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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Journal of Asia-Paciﬁc Entomology

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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 State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China b Bangladesh Sugarcane Research Institute, Ishurdi-6620, Pabna, Bangladesh article info abstract

Article history: Parasitoid wasps from the insect order Hymenoptera can be deployed successfully as biological control Received 3 January 2013 agents for a number of pests, and have previously been introduced for the control of corn pest insect species Revised 25 March 2013 from the Lepidopteran genus Ostrinia. Organs on the ovipositor of parasitoid wasps have mechanical and tac- Accepted 23 April 2013 tile 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 Keywords: wasps has evolved an additional function as a piercing organ that is required for the deposition of eggs within Morphology Sense organ suitable host larvae. The morphology and ultrastructure of sense organs on the ovipositor and sheath of Macrocentrus cingulum Macrocentrus cingulum Brischke (Hymenoptera: Braconidae) are described using scanning and transmission Ovipositor electron microscopy. Three types of sensilla trichodea were shown to be abundant on the outer sheath of the Electron microscopy 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 identified this parasitoid as an ideal candidate for introduction into North America for the control of O. nubilalis populations in corn produc- The parasitic wasp Macrocentrus cingulum Brischke (Hymenoptera: ing areas (Parker, 1931). The efficacy of parasitoid infection of host in- Braconidae) (syn. M. grandii Goidanich) is a polyembryonic endo- sects in corn fields has since been determined to range from 4 to 23% parasitoid of the Asian corn borer, Ostrinia furnacalis (Lepidoptera: in the region (Siegel et al. 1986). Pyralidae) and the European corn borer, O. nubilalis (Edwards and The insect ovipositor is a complex structure associated with the Hopper, 1999; Hu et al., 2003). The native range of M. cingulum includes 8th and 9th abdominal segments in females (Dweck et al., 2008). most of Europe as well as Asia, where it is distributed in Japan, Korea Among the endopterygote insects, members of the order Hymenoptera and China (Watanabe, 1967). Polyembryonic development is advanta- (bees and wasps) have evolved a well-developed ovipositor apparatus geous for rapid propagation of a biological control agent, whereby mul- that performs multiple functions. The ancestral function of insect ovi- tiple offspring can be produced from a single zygote (Xu et al., 2010). positors is solely for the deposition of eggs onto suitable substrates, Furthermore, oviposition by M. cingulum has been shown to be highly which involves the coordination of multiple sensory inputs. The ovipos- specific for species of the genus Ostrinia (Losey et al., 2001), which itors 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

