involved in tactile sensing for the movement of the ovipositor on and through the host cuticle. Sensilla coeloconica are also commonly considered to be chemoreceptors or thermo-hygroreceptors (Altner and Prillinger, 1980) and have rarely been considered as purely mechanoreceptive (Brown and Anderson, 1998). These sensilla have relatively deep sockets for their size, which may provide protection from excessive bending or damage while in contact with the host. Thus, the structures do not protrude far above the ovipositor surface of M. cingulum, yet are likely sufficient for the detection of external stimuli. Sensilla campaniform are also present on the M. cingulum ovipositor, and were observed as raised domes with a smooth surface on the ventral and dorsal valves. These sensilla are innervated by only one neuron which possesses a tubular body, and most likely function in
tactile sensing. Le Ralec and Wajnberg (1990) and Cônsoli et al. (1999) also described campaniform sensilla on the ovipositor of Trichogramma maidis, T. galloi Zucchi and T. pretiosum Riley, and hypothesized that they may act as a mechanoreceptor for positioning of the ovipositor prior to stabbing the host. Nacro and Nenon (2009) similarly described these sensilla on the ovipositors of Aprostocetus procerae (Risbec) and Platygaster diplosisae (Risbec). Shah (2012) described the function of the campaniform sensilla on the ovipositor of Venturia canescens (Gravenhorst), and suggested them to be responsible for pressure regulation during the act of stabbing, and as a mechanoreceptor when the ovipositor bends during oviposition. Additionally, Dweck et al. (2008) reported that campaniform sensilla may be involved in perceiving tactile cues through which Habrobracon hebetor is able to discriminate between different stages of the host. The outer surface of M. cingulum ovipositor sheaths was densely covered with types I, II and III trichodea, which also have been found on the Braconidae wasp H. hebetor (Say) (Dweck et al., 2008), Cotesia sesamiae Cameron 1906 and C. flavipes Cameron 1891 (Obonyo, 2011) and Spathius agrili Yang (Wang et al., 2010) as well as the ichneumon wasp Megarhyssa atrata F. (Nenon et al., 1997), and the trichogrammatid T. maidis (Le Ralec and Wajnberg, 1990). From our TEM of thin sectioned M. cingulum ovipositor sheaths, trichodea were shown not to possess any pore system or sensory neurons, which suggests that they are not involved in olfactory or gustatory functions. Sensilla trichodea may, serve as mechanoreceptors during host searching, with potential roles played in response to air currents and physical vibrations. Stinging mechanism The ovipositor plays an important role in host discrimination in Braconid wasps, and is often used by females to probe a potential host prior to oviposition (Canale and Raspi, 2000). Most parasitoid ovipositor tips contain at least one or more types of sensilla the structures of which are known to be involved in the detection of both chemical and mechanical stimuli. After finding and selecting a suitable larval host, the
Fig. 5. SEM micrographs of third valve of the ovipositor of M. cingulum. Densely covered with microtrichia in inner-surface of tip (A), and median inside wall with microtrichia (B and C).
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Fig. 6. Electron micrograph of the approximately one third regions of the dorsal valve and tip of the dorsal valve. A: SEM image of nonsprouted sensilla campaniform (Ca) and sensilla coeloconica (COE) inside cuticle depression, and B: concentrated sensilla coeloconica (COE) with a blunt tip and central pore at the tip of dorsal valve.
Fig. 9. SEM micrograph of secretory pore (SP) on the tip of ovipositor just below the rachis, “R” for rachis.
Fig. 7. Transverse sections of the dorsal valve lumen of the ovipositor, approximately one third from the distal end show the tubular body (black arrow) of a campaniform sensillum.
