Authors: Benjamin Eggs, Stefan Fischer, Michael Csader, István Mikó, Alexander Rack, Oliver Betz
Categories: Research, Chalcidoidea, Functional morphology, Hymenoptera, Ovipositor, Parasitoid, Pteromalidae
Source: Frontiers in Zoology
Authors: Benjamin Eggs, Stefan Fischer, Michael Csader, István Mikó, Alexander Rack, Oliver Betz
Various chalcidoid wasps can actively steer their terebra (= ovipositor shaft) in diverse directions, despite the lack of terebral intrinsic musculature. To investigate the mechanisms of these bending and rotational movements, we combined microscopical and microtomographical techniques, together with videography, to analyse the musculoskeletal ovipositor system of the ectoparasitoid pteromalid wasp Lariophagus distinguendus (Förster, 1841) and the employment of its terebra during oviposition. The ovipositor consists of three pairs of valvulae, two pairs of valvifers and the female T9 (9th abdominal tergum). The paired 1st and the 2nd valvulae are interlocked via the olistheter system, which allows the three parts to slide longitudinally relative to each other, and form the terebra. The various ovipositor movements are actuated by a set of nine paired muscles, three of which (i.e. 1st valvifer-genital membrane muscle, ventral 2nd valvifer-venom gland reservoir muscle, T9-genital membrane muscle) are described here for the first time in chalcidoids. The anterior and posterior 2nd valvifer-2nd valvula muscles are adapted in function. (1) In the active probing position, they enable the wasps to pull the base of each of the longitudinally split and asymmetrically overlapping halves of the 2nd valvula that are fused at the apex dorsally, thus enabling lateral bending of the terebra. Concurrently, the 1st valvulae can be pro- and retracted regardless of this bending. (2) These muscles can also rotate the 2nd valvula and therefore the whole terebra at the basal articulation, allowing bending in various directions. The position of the terebra is anchored at the puncture site in hard substrates (in which drilling is extremely energy- and time-consuming). A freely steerable terebra increases the chance of contacting a potential host within a concealed cavity. The evolution of the ability actively to steer the terebra can be considered a key innovation that has putatively contributed to the acquisition of new hosts to a parasitoid’s host range. Such shifts in host exploitation, each followed by rapid radiations, have probably aided the evolutionary success of Chalcidoidea (with more than 500,000 species estimated).
The online version contains supplementary material available at 10.1186/s12983-023-00503-1.
The evolution of parasitoidism in Hymenoptera has led to one of the largest species radiations in insects [1–5]. A large proportion of parasitoids belong to the Chalcidoidea, an extremely diverse and ecologically important group (nearly 27,000 species described, over 500,000 species estimated) of mainly minute wasps (average body size range from 1–2 mm) that are omnipresent in almost all terrestrial habitats [6–11]. Most chalcidoids are ectoparasitoids of other insects, usually attacking enclosed host stages with reduced mobility (i.e. egg or larval stages of wood and stem borers, leaf-miners or inhabitants of galls, seeds and fruits) [12], although other life stages are also targeted [13]. The parasitization of hosts living deep within substrates allows the ectoparasitoid larvae to develop within the protection of a concealed environment and without exposure to the host immune system as occurs in endoparasitoids. An evolutionary novelty and presumably a strong driver of diversification in Chalcidoidea is the secondary reversal to monocondylic mandibles (reduction of the posterior condyle accompanied with modified musculature with functional separation), which allow the emerging wasp to bite through a hard substrate by precise cutting movements that overcome the limitations of a single degree of freedom [14]. However, the use of hosts living concealed within hard substrates poses challenges not only for the emerging wasp (i.e. in leaving the substrate), but also for females attempting to parasitize them (i.e. entering the substrate to find a potential host) [15]. In this context, the ovipositor has to fulfil several functional penetration or navigation through the substrate or the target egg/puparium, assessment of the host, discrimination between suitable and previously parasitized hosts, piercing of the host, injection of venom, formation of a feeding tube for host feeding, ovicide or larvicide of the competitors’ eggs or larvae, respectively, marking of the host and find a suitable place for egg laying and oviposition [16]. However, putative evolutionary novelties of the chalcidoid ovipositor system, such as morphological and behavioural adaptations that enable the steering of the terebra (= ovipositor (shaft) sensu [17–27]) and its underlying mechanisms have not been thoroughly investigated hitherto.
