Ovipositor of the braconid wasp Habrobracon hebetor: structural and functional aspects

The Braconidae are a megadiverse and ecologically highly important group of insects. The vast majority of braconid wasps are parasitoids of other insects, usually attacking the egg or larval stages of their hosts. The ovipositor plays a crucial role in the assessment of the potential host and precise egg laying. We used lightand electron-microscopic techniques to investigate all inherent cuticular elements of the ovipositor (the female 9th abdominal tergum, two pairs of valvifers, and three pairs of valvulae) of the braconid Habrobracon hebetor (Say, 1836) in detail with respect to their morphological structure and microsculpture. Based on serial sections, we reconstructed the terebra in 3D with all its inherent structures and the ligaments connecting it to the 2nd valvifers. We examined the exact position of the paired valvilli, which are bilateral concave structures that protrude into the egg canal. In H. hebetor, these structures putatively divert the egg ventrally between the 1st valvulae for oviposition. We discuss further mechanical and functional aspects of the ovipositor in order to increase the understanding of this putative key feature in the evolution of braconids and of parasitoid wasps in general.


Introduction
Most hymenopteran species belong to the guild of parasitoids of other insects (Quicke 1997). The astonishing radiation of the most diverse parasitoid wasp lineages (i.e. Ceraphronoidea, Ichneumonoidea and Proctotrupomorpha; = Parasitoida sensu Peters et al. 2017) has been estimated to have occurred 266-195 million years ago. This process was presumably triggered by continuous adaptations of the parasitoid lifestyle including features such as endoparasitism, miniaturization, and/or modifications of the ovipositor (Peters et al. 2017). Adaptations in oviposition behavior and the morphological structure of the ovipositor might not only have increased the reproductive success of the wasps, but have potentially also enabled them to oviposit in a multitude of different substrates, facilitating the acquisition of new hosts and host ranges (Gauld and Bolton 1988;Quicke 1997). The ovipositor of parasitoid wasps serves a set of functions: penetration of the substrate (if the host is concealed) or of the targeted egg/ puparium, the location, assessment, and piercing of the host, the injection of venom, the killing of the competitors' eggs or larvae, the finding of a suitable place for egg laying, and oviposition (Quicke et al. 1999).
The hymenopteran ovipositor is an anatomical and functional cluster that consists of the following elements: the paired 1 st valvulae (8 th gonapophyses), the 2 nd valvula (fused 9 th gonapophyses), the paired 3 rd valvulae (9 th gonostyli), the paired 1 st valvifers (fusion of the 8 th gonocoxites and gonangula (Vilhelmsen 2000)), the paired 2 nd valvifers (9 th gonocoxites), and the female T9 (9 th abdominal tergum) (Snodgrass 1933;Oeser 1961). All the morphological terms are applied according to the Hymenoptera Anatomy Ontology (HAO; Yoder et al. 2010; Hymenoptera Anatomy Consortium 2021; a table of the terms used and of their definitions is given in Table A1 in the Appendix 1). The 1 st valvifer is connected to the 2 nd valvifer via the intervalvifer articulation and with the female T9 via the tergo-valvifer articulation. Each of the 1 st valvulae is attached to the corresponding 1 st valvifer via the dorsal ramus of the 1 st valvula, whereas the 2 nd valvula is connected to the 2 nd valvifer via the basal articulation and fine ventral rami of the 2 nd valvula (cf. Bender 1943). Both the 1 st and the 2 nd valvulae are firmly interlocked along almost their entire length via a tongue and groove-like system called the olistheter. They form the terebra (= ovipositor shaft) and accommodate the egg canal (Oeser 1961;Quicke et al. 1994).
However, knowledge about structural and functional aspects of the ovipositor system of the ecologically and morphologically extremely diverse and species-rich Braconidae remains limited.
Habrobracon hebetor (Say, 1836) ( Fig. 1a, b) is a gregarious, idiobiont, larval ectoparasitoid of pyralid moths (Lepidoptera) (Paust et al. 2006) and is well known for its use in biological pest control (Paust et al. 2006;Mbata and Shapiro-ilan 2010;Sanower et al. 2018). Dweck et al. (2008) provided the first morphological descriptions of the ovipositor of this species, with a strong focus on the terebra and its sensillar equipment. In the present study, however, we aim to describe thoroughly all the inherent cuticular elements of the ovipositor of H. hebetor in order further to discuss their structural, mechanical, and functional aspects. We have therefore combined scanning electron microscopic (SEM) and light-microscopic studies on both complete cuticular structures and histological serial sections. Serial sections of the terebra have been used to provide a 3D reconstruction that has helped us to understand its morphology especially with regard to all its functionally clustered inherent structures. Finally, we present a structural model of the braconid ovipositor and discuss its mode of function.
to 24 h at room temperature to remove tissues, cleaned in distilled water on a minishaker, and dehydrated stepwise in ethanol. We then dissected the ovipositor out of the metasoma by using thin tungsten needles, mounted the specimen onto a microscope slide, and embedded it in Entellan® (Merck KGaA, Darmstadt, Deutschland).