female M. cingulum abdomen bends forward and stings the host by penetrating the host's integument with the unsheathed ovipositor. Barbs and serrations are located at the distal end of the ventral valve on the M. cingulum ovipositor, which are likely used for piercing the host integument. The proportions and relative density of these serrated structures vary widely among taxa (Rahman et al., 1998) and may have evolved in response to variations in host cuticle thickness and resilience to penetration. In addition to function during piercing the host integument, the serrations may also act to hold the ovipositor in position as oviposition takes place (Le Ralec and Wajnberg, 1990; Brown and Anderson, 1998; Cônsoli et al., 1999; Dweck et al., 2008; Nacro and Nenon, 2009; Wang et al., 2010). In contrast, the dorsal valves of the ovipositor of M. cingulum are blunt and round at the apex and are unsuited for penetrating through host cuticle. However, serial sections through the ovipositor reveal an olistheter-like interlocking of the mediodorsal part of the first valve just prior to the apex, thereby possibly forming a slender envenomation
tube. A similar type of olistheter-like interlocking structure has previously been observed near the ovipositor tip in several other Braconids (Quicke et al., 1994) which suggests this may be a shared ancestral trait of Braconid wasps. The ovipositor of M. cingulum becomes flexible due to the presence of annulations of the ovipositor sheath, which likely allows for flexibility during stinging, and may prevent the ovipositor sheath from interfering with movements of the ovipositor shaft. During the stinging process, as the ovipositor shaft begins to penetrate the host integument, the ovipositor sheath slides along the ovipositor shaft to form a gradually expanding loop. These observations indicate that the ovipositor sheath might serve as support when retracted during stinging, or as a general protection when sheathed over the ovipositor. The inner surface of the ovipositor sheath is also densely covered with microtrichia, which probably serve to clean the ovipositor sensilla of the residual host integument between different stinging actions, and may be important for keeping the ovipositor shaft functional. The microtrichia are most densely concentrated at the tip of the ovipositor sheath, which might rationally have co-evolved to be in proximity to the sensilla that are concentrated at the tip of the ovipositor. Microtrichia have been shown to be dense along the inner surface of the ovipositor sheath in some other Braconid species (Le Ralec and Wajnberg, 1990; Dweck et al., 2008; Nacro and Nenon, 2009) and suggest a shared function within this group of wasps. Secretory pores are commonly observed at the ovipositor tip of Hymenopteran parasitoid wasps (Copland and King, 1972; Shah, 2012), including Habrocytus cerealellae Ashmead (Fulton, 1933) and Nasonia vitripennis Walker (King, 1962; King and Rafai, 1970). Although these pores were also described by Ganesalingam (1972), the probable function was not described. Lyngnes and Sunmore, 1960 proposed that the most probable function of the SP may be to secrete lubricating fluids that reduce friction during stabbing and cuticle penetration.
Fig. 8. Transverse section of the proximal part of sensilla trichodea. A: Sensilla trichodea III, scale bar = 0.5 μm, B: sensilla trichodea II, scale bar = 1 μm, and C: sensilla trichodea I, scale bar = 0.5 μm; the sensillum wall (SW) is a grooved and non-porous thick cuticle and a sensillum lymph (SL), which is not innervated by dendrites.
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Ovipositional mechanism After locating a potential larval host, female M. cingulum mounts and examines the external surface prior to acceptance. If deemed an acceptable host, the host is restrained while the much-dilated abdomen curves underneath her. The ovipositor shaft then extends to its full length using the ovi-sheath, and becomes slightly unsheathed. The eggs are transferred to the tip of the ovipositor via the oviduct by sliding the dorsal and ventral valves, back and forth upon one another, and the ovipositor may be positioned with respect to the host by bending the ovipositor slightly in any direction using the sheath. When the female detects the larva, thrusts are made frequently until the cuticle is penetrated, after which an egg is quickly laid, and the ovipositor is almost immediately withdrawn, straightened out, and sheathed (Parker, 1931). Conclusion Morphological descriptions made from SEM and TEM imaging of the M. cingulum ovipositor provide evidence for the shared evolution of structures necessary for female parasitoids. Three types of sensilla and secretory pores are present and likely function in the coordination of host sensing and penetration as well as egg deposition. Our observations confirm those of previous studies that, the sensilla of coeloconica and campaniform, mainly function as thermo-hygroreceptors for detecting host location, and in providing tactile and chemosensory discrimination between the different species and stages of the host, respectively. Secretory pores provide fluid that reduces friction during stinging and stabbing. Sensilla trichodea act as a mechanoreceptor to respond to air current and vibration during host searching. Acknowledgment This work was supported by the China Agriculture Research System (CARS-02). The authors gratefully acknowledge Dr. Brad Coates, Research Geneticist, USDA-ARS, Iowa, USA for his comments and suggestions on this manuscript. References Altner, H., Prillinger, L., 1980. Ultrastructure of invertebrate chemo-, thermo- and hygroreceptors and its functional significance. In: Bourne, G.H., Danielli, J.F. (Eds.), International Review of Cytology. Academic Press, pp. 69–139. Austin, A.D., Field, S.A., 1997. The ovipositor system of Scelionid and Platygastrid wasps (Hymenoptera: Platygastroidea): comparative morphology and phylogenetic implications. Invertebr. Syst. 11, 1–87. http://dx.doi.org/10.1071/IT95048. 