☆ This is an open-access article distributed under the terms of the Creative Commons ovipositor structures. Additionally, parasitic Hymenopteran wasps have Attribution License, which permits unrestricted use, distribution, and reproduction in specialized sensory organs (Le Ralec et al., 1996)whicharerequiredfor any medium, provided the original author and source are credited. the location, recognition and acceptance of suitable host insects for ovi- ⁎ Corresponding author at: Institute of Plant Protection, Chinese Academy of Agricultural position (Papp, 1974). These functions are coordinated by complex phys- Sciences, No.2 West Yuanmingyuan Road, Beijing 100193, China. Tel.: +86 10 62815945; ical structures that originate from gonad tissues (Snodgrass, 1931, 1933). Fax: +86 10 62896114. E-mail address: [email protected] (Z. Wang). Previous studies of Hymenopteran ovipositor sense organs stylets 1 Tofael AHMED and Tian-tao ZHANG contributed equally to this work. 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.2013.04.015 344 T. Ahmed et al. / Journal of Asia-Pacific Entomology 16 (2013) 343–348 and TEM), have proven useful for defining species-specific morphol- Results ogies (Le Ralec and Wajnberg, 1990; Brown and Anderson, 1998; Rahman et al., 1998; Vilhhelmsen, 2000; Roux et al., 2005; Dweck et Ovipositor morphology al., 2008; Nacro and Nenon, 2009; Wang et al., 2010; Shah, 2012). Ad- ditionally, variation in ovipositor structures has been useful for the The female genitalia of M. cingulum showed that the basic organiza- classification of parasitoids, as well as for determining phylogenetic tion is remarkably uniform among the Hymenopteran insects (Rahman relationships (Austin and Field, 1997) and relating ovipositor struc- et al., 1998). The ovipositors averaged 4.42 ± 0.41 mm in length, and ture with possible function (Quicke and Fitton, 1995; Quicke et al., consisted of ventral and dorsal valves and an ovipositor sheath (Fig. 1). 1995; Dweck et al., 2008). When fused together, the internal lumen functions as a passageway for The present study aims to determine the morphology and ultra- eggs and/or venom. structure of sense organs on the ovipositor stylets and sheaths of The paired ventral valves are independent along most of their M. cingulum Brischke (Hymenoptera: Braconidae), and to confirm length, while dorsal valves were fused along their entire length. The the classification of the different types of sensilla using SEM and tip of the dorsal valve was observed to be enlarged and slightly TEM. The goal of this research was to better understand the morphol- curved to form a characteristic notch like structure located approxi- ogy of the sense organs on this structure, which is used in parasitoid mately 1 mm from the distal tip (Fig. 2). The dorsal and ventral valves host location and oviposition. are interlocked and form a tongue-like structure called the “rachis” (Figs. 2 and 3). The dorsal and ventral valves are parallel with respect Materials and methods 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 Experimental Insects of the ovipositor. Longitudinal thin chitinous flaps form a seal-like structure, which protrudes into the egg canal from the medial ventral The parasitoid wasp, M. cingulum Brischke (Hymenoptera: portion of each of the lower valves (Fig. 3), and may function as a seal Braconidae) was reared in the laboratory on the host insect, O. furnacalis, to prevent the loss of egg/venom from the canal. at a constant 25 °C temperature and a 16:8 light:dark photoperiod as The single segmented ovipositor sheath was flexible and well scler- described by Hu et al. (2003). All laboratory procedures were conducted otized, with transverse annulations from the base along the entire at the Institute of Plant Protection, Chinese Academy of Agricultural length, with the exception of the most distal part (Fig. 4). The sensilla Sciences, Beijing, China. trichodea covered the external surface from base to apex with an evenly spaced distribution. Three types of sensilla trichodea were distinctly ob- Scanning electron microscopy (SEM) served at the apex of the ovipositor sheath. The apex of the ovipositor sheath was pointed and heavily sclerotized, and the inner surface was The ovipositor with sheaths was dissected from newly emerged densely covered with flexible cutaneous microtrichia (Fig. 5). These M. cingulum parasitoids using a fine forceps, and was placed into a microtrichia on the ovipositor sheath were unevenly distributed with pre-fixation solution (2.5% glutaraldehyde in 0.1 M phosphate buffer the highest density at the apex (Fig. 5). 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 Sensory structures on the M. cingulum ovipositor 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 Three types of sensilla were identified on the ventral and dorsal absolute (100%) alcohol for 15–20 min. The ovipositor stylets were valves, as well as on the ovipositor sheath of the M. cingulum ovipositor 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.

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 di- luted 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 Fig. 1. Ovipositor of M. cingulum (total view) 40×; “V” for ventral valve; and “D” for stored on a computer. dorsal valve. T. Ahmed et al. / Journal of Asia-Pacific Entomology 16 (2013) 343–348 345