For semithin serial sections, we anaesthetized female H. hebetor with carbon dioxide. The metasomas were removed and, in order to avoid autolysis, immediately submersed in a primary fixative containing 2.5% glutaraldehyde and 5% sucrose, buffered with 0.1 M cacodylate to pH 7.4. During this fixation, the samples were held in an ice bath at approximately 4 °C for 4 h. Samples were rinsed three times for 10 min in prechilled 0.1 M cacodylate buffer (pH 7.4) and post-fixed by using a solution of 1% osmium tetroxide in 0.1 M cacodylate buffer at 4 °C for 4 h. After being further rinsed in the same buffer, the samples were dehydrated through a graded series of ethanol with steps of 30% for 15 min and 50% for 10 min at 4 °C, three times per step, and of 70% for 10 min, three times at room temperature. The following steps were performed at room temperature. En bloc staining was conducted using a saturated solution of 70% ethanolic uranyl acetate for 12 h on a rotatory shaker. Afterwards, dehydration was continued in 5% steps, three times for 10 min each. The fully dehydrated samples were washed in 100% propylene oxide twice for 1 h , and subsequently infiltrated in Spurr low-viscosity embedding resin (Polysciences Inc., Warrington, PA, USA) via a propylene oxide:resin mixture at ratios of 1:1, 1:3, and 1:7 for 1 h per step and then in pure resin for 17 h on a rotatory shaker. As a last incubation step, the samples were placed in fresh pure resin, embedded in silicon molds, and polymerized at 70 °C for 8 h.
Semithin sections of 600 nm were cut perpendicularly to the terebra of H. hebetor by using an ultramicrotome Leica Ultracut UTC (Leica Microsystems GmbH, Wetzlar, Germany) equipped with a diamond knife (45° knife angle; DuPont Instruments, Wilmington, DE, USA); a series was obtained with 1920 sections. Semithin sections were then mounted on a microscopic slide by using a 'Perfect Loop for Light Microscopy' (Electron Microscopy Sciences, Hatfield, PA, USA), stained with Stevenel's blue (del Cerro et al. 1980) for 40 min at 60 °C, and subsequently washed in distilled water twice for 5 min each. After being dried, the stained sections were embedded in 'Xylolfreies Eindeckmittel' (Engelbrecht Medizin-und Labortechnik GmbH, Edermüde, Germany).
The image stack for the 3D reconstruction was generated using a Zeiss Axioplan (Carl Zeiss Microscopy GmbH, Jena, Deutschland) light microscope, equipped with a Nikon D7100 single-lens reflex digital camera (Nikon K.K., Tokio, Japan) and Helicon Remote software version 3.6.2.w (Helicon Soft Ltd, Kharkiv, Ukraine). Flawed images (missing or folded structures and staining problems) were replaced by a copy of the previous or the following image (this was the case for fewer than 3% of the images) for reconstruction purposes. Adobe Photoshop Lightroom version 6.0 (Adobe Systems, San José, CA, USA) was used for initial image processing (white balancing, color contrasting, black and white conversion), whereas Fiji ; available online at https://imagej.net/Fiji) was employed for cropping, CLAHE filtering, and image stack calibration by using the plugin TrakEM2 . A preliminary least square rigid alignment followed by an elastic alignment of the image stack was performed using the 'Elastic Stack Alignment' plugin ). The aligned image stack was then imported into Amira version 6.0 (FEI Company, Hillsboro, OR, USA). We pre-segmented the 1 st and 2 nd valvulae, the duct of the venom gland and the ligaments that connect the terebra and the 2 nd valvifer in the software's segmentation editor by manually labeling approximately every 15 th virtual slice along the terebra and every 4 th virtual slice in the proximal bulbous region and assigned them to different 'materials'. The labels served as an input for automated segmentation by using the Biomedical Image Segmentation App Biomedisa (available online at https://biomedisa.de) (Lösel et al. 2020). The output of Biomedisa was then partially corrected manually in Amira, and a final surface model was generated.
Schematic drawings of the cross-sections of the terebra were generated in Inkscape version 0.92.4 (Inkscape Community; available online at http://www.inkscape.org/) based on the original light-microscopic images of the semithin sections.
For lateral and ventral habitus images, female wasps were imaged with a Keyence VHX-7000 Digital Microscope (Keyence Corporation, Osaka, Japan) using focus stacking.

Scanning electron microscopy (SEM)
For scanning electron microscopy (SEM), the specimens were air-dried in a desiccator with Silica gel blue (Carl Roth GmbH & Co. KG, Karlsruhe, Deutschland) for at least four days before being mounted with double-sided adhesive tabs onto stubs and sputter-coated with 19 nm pure gold by using an Emitech K550X (Quorum Technologies Ltd, West Sussex, UK). Images were taken with a scanning electron microscope of the type EVO LS 10 (Carl Zeiss Microscopy GmbH, Jena, Germany) and SmartSEM version V05.04.05.00 software (Carl Zeiss Microscopy GmbH, Jena Germany).