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. http://dx.doi.org/10.1016/s0022-1910(98)00072-9. Canale, A., Raspi, A., 2000. Host location and oviposition behaviour in Opius concolor (Hymenoptera: Braconidae). Ent. Prob. 31, 25–32. Cônsoli, F.L., Kitajima, E.W., Parra, J.R.P., 1999. Sensilla on the antenna and ovipositor of the parasitic wasps Trichogramma galloi Zucchi and T. pretiosum Riley (Hym., trichogrammatidae). Microsc. Res. Tech. 45, 313–324. http://dx.doi.org/10.1002/ (sici)1097-0029(19990515/01)45:4/5b313::aid-jemt15>3.0.co;2-4. Copland, M.J.W., King, P.E., 1972. The structure of the female reproductive system in the Torymidae (Hymenoptera: Chalcidoidea). Trans. Roy. Soc. London 124, 191–212. http://dx.doi.org/10.1111/j.1365-2311.1972.tb00363.x. Dweck, H., Gadallah, N., Darwish, E., 2008. Structure and sensory equipment of the ovipositor of Habrobracon hebetor (Say) (Hymenoptera: Braconidae). Micron 39, 1255–1261. http://dx.doi.org/10.1016/j.micron.2008.03.012. Edwards, O.R., Hopper, K.R., 1999. Using superparasitism by a stem borer parasitoid to infer a host refuge. Ecol. Entomol. 24, 7–12. http://dx.doi.org/10.1046/j.13652311.1999.00165.x. Fulton, B.B., 1933. Notes on Habrocytus cerealellae, parasite of the Angoumois grain moth. Ann. Entomol. Soc. Am. 26, 536–553. Ganesalingam, V.K., 1972. Anatomy and histology of the sense organs of the ovipositor of the ichneumonid wasp. Devorgilla canescens. J. Insect Physiol. 18, 1857–1867.
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Sense organs on the ovipositor of Macrocentrus cingulum Brischke (Hymenoptera: Braconidae): their probable role in stinging, oviposition and host selection process☆ Tofael Ahmed a, b, 1, Tian-tao Zhang a, 1, Kang-lai He a, Shu-xiong Bai a, Zhen-ying Wang a,⁎ a b
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China Bangladesh Sugarcane Research Institute, Ishurdi-6620, Pabna, Bangladesh
a r t i c l e
i n f o
Article history: Received 3 January 2013 Revised 25 March 2013 Accepted 23 April 2013 Keywords: Morphology Sense organ Macrocentrus cingulum Ovipositor Electron microscopy
a b s t r a c t Parasitoid wasps from the insect order Hymenoptera can be deployed successfully as biological control agents for a number of pests, and have previously been introduced for the control of corn pest insect species from the Lepidopteran genus Ostrinia. Organs on the ovipositor of parasitoid wasps have mechanical and tactile senses that coordinate the complex movements of egg laying, and the ovipositor of Hymenopteran insects have evolved associated venom glands as part of their stinging defense. The ovipositor of parasitic wasps has evolved an additional function as a piercing organ that is required for the deposition of eggs within suitable host larvae. The morphology and ultrastructure of sense organs on the ovipositor and sheath of Macrocentrus cingulum Brischke (Hymenoptera: Braconidae) are described using scanning and transmission electron microscopy. Three types of sensilla trichodea were shown to be abundant on the outer sheath of the ovipositor, with types II and III being most distal, and the inner surface of the ovipositor covered with microtrichia, more densely near the apex. Sensilla coeloconica are distributed on both ventral and dorsal valves, while campaniform sensilla and secretory pores are only located on the dorsal valve. The olistheter-like interlocking mechanism, as well as the morphology of the ventral and dorsal valve tips and the ventral valve seal may be important in stinging, oviposition and in the host selection process. © 2013 The Authors. Published by Elsevier B.V. on behalf of Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society. All rights reserved.
Introduction The parasitic wasp Macrocentrus cingulum Brischke (Hymenoptera: Braconidae) (syn. M. grandii Goidanich) is a polyembryonic endoparasitoid of the Asian corn borer, Ostrinia furnacalis (Lepidoptera: Pyralidae) and the European corn borer, O. nubilalis (Edwards and Hopper, 1999; Hu et al., 2003). The native range of M. cingulum includes most of Europe as well as Asia, where it is distributed in Japan, Korea and China (Watanabe, 1967). Polyembryonic development is advantageous for rapid propagation of a biological control agent, whereby multiple offspring can be produced from a single zygote (Xu et al., 2010). Furthermore, oviposition by M. cingulum has been shown to be highly specific for species of the genus Ostrinia (Losey et al., 2001), which
☆ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ⁎ Corresponding author at: Institute of Plant Protection, Chinese Academy of Agricultural Sciences, No.2 West Yuanmingyuan Road, Beijing 100193, China. Tel.: +86 10 62815945; Fax: +86 10 62896114. E-mail address: [email protected] (Z. Wang). 1 Tofael AHMED and Tian-tao ZHANG contributed equally to this work.