As in all hymenopterans, the chalcidoid ovipositor consists of the female T9 (9th abdominal tergum; = outer ovipositor plates sensu [17–25]), two pairs of valvifers and three pairs of valvulae derived from the 8th and 9th abdominal segments (7th and 8th metasomal segments). The basally situated valvifers accommodate the operating musculature, whereas all the valvulae are devoid of intrinsic musculature [28–32]. The 1st valvifers (8th gonocoxites [33, 34] or the fusion of the same with the gonangula [30]; = fulcral plates sensu [17–25, 35–37]; = gonangulum, gonangula sensu [26, 27]) are anteriorly continuous with the rami of the 1st valvulae (8th gonapophyses; = stylets sensu [17–25, 35–38]; = lower valves sensu [26, 27]), and their posterior angles articulate dorsally with the female T9 via the tergo-valvifer articulation and ventrally with the 2nd valvifers via the intervalvifer articulation. The 2nd valvifers (9th gonocoxites; = inner ovipositor plates sensu [17–25]) extend as the 3rd valvulae (9th gonostyli; = (articulating/terminal) palps sensu [19, 20, 22, 23, 36]; = ovipositor sheaths sensu [26, 27]) and are ventrally articulated with the 2nd valvula (fusion of the 9th gonapophyses; = (stylet) sheath sensu [17–25, 36–38]; = upper valve sensu [26, 27]) [28, 29], which is asymmetrically split except at the apex in all chalcidoid families [39]. The two overlapping asymmetric halves of the 2nd valvula are connected dorsally by the notal membrane, which extends almost to the apex [17, 19–25, 40]. The interlocked 1st and 2nd valvulae enclose the egg canal and form the terebra, which is embraced by the 3rd valvulae when not in use. The ventral surface of the 2nd valvula is interlocked with both of the 1st valvulae by a sublateral longitudinal tongue called the rhachis, which runs within a corresponding groove called the aulax along the dorsal surface of each of the 1st valvulae. This so-called olistheter system allows the three elements of the terebra to slide longitudinally relative to each other and simultaneously prevents their unwanted separation [29, 39].
In order to reach their hosts and permit greater control over egg placement, several parasitoid wasps are able actively to bend and rotate their terebra in any direction relative to their body axis [41–44], despite the lack of intrinsic terebral musculature. Such terebra movements have also been reported in chalcidoid wasps of the family of Pteromalidae [40, 45–47], a polyphyletic group sensu lato [6, 10, 48, 49] (over 3500 species described [8]). However, little is known about the actuation of the various ovipositor movements, with the mechanisms involved in terebra steering (i.e. bending and rotating) remaining unclear. In this study, we investigated the working mechanisms of the terebra steering movements of Lariophagus distinguendus (Förster, 1841) (Chalcidoidea: Pteromalidae: Pteromalinae), a cosmopolitan synanthropic synovigenic autogenous solitary idiobiont larval and pupal ectoparasitoid of several granivorous coleopteran species [50, 51]. This species exhibits extensive terebra movements during the assessment of a potential host and eventual subsequent egg placement [45, 47]. We aimed (1) to analyse the oviposition process in vivo, (2) to describe the ovipositor of L. distinguendus, including all inherent cuticular elements and muscles, (3) to examine the mechanics and mode of function of the musculoskeletal system, including the actuation of the various ovipositor movements, (4) to investigate the underlying working mechanisms of the terebra steering movements and (5) to discuss their eco-evolutionary significance.