Results and discussion
As in all hymenopterans, the ovipositor of H. hebetor consists of three pairs of valvulae, two pairs of valvifers, and the female T9 (Fig. 1c).
Overall structure of the terebra The 1 st and 2 nd valvulae form the terebra and enclose the egg canal (cf. Figs 2c-g, 3; Suppl. material 1). The terebra of H. hebetor extends far beyond the posterior tip of the metasoma. They are interconnected by a tongue and groove-like system, called the olistheter (oth; Fig. 2a-h). The olistheter comprises two longitudinal ridges that are called the rhachises (rh; Figs 2a, 5c, d) on both sides at the ventral surface of the 2 nd valvula and that fit into corresponding T-shaped grooves termed the aulaxes (au; Figs 2a, 4b, f, h) Figure 2. (next page) Cross sections through the terebra of Habrobracon hebetor (from proximal to distal); schematic drawings of the 1 st and 2 nd valvula (a-i) based on the light microscopic images of the presented semithin sections (a'-i' 600 nm; stained with Stevenel's blue). The drawings are of the same size ratio. The 2 nd valvulae possesses, in the proximal region, two lumina that merge into one in the most distal region (h-i). The bulbs (b) and the valvilli (g) are visible. The orange lines (in e, f) mark the position of the distally pointing ctenoid structures on the dorsal surfaces of the 1 st valvulae, which are in close contact with the ventral surface of the 2 nd valvula. The genital membrane connects the dorsal margins of the 2 nd valvifers (b'-h') c fine cuticular structures arise from the dorsal and ventral parts of the 2 nd valvula and define the lumina of the bulbs (arrow) h1 olistheter-like interlocking system connecting the medial surfaces of the apices of the paired 1 st valvulae (arrow). Final segmented 3D reconstruction based on a semithin section series (600 nm thickness) a*-i* position of each single section marked on the final 3D reconstruction of the terebra. Abbreviations: 1vv = 1 st valvula; 2vv = 2 nd valvula; 3vv = 3 rd valvula; au = aulax; blb = bulb; cr = longitudinal crack of 2 nd valvula; ec = egg canal; fl1 = longitudinal flap of the 1 st valvula; gm = genital membrane; igs = internal guiding structure; l1 = lumen of 1 st valvula; l2 = lumen of 2 nd valvula; lb = lumen of the bulb; lg = ligament; oth = olistheter; rh = rhachis; vd = duct of the venom gland; vl = valvillus. along the dorsal surface of each of the 1 st valvulae. This system allows the 1 st valvulae to slide longitudinally relative to each other when actuated by the corresponding operating muscles (Oeser 1961;Quicke et al. 1994). Distally pointing scale-like structures are found on both the olistheter elements and might reduce the friction forces by reducing the contact surface between the 1 st valvulae and the 2 nd valvula (sc; Fig. 4f ) (Rahman et al. 1998).
In H. hebetor, the cross sections of the terebra differ notably along its length ( Fig. 2a-i). A common oviduct enters the proximal bulbous part of the terebra (Bender 1943;Pampel 1914), where it ends at the base of the egg canal (Quicke 1997). Distally, the egg canal is largely defined by the 1 st valvulae, but with the dorsal side being formed by the 2 nd valvula. The diameter of the terebra decreases from proximal to distal, whereas the diameter of the egg canal remains constant for a long distance from proximal until the valvillus (see subsection '1 st valvulae').

st valvulae
The paired 1 st valvulae of H. hebetor form the ventral half of the terebra (1vv; Figs 1c, 2a-i, 3, 7a). The proximal end of each 1 st valvula is continuous with its dorsal ramus At their apices, the 1 st valvulae of H. hebetor possess several sawteeth, which decrease in size apically (st; Fig. 4a, b, c) (cf. Dweck et al. 2008). They probably serve to penetrate the substrate and the host's skin and tissue. Distally pointing ctenoid structures (rcs; Fig. 4h) arranged in rows can be found on the dorsal surfaces of the 1 st valvulae, which are in close contact with the ventral surface of the 2 nd valvula (orange line; Fig. 2e, f ). These ctenoid structures potentially reduce friction forces by minimizing the contact surface between the 1 st and the 2 nd valvula. The aulaces do not extend all the way to the apex of the 1 st valvulae but end just before the lateral sawteeth occur (au; Fig. 4b). Both 1 st valvulae are separated for the most of their length. However, mediodorsally at their very apex, the two 1 st valvulae become interlocked dorsally by a mechanism similar to that of the olistheter ( Fig. 2h1; also see fig. 4a of Dweck et al. 2008). Such a mechanism has previously been observed in other braconids (Zaglyptogastra (Quicke 1991), Aleiodes, Ligulibracon and Odontobracon (Quicke et al. 1994), andD. longicaudata (van Meer et al. 2020)) and is suggested to be an adaptation to the injection of venom into the host while laying the egg externally (Dweck et al. 2008). In addition, this mechanism might also increase the stability of the apex of the terebra when the host cuticle is pierced (Quicke 2015).