identified this parasitoid as an ideal candidate for introduction into North America for the control of O. nubilalis populations in corn producing areas (Parker, 1931). The efficacy of parasitoid infection of host insects in corn fields has since been determined to range from 4 to 23% in the region (Siegel et al. 1986). The insect ovipositor is a complex structure associated with the 8th and 9th abdominal segments in females (Dweck et al., 2008). Among the endopterygote insects, members of the order Hymenoptera (bees and wasps) have evolved a well-developed ovipositor apparatus that performs multiple functions. The ancestral function of insect ovipositors is solely for the deposition of eggs onto suitable substrates, which involves the coordination of multiple sensory inputs. The ovipositors of Hymenoptera also have associated venom glands, and ducts for the injection of venom into prey insects, or for use as a defense mechanism, and this additional development required the evolution of unique ovipositor structures. Additionally, parasitic Hymenopteran wasps have specialized sensory organs (Le Ralec et al., 1996) which are required for the location, recognition and acceptance of suitable host insects for oviposition (Papp, 1974). These functions are coordinated by complex physical structures that originate from gonad tissues (Snodgrass, 1931, 1933). Previous studies of Hymenopteran ovipositor sense organs stylets and sheaths, by scanning and transmission electron microscopy (SEM
1226-8615/$ – see front matter © 2013 The Authors. Published by Elsevier B.V. on behalf of Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society. All rights reserved. http://dx.doi.org/10.1016/j.aspen.2013.04.015
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and TEM), have proven useful for defining species-specific morphologies (Le Ralec and Wajnberg, 1990; Brown and Anderson, 1998; Rahman et al., 1998; Vilhhelmsen, 2000; Roux et al., 2005; Dweck et al., 2008; Nacro and Nenon, 2009; Wang et al., 2010; Shah, 2012). Additionally, variation in ovipositor structures has been useful for the classification of parasitoids, as well as for determining phylogenetic relationships (Austin and Field, 1997) and relating ovipositor structure with possible function (Quicke and Fitton, 1995; Quicke et al., 1995; Dweck et al., 2008). The present study aims to determine the morphology and ultrastructure of sense organs on the ovipositor stylets and sheaths of M. cingulum Brischke (Hymenoptera: Braconidae), and to confirm the classification of the different types of sensilla using SEM and TEM. The goal of this research was to better understand the morphology of the sense organs on this structure, which is used in parasitoid host location and oviposition. Materials and methods Experimental Insects The parasitoid wasp, M. cingulum Brischke (Hymenoptera: Braconidae) was reared in the laboratory on the host insect, O. furnacalis, at a constant 25 °C temperature and a 16:8 light:dark photoperiod as described by Hu et al. (2003). All laboratory procedures were conducted at the Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China. Scanning electron microscopy (SEM) The ovipositor with sheaths was dissected from newly emerged M. cingulum parasitoids using a fine forceps, and was placed into a pre-fixation solution (2.5% glutaraldehyde in 0.1 M phosphate buffer solution (PBS), pH 7.4) and incubated at 4 °C for 24 h. Tissues were then changed to a post-fixation solution (1% buffered osmium tetraoxide) for overnight incubation at 4 °C. The specimens were then dehydrated by passage through a series of 30, 50, 70, 80, 90, 95 and 99.9% ethyl alcohol for 1 h each, and were then placed in absolute (100%) alcohol for 15–20 min. The ovipositor stylets were then mounted on stubs with carbon coated double sticky tape and were sputter coated with gold particles in a SPI-Module TM Sputter Coater. Examination and photography were performed using a SEM (JEOL JSM-35) with accelerating voltage set at 6.0 kV. Images were recorded digitally and stored electronically. The ovipositor and sensilla length were measured from digital images using Adobe Photoshop version CS3. At least six ovipositors were investigated, and 18 sensilla of each type were examined to calculate mean values.