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 campaniform sensilla types will be described individually in the following text. SEM results from M. cingulum showed that sensilla campaniform (Ca) were present and observed as irregular and depressed cuticle Sensilla coeloconica like structures, from which somewhat smooth raised domes protrud- ed from the center (Fig. 6). These Ca possessed a tubular body just Sensilla coeloconica (COE) were present along the distal two-thirds before entering the cuticle, which was innervated by a dendritic of the ventral valve and along most of the length of the dorsal valve. membrane (Fig. 7). Ca were shown to be located along the ventral However, COE were found to be more densely distributed at the tip of and dorsal valves of the ovipositor, and were most densely distribut- the dorsal valve (Figs. 2 and 6), and occurred in a donut-shaped struc- ed on the lateral side of the distal dorsal valve tip. Furthermore, each ture with a small formation protruding beyond the surface of the stylets. Ca was shown to be associated with one coeloconica sensillum The external protuberance of these sensilla possessed a smooth surface, (Fig. 2). rounded body, and blunt tip that possessed a central pore-like opening. These sensilla could not be detected in ultrathin sections from TEM Sensilla trichodea imaging. 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

Morphological evidence can be used to infer the function of physical structures involved in parasitoid host location and oviposition Fig. 3. Transverse sections of ovipositor stylet, approximately one third from the distal (Quicke et al., 1994; Quicke and Fitton, 1995; Austin and Field, end. The egg canal (EC) surrounded with paired ventral valve (V) and the two fused 1997). From the findings of our SEM and TEM studies of the ovipositor 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 structure, we were able to summarize a possible correlation between makes a seal (SI) like structure. the sensory equipment which was present, and their functions, during 346 T. Ahmed et al. / Journal of Asia-Pacific Entomology 16 (2013) 343–348

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 tactile sensing. Le Ralec and Wajnberg (1990) and Cônsoli et al. M. cingulum ovipositor morphology are discussed in the sections below. (1999) also described campaniform sensilla on the ovipositor of Trichogramma maidis, T. galloi Zucchi and T. pretiosum Riley, and hypoth- Host selection process esized that they may act as a mechanoreceptor for positioning of the ovipositor prior to stabbing the host. Nacro and Nenon (2009) similarly Three types of sensory organs and secretory pores were identified described these sensilla on the ovipositors of Aprostocetus procerae on the ventral and dorsal valves, and on the ovipositor sheath of the (Risbec) and Platygaster diplosisae (Risbec). Shah (2012) described the M. cingulum ovipositor, based on SEM and TEM observations. These function of the campaniform sensilla on the ovipositor of Venturia types of sense organs have been described in other parasitic Hyme- canescens (Gravenhorst), and suggested them to be responsible for noptera (Le Ralec and Wajnberg, 1990; Brown and Anderson, 1998; pressure regulation during the act of stabbing, and as a mechanorecep- van Lenteren et al., 2007; Dweck et al., 2008; Nacro and Nenon, tor when the ovipositor bends during oviposition. Additionally, Dweck 2009; Shah, 2012). Furthermore, the observation that these struc- et al. (2008) reported that campaniform sensilla may be involved in tures are conserved among species suggests they may have a com- perceiving tactile cues through which Habrobracon hebetor is able to mon function and that they may be necessary for parasitism. discriminate between different stages of the host. Sensilla coeloconica were found on the distal two-thirds of the The outer surface of M. cingulum ovipositor sheaths was densely cov- ventral valve, and on almost the entire length of the dorsal valve of ered with types I, II and III trichodea, which also have been found on the M. cingulum. Such type of sensilla has also been described on the anal- Braconidae wasp H. hebetor (Say) (Dweck et al., 2008), Cotesia sesamiae ogous structures of the parasitoid wasp species Trybliographa rapae Cameron 1906 and C. flavipes Cameron 1891 (Obonyo, 2011)and (Brown and Anderson, 1998), Leptopilina heterotoma Nordlander Spathius agrili Yang (Wang et al., 2010) as well as the ichneumon wasp (van Lenteren et al., 2007), Aprostocetus procerae (Nacro and Nenon, Megarhyssa atrata F. (Nenon et al., 1997), and the trichogrammatid 2009) and Venturia canescens (Shah, 2012). Sensilla are known to T. maidis (Le Ralec and Wajnberg, 1990). From our TEM of thin sectioned function in the detection of external stimuli, and are likely involved M. cingulum ovipositor sheaths, trichodea were shown not to possess any in tactile sensing for the movement of the ovipositor on and through pore system or sensory neurons, which suggests that they are not the host cuticle. Sensilla coeloconica are also commonly considered to involved in olfactory or gustatory functions. Sensilla trichodea may, be chemoreceptors or thermo-hygroreceptors (Altner and Prillinger, serve as mechanoreceptors during host searching, with potential roles 1980) and have rarely been considered as purely mechanoreceptive played in response to air currents and physical vibrations. (Brown and Anderson, 1998). These sensilla have relatively deep sockets for their size, which may provide protection from excessive Stinging mechanism bending or damage while in contact with the host. Thus, the structures do not protrude far above the ovipositor surface of M. cingulum, The ovipositor plays an important role in host discrimination in yet are likely sufficient for the detection of external stimuli. Braconid wasps, and is often used by females to probe a potential host Sensilla campaniform are also present on the M. cingulum ovipos- prior to oviposition (Canale and Raspi, 2000). Most parasitoid ovipositor itor, and were observed as raised domes with a smooth surface on the tips contain at least one or more types of sensilla the structures of which ventral and dorsal valves. These sensilla are innervated by only one are known to be involved in the detection of both chemical and me- neuron which possesses a tubular body, and most likely function in chanical 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). T. Ahmed et al. / Journal of Asia-Paciﬁc Entomology 16 (2013) 343–348 347