A single valvillus situated on the inner surface of each 1 st valvulae protrudes inside the egg canal (vl; Figs 2g, 4d, e; cf. Dweck et al. 2008). The valvillus is a bilaterally concave structure lying in the distal third of the terebra and occupies the whole diameter of the egg canal. Valvilli can be found in the Ichneumonoidea and in various families of the Apocrita (Snodgrass 1933;Quicke et al. 1992;Rahman et al. 1998). They are postulated to serve as a stop and release mechanism for the egg by maintaining the egg in position within the terebra and blocking the egg canal in Ichneumonoidea (Rogers 1972;Rahman et al. 1998;Boring et al. 2009), or for venom pumping in Apocrita (Quicke et al. 1992). However, in the ectoparasitoid H. hebetor, the eggs are observed to advance and even partially emerge ventrally at the base of the terebra, i.e. in between the 1 st valvulae and near the genital opening Wührer et al. 2009, see also Shaw 2017). Further distally, the valvilli seem to divert the egg ventrally between the 1 st valvulae and to press it out completely, since the egg does not emerge at the tip of the terebra but rather ventrally in between the 1 st valvulae approximately at the region at which the valvilli are located Wührer et al. 2009). We therefore suggest that the valvilli guide the relatively large egg ventrally out in between the 1 st valvulae. The latter are capable of being widely spread in this region because of the olistheter mechanism (Shaw 2017). In cross sections further apically to the valvilli, an egg canal is rarely visible or is absent (Fig. 2h, i), which suggests that at that point the egg has already left the terebra. In addition, the apical interlocking in between the two 1 st valvulae (red arrow; Fig. 2h1), which is similar to that of the olistheter, prevents the canal from expanding at the very apex. Proximal to the valvillus, the walls of the egg canal carry leaf-like ctenidia (ct; Fig. 4g, h), which are arranged in rows and are directed towards the distal end of the terebra. These rows of ctenidia point distally in the direction of egg movement and presumably prevent the regression of the egg during the oviposition process (Austin and Browning 1981). Setiform structures (= subctenidial setae sensu Rahman et al. 1998) are also found at the inner walls of the 1 st valvulae lying distally to the valvilli. They are arranged in distinct rows.
Each 1 st valvula contains a lumen (l1, Fig. 2a-i) whose cuticular walls differ along its length. Proximally, the cuticle is thin but becomes thicker towards the middle and diminishes again apical to the valvillus (Fig. 2a-i). In cross section, the shape of the 1 st valvula differs between the basal region and the rest of the terebra. In the basal part, it is triangular in shape (1vv; Fig. 2a), whereas further distally, it appears more oval (1vv; Fig. 2b-i). A longitudinal flap extends along the mediodorsal edge for most of the length of the 1 st valvulae and is clearly recognizable in cross sections (fl1; Fig. 2a-f ). This flap is highly prominent in the proximal part of the terebra but vanishes further apically (fl1; Fig. 2g-i). It might seal the egg canal to prevent the leaking of venom, since the pressure of the venom has been suggested to squeeze the two flaps together and therefore to seal the gap (Quicke et al. 1994;Shaw 2017). It has been observed in almost all the examined braconid species (Quicke and van Achterberg 1990;Quicke et al. 1994).
The apex of the 2 nd valvula is not serrated but is slightly enlarged before it narrows towards the tip (Figs 5a, c, 7a). In contrast to many ichneumonid and other braconid species (cf. Boring et al. 2009;Shah et al. 2012;Eggs et al. 2018), the 2 nd valvula of H. hebetor does not feature a prominent apical notch. Campaniform sensilla can be found in this area (cs; Fig. 5f ) (for a discussion of the sensillary equipment of the terebra of H. hebetor, see Dweck et al. 2008). Similar to the aulaces on the 1 st valvulae, the rhachises (rh; Fig. 5c, d) do not extend all the way to the apex but end at about the same distance away from the apex as seen for the aulaces (arrow; Fig. 5c). The apical half of the ventral side of the 2 nd valvula forms the dorsal wall of the egg canal and is, similar to the 1 st valvulae, covered by rows of ctenidia directed distally (ct; Fig. 5e). As previously discussed for the 1 st valvulae, these structures might prevent the regression of the egg during oviposition (cf. Rahman et al. 1998). Medioproximally, the bulbs feature ligaments (lg; Figs 2a, 3a, b, d) that connect the 2 nd valvula with the anterior section of the 2 nd valvifer. The ligament marks the region at which parts of the 2 nd valvifer merge into the anterior part of the 2 nd valvula. The bulbs also contain a lumen (lb; Fig. 2b). The proximal end of the 2 nd valvula bears the processus articularis (pa; Figs 3b, 7h) laterally and the processus musculares (pm; Figs 3b, 7h) at the anterior peak-like structure of the 2 nd valvula (red arrow; Fig. 3a, b). However, the medial 2 nd valvifer-2 nd valvula muscle (M-2vfl-2vlv) that might stabilize the 2 nd valvifer and that was newly described in the braconid D. longicaudata by Meer et al. (2020) was absent in our serial sections. There are two openings (black arrows; Fig. 3a, c) at the proximal side of the bulbs. The duct of the venom gland enters the dorsoproximal area of the bulbs on the left side only (vd; Figs 2a, b, 3, Suppl. material 2) (cf. Bender 1943, who investigated the anatomy and histology of the female reproductive organs of the closely related Habrobracon juglandis (Ashmead, 1889)). Further distally, the closed duct of the venom gland seems to disappear and to merge with the egg canal formed by the valvulae (Suppl. material 2). In this area, the venom presumably flows into the egg channel that is formed by both the 1 st and 2 nd valvulae with the longitudinal flaps of the 1 st valvulae acting as a seal (fl1; Fig. 2a-f ).