Results Ovipositor morphology The female genitalia of M. cingulum showed that the basic organization is remarkably uniform among the Hymenopteran insects (Rahman et al., 1998). The ovipositors averaged 4.42 ± 0.41 mm in length, and consisted of ventral and dorsal valves and an ovipositor sheath (Fig. 1). When fused together, the internal lumen functions as a passageway for eggs and/or venom. The paired ventral valves are independent along most of their length, while dorsal valves were fused along their entire length. The tip of the dorsal valve was observed to be enlarged and slightly curved to form a characteristic notch like structure located approximately 1 mm from the distal tip (Fig. 2). The dorsal and ventral valves are interlocked and form a tongue-like structure called the “rachis” (Figs. 2 and 3). The dorsal and ventral valves are parallel with respect to one another, and the former fits into a grove of the latter called the “aulax” (Fig. 3). This interlocking system does not extend to the apex of the ovipositor. Longitudinal thin chitinous flaps form a seal-like structure, which protrudes into the egg canal from the medial ventral portion of each of the lower valves (Fig. 3), and may function as a seal to prevent the loss of egg/venom from the canal. The single segmented ovipositor sheath was flexible and well sclerotized, with transverse annulations from the base along the entire length, with the exception of the most distal part (Fig. 4). The sensilla trichodea covered the external surface from base to apex with an evenly spaced distribution. Three types of sensilla trichodea were distinctly observed at the apex of the ovipositor sheath. The apex of the ovipositor sheath was pointed and heavily sclerotized, and the inner surface was densely covered with flexible cutaneous microtrichia (Fig. 5). These microtrichia on the ovipositor sheath were unevenly distributed with the highest density at the apex (Fig. 5).
Sensory structures on the M. cingulum ovipositor Three types of sensilla were identified on the ventral and dorsal valves, as well as on the ovipositor sheath of the M. cingulum ovipositor
Transmission electron microscopy (TEM) The ovipositor stylets were dissected as described previously and was then placed into a pre-fixation solution (2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2) for 4 h at 4 °C. Fixation took place overnight in fixation buffer 25% glutaraldehyde and 10% acrolein diluted in a 0.1 M cacodylate buffer (pH 7.2). Samples were briefly washed with fixation buffer, placed into post-fixation buffer (1% osmium tetroxide in 0.1 M cacodylate buffer) for 1.5 h and then dehydrated as described previously. After dehydration, the fixed specimens were treated with propylene oxide, and were then transferred to embedding molds with Spur's resin and polymerized in an oven set at 60 °C for 24 h. Ultrathin sections were cut with an ultramicrotome (Reichert, OMU-3) using a diamond blade and were mounted on 50 mesh pioloform coated grids, stained with uranyl acetate and lead citrate, and finally observed in a TEM (JEOL-1200 EX) with accelerating voltage set at 6.0 kV, and images were then recorded digitally and stored on a computer.
Fig. 1. Ovipositor of M. cingulum (total view) 40×; “V” for ventral valve; and “D” for dorsal valve.
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Fig. 2. SEM micrograph of the tip of the ovipositor M. cingulum showing ventral (V) and dorsal (D) valves. Sensilla campaniform (Ca) on dorsal valve and sensilla coeloconica (COE) distributed over both valves. Barb-like apophyses (A) on ventral valve and rachis (R) on dorsal valve are also apparent.
shaft by SEM and TEM imaging. The structure and distribution of these sensilla types will be described individually in the following text.
Sensilla coeloconica Sensilla coeloconica (COE) were present along the distal two-thirds of the ventral valve and along most of the length of the dorsal valve. However, COE were found to be more densely distributed at the tip of the dorsal valve (Figs. 2 and 6), and occurred in a donut-shaped structure with a small formation protruding beyond the surface of the stylets. The external protuberance of these sensilla possessed a smooth surface, rounded body, and blunt tip that possessed a central pore-like opening. These sensilla could not be detected in ultrathin sections from TEM imaging.
Sensilla campaniform SEM results from M. cingulum showed that sensilla campaniform (Ca) were present and observed as irregular and depressed cuticle like structures, from which somewhat smooth raised domes protruded from the center (Fig. 6). These Ca possessed a tubular body just before entering the cuticle, which was innervated by a dendritic membrane (Fig. 7). Ca were shown to be located along the ventral and dorsal valves of the ovipositor, and were most densely distributed on the lateral side of the distal dorsal valve tip. Furthermore, each Ca was shown to be associated with one coeloconica sensillum (Fig. 2). Sensilla trichodea Sensilla trichodea (ST) were identified along the entire external surface of the ovipositor sheath through SEM imaging. ST type I were inserted into a socket which was elevated above the cuticle. Each ST rose from a conspicuous base and gradually tapered to a pointed tip. These sensilla were observed to have shallow longitudinal grooves that formed a spiral pattern around its surface. This sensillum also exhibits a thick cuticle and non-porous wall which was not innervated by dendrites, as seen in the TEM reprint (Fig. 8). ST type III were the shortest trichodea sensilla observed by SEM, whereas ST type II were longer than type III sensilla, but shorter than type I (Fig. 4). Both types II and III were similar to type I but were distinctly located at the distal end of the ovipositor sheath. Overall lengths of these three types I, II, and III were 65.65 ± 0.56, 38.82 ± 0.42, and 20.43 ± 0.48 μm, respectively. Secretory pores on the M. cingulum ovipositor Secretory pores (SP) were elongated and oval shaped with an eye-like appearance, having a central longitudinal slit, surrounded by a raised portion of cuticle (Figs. 2 and 9). These types of pores were observed only near the tip of the dorsal valve and below the rachis (R; Fig. 9), where they occurred in pairs on the dorsal valve. Each SP occurred longitudinally, and ran approximately parallel to the rachis on the tip of the dorsal valve. Discussion
Fig. 3. Transverse sections of ovipositor stylet, approximately one third from the distal end. The egg canal (EC) surrounded with paired ventral valve (V) and the two fused dorsal valves (D). The ventral and dorsal valves interlocking with rachis (R) which fit into aulax (A), and “L” for valve lumen and thin longitudinal chitinous flap that makes a seal (SI) like structure.