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.

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 Fig. 7. Transverse sections of the dorsal valve lumen of the ovipositor, approximately might serve as support when retracted during stinging, or as a general one third from the distal end show the tubular body (black arrow) of a campaniform protection when sheathed over the ovipositor. sensillum. The inner surface of the ovipositor sheath is also densely covered with microtrichia, which probably serve to clean the ovipositor sensilla female M. cingulum abdomen bends forward and stings the host by of the residual host integument between different stinging actions, and penetrating the host's integument with the unsheathed ovipositor. may be important for keeping the ovipositor shaft functional. The Barbs and serrations are located at the distal end of the ventral microtrichia are most densely concentrated at the tip of the ovipositor valve on the M. cingulum ovipositor, which are likely used for piercing sheath, which might rationally have co-evolved to be in proximity to the host integument. The proportions and relative density of these the sensilla that are concentrated at the tip of the ovipositor. Microtrichia serrated structures vary widely among taxa (Rahman et al., 1998) have been shown to be dense along the inner surface of the ovipositor and may have evolved in response to variations in host cuticle thick- sheath in some other Braconid species (Le Ralec and Wajnberg, 1990; ness and resilience to penetration. In addition to function during Dweck et al., 2008; Nacro and Nenon, 2009) and suggest a shared func- piercing the host integument, the serrations may also act to hold tion within this group of wasps. the ovipositor in position as oviposition takes place (Le Ralec and Secretory pores are commonly observed at the ovipositor tip of Wajnberg, 1990; Brown and Anderson, 1998; Cônsoli et al., 1999; Hymenopteran parasitoid wasps (Copland and King, 1972; Shah, 2012), Dweck et al., 2008; Nacro and Nenon, 2009; Wang et al., 2010). including Habrocytus cerealellae Ashmead (Fulton, 1933)andNasonia In contrast, the dorsal valves of the ovipositor of M. cingulum are vitripennis Walker (King, 1962; King and Rafai, 1970). Although these blunt and round at the apex and are unsuited for penetrating through pores were also described by Ganesalingam (1972), the probable func- host cuticle. However, serial sections through the ovipositor reveal an tion was not described. Lyngnes and Sunmore, 1960 proposed that the olistheter-like interlocking of the mediodorsal part of the first valve most probable function of the SP may be to secrete lubricating fluids just prior to the apex, thereby possibly forming a slender envenomation 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. 348 T. Ahmed et al. / Journal of Asia-Paciﬁc Entomology 16 (2013) 343–348

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