Proximally, the 2 nd valvula features a distinct longitudinal crack at the ventral side along the middle, which is clearly visible in cross-section (cr; Fig. 2c-g), presumably indicating the paired origin of the 2 nd valvulae. At the basal part of the 2 nd valvula, fine cuticular structures (arrow; Fig. 2c) arise from it dorsal and ventral parts and define the two lumina (l2; Fig. 2c-g) that run almost the entire length of the 2 nd valvula and that fuse at the apex (Fig. 2h, i). Proximally, the ventral part of the 2 nd valvula gradually changes shape and forms a U-shaped structure that extends distally into the egg canal (Suppl. material 2). This internal structure (igs; Fig. 2c-e) presumably helps in guiding the egg by forming a temporary egg canal. Without this internal guiding structure, the diameter of the egg canal would be large in this proximal region; this might lead to a lowered internal pressure and thus to problems when the egg is pushed further distally.

rd valvulae
The paired 3 rd valvulae of H. hebetor originate at the distal end of the 2 nd valvifers and extend far beyond the posterior tip of the metasoma towards the tip of the terebra (Figs 1b,c,7a,e). Each is U-shaped in cross-section (3vv; Figs 2g'-i', 6a) and they completely ensheath and protect the terebra in the resting position (3vv, trb; Figs 2g'-i', 6a) (cf. Bender 1943;Dweck et al. 2008). The distal third of the 3 rd valvulae is enlarged (Figs 6a, 7a), and their lateral surfaces differ over the course of their length: proximally, the 3 rd valvulae are annulated by fine transverse furrows (arrow; Fig. 6c; cf. Vilhelmsen 2003;Eggs et al. 2018), whereas the enlarged distal part lacks these structures (arrow; Fig. 6d). Trichomes, which Dweck et al. (2008) have described as trichoid sensilla, cover most of the external surface of the 3 rd valvulae (Fig. 6a). The density of the trichomes varies along the length of the 3 rd valvulae and is highest at the apex (Fig. 6a).
The inner surface of the 3 rd valvulae facing the terebra is densely covered by trichomes (t; Fig. 6b, e), particularly at the distal enlarged part (Fig. 6a, e). These structures might be involved in cleaning the terebra sensilla between oviposition episodes (Quicke et al. 1999;Vilhelmsen 2003). Observations have shown that the 3 rd valvulae also play a role in stabilizing the terebra during oviposition Wührer et al. 2009;Vilhelmsen 2003;Cerkvenik et al. 2017;Eggs et al. 2018;van Meer et al. 2020).

st valvifer
In lateral view, the paired 1 st valvifers of H. hebetor have a compact triangular shape with rounded edges (1vf; Figs 1c, 7a, c-e, 8). The intervalvifer articulation (iva; Figs 1c, 7a, c-f ), a rotational joint, is located at the rounded posteroventral side and connects the 1 st valvifer to the 2 nd valvifer. The ventral edge of the 1 st valvifer is placed laterally of the 2 nd valvifer and seems to be in contact with a sensillar patch (sp; Figs 1c, 7b, d-f ) that extends dorsally at the anterior beginning of the the dorsal flange of the 2 nd valvifer (df2; Fig. 7d, f ). The tergo-valvifer articulation (tva; Fig. 7a, c-e, g) connects the 1 st valvifer to the female T9. A ridge, called the interarticular ridge of the 1 st valvifer (iar;Figs 1c,8), extends in between the two articulations. This ridge might mechanically stabilize the 1 st valvifer and prevent it from extensive deformation. At its anterodorsal corner (arrow; Fig. 8), the 1 st valvifer is fused with the dorsal ramus of the 1 st valvula (dr1;Figs 1c,7c,h,8), which is continuous with the 1 st valvula.