We combined behavioural analyses involving high-resolution video recordings with morphological investigations based on microscopical and microtomographical techniques. These studies have enabled us to present a thorough morphological and mechanical analysis of the musculoskeletal ovipositor system that steers the various movements executed by the female L. distinguendus (Fig. 1) during oviposition. In particular, we focused on the employment of the terebra and on its form, structure and material properties.Fig. 1Habitus image of a female Lariophagus distinguendus (lateral view). Abbreviations: Terebra
Morphological terms are applied according to the Hymenoptera Anatomy Ontology (HAO; [52–54]; a table of all 210 terms relevant to the hymenopteran ovipositor system, their definitions and 513 synonyms commonly found in literature is given in Table 2 in the Appendix 1).
In cases in which our findings have been confirmed by other studies, these are indicated below with ‘cf.’.
Previous studies describing the behavioural sequences of the attempts of L. distinguendus [45, 50, 55, 56] and other pteromalids [40, 46, 57] to oviposit have been unable to provide an analysis of the events that take place within the cavity of the substrate. Therefore, we mainly focus on the employment of the terebra and its movements in the following (Fig. 2; Additional file 1).Fig. 2Oviposition process of Lariophagus distinguendus. a Female wasps search for potential hosts that live in grains of the common wheat Triticum aestivum. b–p Single frames of high-resolution video recordings of a female L. distinguendus parasitizing a larva of Sitophilus granaries in an artificial chamber (cf. Additional file 1). The wasp and the beetle larva were separated by a piece of blotting paper. After the wasp finds a host (b), it brings its terebra into drilling position by a downward bending of the metasoma (c) and then, once the apex of the terebra is engaged in the substrate, it lifts the metasoma and the 3rd valvulae upwards (d). Following penetration of the substrate, the wasp permanently paralyzes the host larva by venom injection and then usually forms a feeding tube for host feeding. During the subsequent assessment of the host and the search for a suitable place for oviposition, the wasp is able to actively bend (e–l) its terebra in various directions and also to rotate it to a certain degree (o). The individual movements of the single valvulae can be observed (m–p). The 1st valvulae is frequently protracted far beyond the apex of the 2nd valvula (marked with an arrowhead in i, k, m, n, o). Finally, an egg is laid (p). Rapid alternating movements of the 1st valvulae can be observed during substrate drilling, host envenomation and egg laying. Abbreviations: 1st valvula; 2nd valvula; 3rd valvula; Terebra
Search for the host's habitat: L. distinguendus parasitizes concealed granivorous host larvae (Fig. 2a). The parasitoids mainly use volatile chemicals to locate the habitat of their faecal cues from the host itself and herbivory-associated chemicals in the seed induced by the mechanical damage caused by the host larvae [51, 55, 58].
Search for an infested substrate: Once L. distinguendus finds the host’s location (infested grains; blotting paper with the host faeces in our experimental setup), the wasp starts to walk on the substrate followed by antennal drumming with the flagellum directed towards the ground (Additional file 1, min. 05–0:07; cf. [45, 47, 50, 55, 56]). The female parasitoid is able to discriminate between healthy and infested grains [59].