Morphological evidence can be used to infer the function of physical structures involved in parasitoid host location and oviposition (Quicke et al., 1994; Quicke and Fitton, 1995; Austin and Field, 1997). From the findings of our SEM and TEM studies of the ovipositor structure, we were able to summarize a possible correlation between the sensory equipment which was present, and their functions, during
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Fig. 4. SEM micrographs of third valve of the ovipositor of M. cingulum. A: micrograph of the pointed tip with three types of sensilla trichodea (ST I, ST II and ST III), B: sclerotized median wall with trichodea, and C: lateral wall with spring like annulation structure.
host selection and egg deposition in parasitoid actions. These aspects of M. cingulum ovipositor morphology are discussed in the sections below. Host selection process Three types of sensory organs and secretory pores were identified on the ventral and dorsal valves, and on the ovipositor sheath of the M. cingulum ovipositor, based on SEM and TEM observations. These types of sense organs have been described in other parasitic Hymenoptera (Le Ralec and Wajnberg, 1990; Brown and Anderson, 1998; van Lenteren et al., 2007; Dweck et al., 2008; Nacro and Nenon, 2009; Shah, 2012). Furthermore, the observation that these structures are conserved among species suggests they may have a common function and that they may be necessary for parasitism. Sensilla coeloconica were found on the distal two-thirds of the ventral valve, and on almost the entire length of the dorsal valve of M. cingulum. Such type of sensilla has also been described on the analogous structures of the parasitoid wasp species Trybliographa rapae (Brown and Anderson, 1998), Leptopilina heterotoma Nordlander (van Lenteren et al., 2007), Aprostocetus procerae (Nacro and Nenon, 2009) and Venturia canescens (Shah, 2012). Sensilla are known to function in the detection of external stimuli, and are likely involved in tactile sensing for the movement of the ovipositor on and through the host cuticle. Sensilla coeloconica are also commonly considered to be chemoreceptors or thermo-hygroreceptors (Altner and Prillinger, 1980) and have rarely been considered as purely mechanoreceptive (Brown and Anderson, 1998). These sensilla have relatively deep sockets for their size, which may provide protection from excessive bending or damage while in contact with the host. Thus, the structures do not protrude far above the ovipositor surface of M. cingulum, yet are likely sufficient for the detection of external stimuli. Sensilla campaniform are also present on the M. cingulum ovipositor, and were observed as raised domes with a smooth surface on the ventral and dorsal valves. These sensilla are innervated by only one neuron which possesses a tubular body, and most likely function in
tactile sensing. Le Ralec and Wajnberg (1990) and Cônsoli et al. (1999) also described campaniform sensilla on the ovipositor of Trichogramma maidis, T. galloi Zucchi and T. pretiosum Riley, and hypothesized that they may act as a mechanoreceptor for positioning of the ovipositor prior to stabbing the host. Nacro and Nenon (2009) similarly described these sensilla on the ovipositors of Aprostocetus procerae (Risbec) and Platygaster diplosisae (Risbec). Shah (2012) described the function of the campaniform sensilla on the ovipositor of Venturia canescens (Gravenhorst), and suggested them to be responsible for pressure regulation during the act of stabbing, and as a mechanoreceptor when the ovipositor bends during oviposition. Additionally, Dweck et al. (2008) reported that campaniform sensilla may be involved in perceiving tactile cues through which Habrobracon hebetor is able to discriminate between different stages of the host. The outer surface of M. cingulum ovipositor sheaths was densely covered with types I, II and III trichodea, which also have been found on the Braconidae wasp H. hebetor (Say) (Dweck et al., 2008), Cotesia sesamiae Cameron 1906 and C. flavipes Cameron 1891 (Obonyo, 2011) and Spathius agrili Yang (Wang et al., 2010) as well as the ichneumon wasp Megarhyssa atrata F. (Nenon et al., 1997), and the trichogrammatid T. maidis (Le Ralec and Wajnberg, 1990). From our TEM of thin sectioned M. cingulum ovipositor sheaths, trichodea were shown not to possess any pore system or sensory neurons, which suggests that they are not involved in olfactory or gustatory functions. Sensilla trichodea may, serve as mechanoreceptors during host searching, with potential roles played in response to air currents and physical vibrations. Stinging mechanism The ovipositor plays an important role in host discrimination in Braconid wasps, and is often used by females to probe a potential host prior to oviposition (Canale and Raspi, 2000). Most parasitoid ovipositor tips contain at least one or more types of sensilla the structures of which are known to be involved in the detection of both chemical and mechanical stimuli. After finding and selecting a suitable larval host, the
Fig. 5. SEM micrographs of third valve of the ovipositor of M. cingulum. Densely covered with microtrichia in inner-surface of tip (A), and median inside wall with microtrichia (B and C).