nd valvifer
The paired 2 nd valvifers of H. hebetor are elongated in the longitudinal axis (2vf; Figs 1c, 7a, e). The anteromedial socket-like part of the 2 nd valvifer is connected to the laterally placed bulbs of the 2 nd valvula (blb; Figs 1c, 3, 7e, h) via the ball-and-socket-like basal articulation (ba ; Figs 1c, 7h). The posterior ends of both the 2 nd valvifers are connected to the 3 rd valvulae (3vv; Figs 1c, 7a, e). At their posterodorsal ends, the two 2 nd valvifers are connected by a median bridge (mb2; suggested position indicated in Fig. 1c). A massive dorsal spike (ds; Fig. 7e), a structure that has not as yet been described in other parasitoid wasps, is present at the posterior end of the 2 nd valvifer and potentially serves as an apodeme. In addition, a flexible cuticular area, a conjunctiva called the genital membrane (gm; Fig. 2d'), connects the ventral margins of the 2 nd valvifers arching above the 2 nd valvula.  (a-d) and light microscopic (e-i) images of the ovipositor of Habrobracon hebetor a overview of the ovipositor (lateral aspect; visible pore-like structures are presumably artefacts of detached trichomes) c 1 st valvifer exhibiting the interarticular ridge and the hook-shaped lobe of the 2 nd valvifer. The 1 st valvifer is continuous with the dorsal rami of the 1 st valvula and is articulated with the 2 nd valvifer and the female T9 via the intervalvifer articulation and the tergo-valvifer articulation, respectively d sensillar patch of the 2 nd valvifer (made visible by partly removal of the 1 st valvifer) b, f sensillar patch of the 2 nd valvifer e overview of the 2 nd valvifer and female T9. The arrow shows the dorsal hump of the T9 g tergo-valfiver articulation between the 1 st valvifer and female T9 h detail image of e. The laterally placed bulbs of the most proximal part of the 2 nd valvula are articulated with the paired 2 nd valvifers via the basal articulation i sensilla in a row at the dorsal margin of the 2 nd valvifer. Abbreviations: 1vf =1 st valvifer; 1vv = 1 st valvula; 2vf = 2 nd valvifer; 2vv = 2 nd valvula; 3vv = 3 rd valvula; ar9 = anterior ridge of T9; ba = basal articulation; blb = bulb; df2 = dorsal flange of 2 nd valvifer; dm2 = dorsal margin of the 2 nd valvifer; dr1 = dorsal ramus of the 1 st valvula; ds = dorsal spike of the 2 nd valvifer; hsl = hook-shaped lobe of the 2 nd valvifer; iar = interarticular ridge of the 1 st valvifer; iva = intervalvifer articulation; sp = sensillar patch of the 2 nd valvifer; sr = sensillar row of the 2 nd valvifer; pa = processus articularis; pm = processus musculares; T9 = female T9; trb = terebra; tva = tergo-valvifer articulation. At its anterodorsal corner, the 2 nd valvifer extends upwards in a hook-shaped lobe (hsl; Fig. 7a, c, e; sensu Snodgrass 1933), and features the elongated anterodorsal ridge of the 2 nd valvifer, the so called dorsal margin of the 2 nd valvifer (dm2; Fig. 7c, d, h). The dorsal projection of the 2 nd valvifer, a tongue-like structure situated on the dorsal margin of the 2 nd valvifer, is continuous with the olistheter. The corresponding groove is located on the dorsal side of the dorsal ramus of the 1 st valvula (dr1; Fig. 7c, h; cf. fig.  4h1 of Eggs et al. 2018) and enables its back and forth movement. This hook-shaped lobe might guide and stabilize the 1 st valvifer during its posterior pivoting but might also allow for a larger arc of movement of the 1 st valvifer and therefore a greater retraction distance of the 1 st valvulae (cf. Eggs et al. 2018).
Two main ridges are found on the 2 nd valvifer, i.e. (1) the dorsal flange of the 2 nd valvifer (df2; Fig. 7d, f ), which expands from the sensillar patch in the direction of the hook-shaped lobe and posteriorly from the sensillar patch to the origin of the 3 rd valvulae (Fig. 7e), and (2) the dorsal margin of the 2 nd valvifer (dm2; Fig. 7c, d, h). The two cuticular ridges might have a stabilizing function to prevent deformation. The 2 nd valvifer of H. hebetor does not feature a basal line (e.g. in contrast to the ichneumonid Venturia canescens (Gravenhorst, 1829), see fig. 4e of Eggs et al. 2018), a ridge that extends from the pars articularis to the dorsal flange of the 2 nd valvifer.
Clusters of sensillae ("styloconic sensillae" according to Dweck et al. 2008) occur in two regions. The first cluster, called the sensillar patch (sp;Figs 1c,7b,, is situated ventrally of the intervalvifer articulation and is covered by the 1 st valvifer laterally. The second cluster occurs at the dorsal margin of the 2 nd valvifer (sr; Figs 1c, 7h, i). These sensilla are arranged in a row and are in contact with the dorsal ramus of the 1 st valvula. The two sensilla clusters presumably monitor the pro-and retraction movements of the 1 st valvifers and the attached 1 st valvulae, respectively. The density of sensilla in the patch is much higher than that on the dorsal margin of the 2 nd valvifer.