Penetration of the substrate: Once the female wasp has selected a small spot with its antennae, it brings its terebra into the drilling position by a downward bending of the metasoma so that its tip taps the surface. The terebra is guided and stabilized by the 3rd valvulae in order to prevent buckling despite axial compressive forces occurring during the initial puncturing of the substrate, i.e. the pericarp of the grain. Once the apex of the terebra is engaged in the substrate, the metasoma with the 2nd valvifer and the attached 3rd valvulae are lifted upwards out of the way (Fig. 2b–d; Additional file 1, min. 06–0:11; cf. [40, 45, 47, 50, 55, 56]). The initial puncturing (i.e. pericarp surface penetration) is necessary for the 1st and 2nd valvulae to be anchored in the substrate so that the subsequent ‘push-pull’ mechanism can be initiated. Thereby, the wasp exhibits alternate reciprocal movements of the paired 1st valvulae, which can be seen as trembling movements of the posteroventral part of the metasoma (i.e. the 2nd valvifers and the female T9). Only one of the 1st valvulae is pushed into the substrate at a time, while the other 1st valvula and the 2nd valvula, which are anchored in the substrate, are simultaneously pulled [60–62]. The apical sawteeth thereby increase the friction with the surrounding substrate. The tension in the two anchored ‘stationaryʼ elements increases their bending stiffness and, hence, they can serve as guides for the particular 1st valvula being pushed into the substrate [44, 60]. Small pushing movements of the 2nd valvula caused by the relative movements of the 2nd valvifers cannot be excluded (cf. [32]). The simultaneous pushing and pulling of the various terebral elements minimizes the net compressive force on the substrate and thus the chance of buckling of the terebra [32, 44, 60]. The ‘push-pull’ mechanism enables drilling without torque and with very low axial load, although these cannot be completely avoided [44, 62]. During the drilling process (Additional file 1, min. 12–0:20; cf. [47]), the wasp combines the ‘push-pull’ mechanism with slight rotations of the terebra [44, 60]. Moreover, a fluid is constantly secreted at the apex and also along the shaft of the terebra. This secretion putatively prevents particles from entering the terebra but might also act as a (cooling) lubricant (cf. [63, 64]).
Search for a potential host within the substrate: As soon as the wasp has penetrated the grain in which a potential host larva is living, it attempts to locate the host larva in its concealed cavity with its terebra (Additional file 1, min. 02–4:32). Thereby, the metasoma is frequently rotated by up to 35° from the longitudinal body axis of the wasp (cf. [45]); this influences the orientation of the terebra. However, the wasp also expresses steering movements of the terebra in several directions that are independent of the orientation of the metasoma (see subchapter ‘Mechanisms of terebra bending and rotation’ below).
Penetration of the targeted host's skin: Once the wasp has succeeded in reaching its host, it pushes its terebra straight down to its fullest extent and penetrates the skin of the beetle larva several times with rapid stabbing movements of the terebra (Additional file 1, min. 21–0:27, 33–2:47; cf. [47, 55]) achieved by fast alternate movements of the 1st valvulae.
Injection of venom: The host larva is usually pierced several times (cf. [45, 47]), with the 1st valvulae performing fast alternate movements. Venom is injected into the host’s body and permanently paralyses the host (Additional file 1, min. 21–0:27, 33–2:47; cf. [47]) thereby preventing its further development. This is crucial for ectoparasitoids, since movements of the host larva within a small cavity might damage the externally attached parasitoid [65].
Assessment of the host: The permanent paralysis of the host larva presumably allows an easier and more accurate assessment to be carried out by the female wasp, which can now actively steer its terebra (Fig. 2e–l; Additional file 1, min. 30–1:10, 48–3:17; cf. [47]; see subchapter ‘Mechanisms of terebra bending and rotation’ below). However, some passive bending of the terebra might also occur because of its deflection on the host surface. A small actively actuated bending of the apex of the terebra would therefore be sufficient to indicate the direction of the bending movement. The assessment of the host is not primarily carried out by the terebra tapping of the host surface, but by the puncture and the assessment of the host’s haemolymph (cf. [66, 67]).
Formation of a feeding tube for host feeding: In most parasitization attempts, the female wasps create a feeding tube. Thereby, a secretion, which is produced by the large colleterial glands [68], oozes from the entire terebra [66, 69]. The terebra is moved up and down and is also putatively rotated to a certain degree to ensure an even distribution of the secretion, which hardens in the air and remains for a couple of minutes, forming a feeding tube (cf. [47]). As a result of capillary forces, the haemolymph of the host flows upwards within the tube. The wasp now appears to lick the end of the feeding tube. The absorbed haemolymph serves both as protein-rich nutrition that is needed for egg maturation [70] and allows an assessment of the quality of the potential host [66, 67].
Ovicide/larvacide of the competitors' eggs/larvae: In our artificial setup, we have not tested whether the female wasps attempt to kill their conspecifics’ eggs or larvae. Ovicidal and larvicidal behaviour has not as yet been observed in L. distinguendus.