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Fig. 6. Electron micrograph of the approximately one third regions of the dorsal valve and tip of the dorsal valve. A: SEM image of nonsprouted sensilla campaniform (Ca) and sensilla coeloconica (COE) inside cuticle depression, and B: concentrated sensilla coeloconica (COE) with a blunt tip and central pore at the tip of dorsal valve.
Fig. 9. SEM micrograph of secretory pore (SP) on the tip of ovipositor just below the rachis, “R” for rachis.
Fig. 7. Transverse sections of the dorsal valve lumen of the ovipositor, approximately one third from the distal end show the tubular body (black arrow) of a campaniform sensillum.
female M. cingulum abdomen bends forward and stings the host by penetrating the host's integument with the unsheathed ovipositor. Barbs and serrations are located at the distal end of the ventral valve on the M. cingulum ovipositor, which are likely used for piercing the host integument. The proportions and relative density of these serrated structures vary widely among taxa (Rahman et al., 1998) and may have evolved in response to variations in host cuticle thickness and resilience to penetration. In addition to function during piercing the host integument, the serrations may also act to hold the ovipositor in position as oviposition takes place (Le Ralec and Wajnberg, 1990; Brown and Anderson, 1998; Cônsoli et al., 1999; Dweck et al., 2008; Nacro and Nenon, 2009; Wang et al., 2010). In contrast, the dorsal valves of the ovipositor of M. cingulum are blunt and round at the apex and are unsuited for penetrating through host cuticle. However, serial sections through the ovipositor reveal an olistheter-like interlocking of the mediodorsal part of the first valve just prior to the apex, thereby possibly forming a slender envenomation
tube. A similar type of olistheter-like interlocking structure has previously been observed near the ovipositor tip in several other Braconids (Quicke et al., 1994) which suggests this may be a shared ancestral trait of Braconid wasps. The ovipositor of M. cingulum becomes flexible due to the presence of annulations of the ovipositor sheath, which likely allows for flexibility during stinging, and may prevent the ovipositor sheath from interfering with movements of the ovipositor shaft. During the stinging process, as the ovipositor shaft begins to penetrate the host integument, the ovipositor sheath slides along the ovipositor shaft to form a gradually expanding loop. These observations indicate that the ovipositor sheath might serve as support when retracted during stinging, or as a general protection when sheathed over the ovipositor. The inner surface of the ovipositor sheath is also densely covered with microtrichia, which probably serve to clean the ovipositor sensilla of the residual host integument between different stinging actions, and may be important for keeping the ovipositor shaft functional. The microtrichia are most densely concentrated at the tip of the ovipositor sheath, which might rationally have co-evolved to be in proximity to the sensilla that are concentrated at the tip of the ovipositor. Microtrichia have been shown to be dense along the inner surface of the ovipositor sheath in some other Braconid species (Le Ralec and Wajnberg, 1990; Dweck et al., 2008; Nacro and Nenon, 2009) and suggest a shared function within this group of wasps. Secretory pores are commonly observed at the ovipositor tip of Hymenopteran parasitoid wasps (Copland and King, 1972; Shah, 2012), including Habrocytus cerealellae Ashmead (Fulton, 1933) and Nasonia vitripennis Walker (King, 1962; King and Rafai, 1970). Although these pores were also described by Ganesalingam (1972), the probable function was not described. Lyngnes and Sunmore, 1960 proposed that the most probable function of the SP may be to secrete lubricating fluids that reduce friction during stabbing and cuticle penetration.