Female T9
The female T9 is unpaired and elongated (T9;Figs 1c,7a,c,d,e). At its anterodorsal corner, it is connected to the 1 st valvifer via the tergo-valvifer articulation (tva; Figs 1c, 7a, c-e, g). Dorsally, it features the anterior ridge almost throughout its length (ar9; Fig. 7e), and posteriorly, it bears a hump-shaped structure (arrow; Fig. 7e). The female T9 mostly lies inside the abdomen, and only the posterolateral part that faces the outside is covered with hairs.

Mode of function of the ovipositor
Functional models of the actuation and movement mechanisms based on thorough analyses of the musculoskeletal system of an ichneumonid and a braconid wasp have recently been described (Eggs et al. 2020, van Meer et al. 2020 and are summarized in the following. Although, in our study, we have not considered the muscles of the system, we have basically found the same arrangement of cuticular elements in the ovipositor system of H. hebetor as described in both of the above-mentioned studies. Hence, we assume analogous functional morphological conditions, although we point out any possible H. hebetor-specific modifications. The ovipositor movements are mainly actuated by two pairs of antagonistically working muscles (further described below), i.e. (1) the depression (i.e. downward rotation to the active position) and elevation (i.e. upward rotation back to the resting position) of the terebra, and (2) the pro-and retraction of the 1 st valvulae. Smaller muscles, i.e. the 1 st valvifer-genital membrane muscle or the posterior T9-2 nd valvifer muscle, might predominantly serve to stabilize the ovipositor system during oviposition.
(1) Depression and elevation of the terebra: The basal articulation is composed of the processus articularis (pa; Figs 3b, 7h) at the 2 nd valvulae and the pars articularis at the 2 nd valvifer. The pars articularis is a small area of anteroventral corners of the 2 nd valvifer, whereas the processus articularis is the respective structure of the bulb. The posterior 2 nd valvifer-2 nd valvula muscle depresses the terebra, i.e. rotates it downwards to the active position from its resting position between the paired 3 rd valvulae. The tendon of this muscle inserts at the processus musculares (pm; Figs 3b, 7h), which is situated at the peak-like posterior part of the 2 nd valvula (arrow; Fig. 3a, b) and thus increases the moment arm. However, the moment arm most probably changes over the range of motion of the terebra. In H. hebetor, we assume that the virtual line that can be drawn perpendicularly to the length axis of the terebra through the ligaments (lg;Figs 2a,3a,b,d) lying anterolaterally on the bulbs (blb; Figs 2b, 5a, b, 7e, h) most likely forms the rotation axis (= joint axis, pivot point or fulcrum; black circle; Fig. 3b), since the ligaments form the connections of the 2 nd valvula with the anterior parts of the 2 nd valvifers and can only stretch to a limited extent. Van Meer et al. (2020) postulate that, in the braconid D. longicaudata, the rotation axis lies directly anterior to the bulbs. In addition, these authors have observed that, during terebra depression (towards an active probing position), the lateral bulbs are pulled out of the socket-like anterior parts of the 2 nd valvifers, which are pushed slightly apart. The ball-and-socket-like connection is therefore assumed mainly to stabilize the terebra in its resting position. The antagonistically acting anterior 2 nd valvifer-2 nd valvula muscle inserts at the processus musculares and elevates the terebra, i.e. rotates it back upwards towards the resting position.
(2) Pro-and retraction of the 1 st valvulae: The 1 st valvifer, 2 nd valvifer, and the female T9 form a mechanical cluster of functionally interconnected elements (for detailed functional models see fig. 5 of Eggs et al. 2018 andfig. 8 of van Meer et al. 2020). The dorsal and the antagonistically acting ventral T9-2 nd valvifer muscle change the relative position of the 2 nd valvifer and the female T9. Both of these structures are connected with the 1 st valvifer via the intervalvifer and the tergo-valvifer articulation (Fig. 7c), respectively. Moreover, both are rotational joints that allow rotation in the sagittal plane only. The 1 st valvifer acts as a lever (Fig. 8) that transfers its movements to the dorsal ramus of the 1 st valvula (dr1; Figs 7c, h, 8). Contraction of the dorsal T9-2 nd valvifer muscle leads to an anterior rotation of the 1 st valvifer around the intervalvifer articulation. The 1 st valvifer acts as a lever that transfers these movements to the dorsal ramus of the 1 st valvula, thus causing the 1 st valvula to slide distally relative to the 2 nd valvula, i.e. to protract. Vice versa, contraction of the antagonistic ventral T9-2 nd valvifer muscle leads to a posterior rotation of the 1 st valvifer, causing the 1 st valvula to slide proximally to the 2 nd valvula, i.e. to retract (Eggs et al. 2018;van Meer et al. 2020). The hook-shaped lobe of the 2 nd valvifer (hsl; Fig. 7a, c, e) might allow a larger arc of movement of the 1 st valvifer and therefore a larger retraction distance of the 1 st valvulae. During the retraction of the 1 st valvula, the dorsal ramus of the 1 st valvula (dr1; Fig.  7c, h) can slide along the dorsal projection of the 2 nd valvifer almost until the posterior end of the hook-shaped lobe (hsl; Fig. 7a, c, e).