Search for a suitable place for oviposition: If the female wasp deems the host larva to be of good quality, it searches for a suitable oviposition site on the host surface. It appears to estimate the available space within the cavity to ensure that the growing larva has enough room for development (Fig. 2e–l; Additional file 1, min. 19–3:34; cf. [47]).
Oviposition: Rapid longitudinal alternate movements of the paired 1st valvulae serve to pass the egg along the terebra (Fig. 2m–p; Additional file 1, min. 56–2:26, 36–4:00; cf. [47, 71]). The diameter of the egg is significantly larger compared with that of the egg canal. The egg is thus strongly deformed during ovipositing. It does not emerge at the very apex of the terebra but is pushed out ventrally between the two paired 1st valvulae in a region about 100–200 µm proximal to the apex (Fig. 2p). Finally, the egg is attached to the surface of the host. In a few cases, it was also observed to be attached to the surface of the cavity near the host larva. Finally, the wasp withdraws its terebra. Female L. distinguendus only lay one egg per host [45, 55].
The musculoskeletal ovipositor system of L. distinguendus consists of three pairs of valvulae, two pairs of valvifers, the female T9, three paired articulations and a set of nine paired muscles.
Because of its bilateral bauplan, all the ovipositor elements and muscles are paired apart from the distal region of the 2nd valvula and the female T9. Paired morphological structures are only described in the singular form in the following, i.e. the elements of the left side only, although they have a mirror image on the right side.
The anatomy of the venom system and of the female internal reproductive system is not discussed thoroughly in the following (for chalcidoids, see [19–25, 35–38, 72–79]; for parasitoid hymenopterans in general, see [26, 27]).
1st valvula (1vv; Figs. 2m–p, 3a, b, f, 4a–d, g–k, and 5a, c): Basally, the thin 1st valvula is continuous with the 1st valvifer via its dorsal ramus (dr1; Figs. 3d, e, g, 5a, c, d, and 6c, j). The 1st valvula has a crescent-shaped cross-section over most of its length (1vv; Fig. 4c). The aulax (au; Figs. 3a and 4g, i, k) of L. distinguendus does not reach the apex of the 1st valvula but tapers off around 50 µm before it. The distal end of the aulax features a coeloconic sensillum (cs; Figs. 3a and 4i, j; sensu [80]), presumably monitoring the position of the 1st valvula relative to the 2nd valvula (cf. [81]). Further sensilla can be seen at regular intervals on the lateral sides (blue ‘notches’ in Fig. 3f), which might have a mechano- and/or chemosensory function. However, the sensillar equipment of the terebra was not further investigated in this study (but see [82–84]). Dorsomedially to the aulax, the medial wall of each 1st valvula is thickened (Fig. 4c). The ventral part of the medial wall is thin and formed into a large membranous fold (when at rest) that projects inwards and overlaps ventrally (Fig. 4a–c; cf. [39]). These thin chitinous folds are considered effectively to seal the crack between the paired 1st valvulae in order to prevent the loss of venom and/or oviposition fluids [39]. The 1st valvula laterally bears two small sawteeth (st1; Fig. 3a) that are of decreasing size at its apex and that are most probably used to penetrate the substrate and the host’s skin. On the dorsomedial side of their apices, the 1st valvulae are connected by the olistheter-like interlock of the 1st valvulae (il1; Fig. 4h, i, k), presumably preventing them from being torn apart during the initial puncturing of the substrate and during drilling. The egg exits the egg canal proximad to these structures and ventrally between the paired 1st valvulae (Fig. 2p; Additional file 1, min. 56–2:26, 36–4:00). Such interlocking structures are also found in other