Fig. 8. Transverse section of the proximal part of sensilla trichodea. A: Sensilla trichodea III, scale bar = 0.5 μm, B: sensilla trichodea II, scale bar = 1 μm, and C: sensilla trichodea I, scale bar = 0.5 μm; the sensillum wall (SW) is a grooved and non-porous thick cuticle and a sensillum lymph (SL), which is not innervated by dendrites.
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Ovipositional mechanism After locating a potential larval host, female M. cingulum mounts and examines the external surface prior to acceptance. If deemed an acceptable host, the host is restrained while the much-dilated abdomen curves underneath her. The ovipositor shaft then extends to its full length using the ovi-sheath, and becomes slightly unsheathed. The eggs are transferred to the tip of the ovipositor via the oviduct by sliding the dorsal and ventral valves, back and forth upon one another, and the ovipositor may be positioned with respect to the host by bending the ovipositor slightly in any direction using the sheath. When the female detects the larva, thrusts are made frequently until the cuticle is penetrated, after which an egg is quickly laid, and the ovipositor is almost immediately withdrawn, straightened out, and sheathed (Parker, 1931). Conclusion Morphological descriptions made from SEM and TEM imaging of the M. cingulum ovipositor provide evidence for the shared evolution of structures necessary for female parasitoids. Three types of sensilla and secretory pores are present and likely function in the coordination of host sensing and penetration as well as egg deposition. Our observations confirm those of previous studies that, the sensilla of coeloconica and campaniform, mainly function as thermo-hygroreceptors for detecting host location, and in providing tactile and chemosensory discrimination between the different species and stages of the host, respectively. Secretory pores provide fluid that reduces friction during stinging and stabbing. Sensilla trichodea act as a mechanoreceptor to respond to air current and vibration during host searching. Acknowledgment This work was supported by the China Agriculture Research System (CARS-02). The authors gratefully acknowledge Dr. Brad Coates, Research Geneticist, USDA-ARS, Iowa, USA for his comments and suggestions on this manuscript. References Altner, H., Prillinger, L., 1980. Ultrastructure of invertebrate chemo-, thermo- and hygroreceptors and its functional significance. In: Bourne, G.H., Danielli, J.F. (Eds.), International Review of Cytology. Academic Press, pp. 69–139. Austin, A.D., Field, S.A., 1997. The ovipositor system of Scelionid and Platygastrid wasps (Hymenoptera: Platygastroidea): comparative morphology and phylogenetic implications. Invertebr. Syst. 11, 1–87. http://dx.doi.org/10.1071/IT95048. 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. http://dx.doi.org/10.1016/s0022-1910(98)00072-9. Canale, A., Raspi, A., 2000. Host location and oviposition behaviour in Opius concolor (Hymenoptera: Braconidae). Ent. Prob. 31, 25–32. Cônsoli, F.L., Kitajima, E.W., Parra, J.R.P., 1999. Sensilla on the antenna and ovipositor of the parasitic wasps Trichogramma galloi Zucchi and T. pretiosum Riley (Hym., trichogrammatidae). Microsc. Res. Tech. 45, 313–324. http://dx.doi.org/10.1002/ (sici)1097-0029(19990515/01)45:4/5b313::aid-jemt15>3.0.co;2-4. Copland, M.J.W., King, P.E., 1972. The structure of the female reproductive system in the Torymidae (Hymenoptera: Chalcidoidea). Trans. Roy. Soc. London 124, 191–212. http://dx.doi.org/10.1111/j.1365-2311.1972.tb00363.x. Dweck, H., Gadallah, N., Darwish, E., 2008. Structure and sensory equipment of the ovipositor of Habrobracon hebetor (Say) (Hymenoptera: Braconidae). Micron 39, 1255–1261. http://dx.doi.org/10.1016/j.micron.2008.03.012. Edwards, O.R., Hopper, K.R., 1999. Using superparasitism by a stem borer parasitoid to infer a host refuge. Ecol. Entomol. 24, 7–12. http://dx.doi.org/10.1046/j.13652311.1999.00165.x. Fulton, B.B., 1933. Notes on Habrocytus cerealellae, parasite of the Angoumois grain moth. Ann. Entomol. Soc. Am. 26, 536–553. Ganesalingam, V.K., 1972. Anatomy and histology of the sense organs of the ovipositor of the ichneumonid wasp. Devorgilla canescens. J. Insect Physiol. 18, 1857–1867.
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