In the context of the described movements, the 1 st valvifer acts as a one-armed class 3 lever (force arm smaller than load arm). In our lever model (Fig. 8), we use the 2 nd valvifer (2vf; Fig. 1c) as a frame of reference. However, in reality, all involved cuticular elements can move relative to each other. The anatomical inlever (c; Fig. 8) equals the distance between the intervalvifer articulation and the tergo-valvifer articulation (where the input force is applied; F (in protraction) , F (in retraction) ; Fig. 8). The distance between the intervalvifer articulation and the beginning of the dorsal ramus of the 1 st valvula at the anterodorsal end of the 1 st valvifer equals the anatomical outlever (d; Fig. 8). The ratio of effective outlever (d' ; Fig. 8) and the effective inlever (c' ; Fig. 8) are indicative for the potential maximum velocity, the mechanical deflection, and the amount of force transmission to the 1 st valvula. An increase of the d':c' ratio results in an increase of the potential maximum velocity and mechanical deflection but entails a smaller force output (F (out protraction) , F (out retraction) ; Fig. 8) of the 1 st valvulae. In resting position, the anatomical in-and outlever are both very similar to their respective effective levers, thereby creating high torques at the intervalvifer articulation and ensuring an optimal force transmission when pro-or retracting the 1 st valvulae. During oviposition, the left and the right 1 st valvulae slide back and forth alternately at a high frequency. These valvula movements are crucial for drilling and precise egg laying (Vilhelmsen 2000;Cerkvenik et al. 2017;van Meer et al. 2020).
The shape of the 1 st valvifer varies between the various hymenopteran superfamilies (Oeser 1961). Ichneumonoid species such as the braconid H. hebetor in the present study possess a 1 st valvifer with a rounded compact shape (Snodgrass 1933;Eggs et al. 2018), in contrast to the elongated and bow-shaped 1 st valvifers of members of the superfamily Chalcidoidea (Copland and King 1972a, 1972b, 1972c, 1973, the triangularly shaped 1 st valvifers of Apis mellifera (Linnaeus, 1758) and other aculeate species (Snodgrass 1933;Oeser 1961;Matushkina 2011;Matushkina and Stetsun 2016;Stetsun and Matushkina 2020;Graf et al. 2021), and the highly diverse 1 st valvifers of basal hymenopterans (e.g. the robust-appearing 1 st valvifers of Tentredinidae (Snodgrass 1933;Vilhelmsen 2000) or the triangular 1 st valvifers in some Xyelidae (Vilhelmsen 2000). The ecomorphological consequences of these morphological differences remain to be explored in future systematic comparative analyses with respect to the parasitization of other hosts in different substrates and habitats.
The two sensilla clusters found on the 2 nd valvifer of H. hebetor (sp, sr; Fig. 7b, d-f, i). probably play an important role in monitoring the pro-and retraction of the 1 st valvulae, since their accurate actuation is of major importance for successful egg deposition (van Meer et al. 2020). Unlike in H. hebetor or V. canescens (Eggs et al. 2018), the sensilla patch at the intervalvifer articulation of other parasitoid wasps can be extremely reduced, e.g. in Pteromalidae (Chalcidoidea) with only three single sensilla (Copland and King 1972b). The question remains as to whether both the density and number of sensilla are linked to the importance of the control of the movements involved in oviposition, and whether this correlates with the shape of the 1 st valvifer.

Conclusion
All the cuticular elements of the ovipositor of Habrobracon hebetor play a crucial role for successful oviposition. The 2 nd valvifer and the female T9 exhibit many muscle insertions, the 1 st valvifer acts as a lever that transmits movements to the 1 st valvulae, and the terebra serves as a device for precise venom injection, host assessment, and accurate egg laying. All the cuticular elements feature many distinct structures in addition to the micro sculpture that is crucial for the performance of these tasks. Our work also has shown that a 3D reconstruction based on a histological section series preserves useful information about the exact morphology and position of inherent structures thereby enabling us to draw conclusions about their function. Future comparative examination of the inherent ovipositor elements, their morphological structure, and the underlying mechanical and functional aspects has the potential to increase our understanding of a putative key feature in the evolution of parasitoid hymenopterans, a feature that probably has significantly impacted the evolutionary success of braconid wasps (more than 18,000 described (Quicke 2015) and about 43,000 estimated species (Jones et al. 2009)) and of parasitoid hymenopterans in general (115,000 described and 680,000 estimated species (Heraty 2009)). Table A1. Morphological terms relevant to the hymenopteran ovipositor system. The terms (abbreviations used in this article in brackets) are used and defined according to the Hymenoptera Anatomy Ontology Portal (HAO) (Yoder et al. 2010; Hymenoptera Anatomy Consortium 2021) and the according Uniform Resource Identifiers (URI) are listed.