pteromalids and some species of Aphelinidae, Chalcididae, Eulophidae, Eurytomidae, Ormyridae, Tanaostigmatidae and Trichogrammatidae [39]. In all chalcidoids, the ventral ramus of the 1st valvula is completely reduced [29] and the valvilli inside the egg canal are absent [85].Fig. 3Ovipositor of Lariophagus distinguendus. a–e SEM images of the various ovipositor elements (left is anterior). a Apex of the terebra comprising the 2nd valvula and the paired 1st valvulae (dorsolateral view). The 2nd valvula is longitudinally split but fused at the apex, featuring seven sawteeth. The 1st valvula features two small apical sawteeth. Its aulax terminates pre-apically and bears a coeloconic sensillum at its apical end (for cross section cf. Fig. 4i, j). Both the 1st and 2nd valvulae bear various types of sensilla. b Upon removal of the 2nd valvula, the aulaces of the inner surface of the 1st valvula become visible (dorsal view), featuring distally directed scale-like structures. c Upon removal of the 1st valvula, the rhachis at the ventral side of the 2nd valvula becomes visible (ventral view), featuring distally directed scale-like structures similar to those of the aulax. The egg canal is formed by both the 1st and 2nd valvulae and bears microsculpture consisting of distally oriented ctenidia. d Anterior part of the ovipositor (lateral view). The 1st valvifer is continuous with the dorsal ramus of the 1st valvula. It is connected with the 2nd valvifer and the female T9 via the intervalvifer and tergo-valvifer articulation, respectively. The 2nd valvifer possesses a post-ramus flap and two clusters of the sensillar patch located anteriorly to the intervalvifer articulation and the sensillar row along its dorsal margin (e). f WFM image of the apical part of the terebra of L. distinguendus (dorsal view, left is anterior; only the images of the DAPI and Cy5 wavelength filters are superimposed here). The cuticle of the aulaces and the sawteeth of the 2nd valvula are heavily sclerotized (as indicated by their red autofluorescence). g, h Superimposed CLSM images of the basal part of the ovipositor of L. distinguendus (dorsal view, left is anterior; cf. Additional file 2). The cuticle of the valvulae and the valvifers is sclerotized, whereas the ctenidia show a high content of resilin (as indicated by their blue autofluorescence; h). Abbreviations: 1st valvifer; 1st valvula; 2nd valvifer; 2nd valvula; 3rd valvula; Aulax; Basal articulation; Bulb; Coeloconic sensillum; Ctenidium; Dorsal ramus of the 1st valvula; Furcula; Intervalvifer articulation; Laminated bridge; m-p-2vf-2vv: Posterior 2nd valvifer-2nd valvula muscle; Post-ramus flap; Rhachis; Scale-like structure; Sensillar patch of the 2nd valvifer; Sensillar row of the 2nd valvifer; Sawtooth of the 1st valvula; Sawtooth of the 2nd valvula; t-m-d-T9-2vf-a: Tendon of the dorsal 2nd valvifer-T9 muscle part a; T9: Female T9 (9th abdominal tergum); Tergo-valvifer articulationFig. 4Terebra of Lariophagus distinguendus. a–d Light microscopical images of semithin cross sections through the terebra (from proximal to distal; scale bar in d applies to all light micrographs; positions of the sections are indicated in Fig. 6c; cf. Additional file 3). The bulbs and the laminated bridge are visible proximally. The 2nd valvula is connected with the paired 1st valvulae via the olistheter system. e–k TEM images of the terebra of L. distinguendus. The cuticle of the valvulae is remarkably homogenous. e Parts of the laminated bridge on the proximal part of the 2nd valvula (cf. a). f Notal membrane (cf. c). g Olistheter system comprising the rhachis of the 1sr valvula and the aulax of the 2nd valvula. h–k Apical part of the terebra. The olistheter-like interlock of the 1st valvulae on their dorsomedial surfaces (h, I, k) and the coeloconic sensillum at the apical end of one aulax are visible (j; for overview image, cf. Fig. 3a). Abbreviations: 1st valvula; 2nd valvifer; 2nd valvula; 3rd valvula; Aulax; Bulb; Coeloconic sensillum; Dendrite; Egg canal; Interlock of the 1st valvulae; Laminated bridge; Lateral extensions of the 2nd valvula; Lumen of the 2nd valvula; m-1vf-gm: 1st valvifer-genital membrane muscle; Notal membrane; Olistheter; Rhachis; Sawtooth of the 2nd valvula; Ventral wall of the 2nd valvulaFig. 5Ovipositor muscles supporting the venom and reproductive system of Lariophagus distinguendus. a–c SR-µCT images of a virtual slices through the anterior part of the ovipositor (a,b sagittal view, left is anterior, c transversal view) highlighting the muscles supporting the venom and reproductive system, the venom gland reservoir and its orifice, the Dufour’s gland and its duct, and the common oviduct (the colour labels correspond to Fig. 6 and Additional file 4). d Light microscopical image of a semithin cross section through the ovipositor. The ventral 2nd valvifer-venom gland reservoir muscle with its Z lines is clearly visible. Abbreviations: 1st valvula; 2nd valvifer; 2nd valvula; Common oviduct; Dg: Dufour’s gland; Dgd: Dufour’s gland duct; Dorsal projection of the 2nd valvifer; Dorsal ramus of the 1st valvula; Laminated bridge; m-d-2vf-vr: Dorsal 2nd valvifer-venom gland reservoir muscle; m-v-2vf-vr: Ventral 2nd valvifer-venom gland reservoir muscle; m-v-2vf-vr-a: Ventral 2nd valvifer-venom gland reservoir muscle part a; m-v-2vf-vr-b: Ventral 2nd valvifer-venom gland reservoir muscle part b; orifice of the venom gland reservoir; Venom gland reservoir of the 2nd valviferFig. 6Musculoskeletal ovipositor system of Lariophagus distinguendus. Segmented 3D model based on SR–µCT data (perspective view; only the left side of the paired ovipositor elements are depicted; cf. Additional file 4). a Orientation of the ovipositor within the metasoma (lateral view, left is anterior; the metasoma is semi-transparent). b–e Cuticular elements, articulations and muscles of the ovipositor (b lateral view, left is anterior; c dorsal view, left is posterior, positions of sections in Fig. 4a–d are indicated here; d medial view, left is posterior; e frontal view). The ovipositor consists of the following cuticular structures (b): 1st valvifer, 1st valvula, 2nd valvifer, 2nd valvula, 3rd valvula and female T9 (9th abdominal tergum). The 1st valvifer is articulated with the 2nd valvifer and the female T9 via the intervalvifer and the tergo-valvifer articulation, respectively (b). It is continuous with the dorsal ramus of the 1st valvula (c). The 1st and 2nd valvulae form the terebra (1st and 2nd valvulae are not distinguished here). The various ovipositor movements are actuated by a set of nine muscles (d): 1st valvifer-genital membrane muscle, dorsal 2nd valvifer-venom gland reservoir muscle, ventral 2nd valvifer-venom gland reservoir muscle part a/b, anterior 2nd valvifer-2nd valvula muscle, posterior 2nd valvifer-2nd valvula muscle, dorsal T9-2nd valvifer muscle part a/b, ventral T9-2nd valvifer muscle, posterior T9-2nd valvifer muscle and T9-genital membrane muscle. f Anterior part of the ovipositor (dorsomedial view, left is posterior) highlighting the basal articulation and the three muscles connected to the venom gland reservoir. g Base of the terebra featuring the laterally placed bulbs, the laminated bridge and the insertion sites of the anterior and posterior 2nd valvifer-2nd valvula-muscles (i.e. the processus articularis and the processus musculares, respectively) and their orientation (left is anterior; g lateral view, h dorsal view). i Ventral part of the 1st valvifer (posterior view) highlighting the bifurcated posteroventral corner forming one part of the intervalvifer articulation, and the horizontal ridge that is part of the tergo-valvifer articulation. j 1st valvifer (lateral view, left is anterior) with dorsal ramus of the 1st valvula. Acting muscle forces are visualized by solid red arrows. Under the simplified assumption that the 2nd valvifer, which acts as the frame of reference, and the female T9 are guided and cannot twist but only slide towards or against each other along the anterior–posterior axis, the input force vectors Fm-d-T9-2vf(x)-in and Fm-v-T9-2vf(x)-in act in the same plane only at the tergo-valvifer articulation. The distance between the tergo-valvifer articulation (where the force is applied) and the intervalvifer articulation (pivot point/joint axis) is the anatomical inlever a, the effective (= mechanical) inlever is a'; for torques, see equations (eqs.) 1, 2. The 1st valvifer acts as a lever with the anatomical outlever b being the distance between the intervalvifer articulation and the point at which the 1st valvifer continues as dorsal ramus of the 1st valvula, the effective outlever is b', resulting in pro- or retraction forces at the dorsal ramus of the 1st valvula Fm-d-T9-2vf-out and Fm-v-T9-2vf-out; see eqs. 3, 4. Abbreviations: 1st valvifer; 1st valvula; 2nd valvifer; 2nd valvula; 3rd valvula; Basal articulation; Bulb; Dorsal ramus of the 1st valvula; F: Force; F(x): Horizontal vector component of a force; Interarticular ridge of the 1st valvifer; Intervalvifer articulation; Laminated bridge; m-1vf-gm: 1st valvifer-genital membrane muscle; m-a-2vf-2vv: Anterior 2nd valvifer-2nd valvula muscle; m-d-2vf-vr: Dorsal 2nd valvifer-venom gland reservoir muscle; m-d-T9-2vf-a: Dorsal T9-2nd valvifer muscle part a; m-d-T9-2vf-b: Dorsal T9-2nd valvifer muscle part b; m-p-2vf-2vv: Posterior 2nd valvifer-2nd valvula muscle; m-p-T9-2vf: Posterior T9-2nd valvifer muscle; m-T9-gm: T9-genital membrane muscle; m-v-2vf-vr-a: Ventral 2nd valvifer-venom gland reservoir muscle part a; m-v-2vf-vr-b: Ventral 2nd valvifer-venom gland reservoir muscle part b; m-v-T9-2vf: Ventral T9-2nd valvifer muscle; Median bridge of the 2nd valvifers; Metasoma; Medial ridge of the 2nd valvifer; Post-ramus flap; T9: Female T9 (9th abdominal tergum): Tergo-valvifer articulation; Terebra
2nd valvula (2vv; Figs. 2m–p, 3a, c, f–h, 4a–g, i, k, and 5a–c): Proximally, the bulbs of the 2nd valvula (blb; Figs. 3g, h, 4a, and 6e–h) are basally articulated with the 2nd valvifer via the basal articulation (ba; Figs. 3h and 6f). At its basal part, the 2nd valvula bears the processus articuaris laterally on the bulbs, and the processus musculares dorsally on the anteriorly directed horn-like processes of the bulbs. On its ventral side, the 2nd valvula bears the rhachises (rh; Figs. 3c and 4g). The 2nd valvula of L. distinguendus consists of two longitudinally split, asymmetrically overlapping and more-heavily sclerotized halves (2vv; Figs. 3a, f–h and 4a–c; Additional file 2) that are thickened medially (2vv; Fig. 4c). The two halves are dorsally connected for most of their length by a conjunctiva called the notal membrane (nm; Fig. 4f) [17, 19–25, 40] but are fused at the apex (2vv; Figs. 3a and 4d, i, k). Proximally, the notal membrane is modified into a transversely striate band called the laminated bridge (lb; Figs. 3g, h, 4a, b, e, 5a–c, and 6c, f, h) [19, 20, 22, 40]. The modified 2nd valvula with its longitudinally split and overlapping halves presumably permit a greater distortion of the valvula and appear to be a synapomorphy for all Chalcidoidea, except for Mymaridae [39]. The ventral side is formed by the ventral wall of the 2nd valvula (vw2; Fig. 4a–c, f; sensu [86]), which extends from the base almost to the apex. This creates a lumen (lu2; Fig. 4a–d, f, i, k). The rhachises are attached to this lamella-like process over most of their length, except for the apex. Ventrolaterally to the rhachises lie lateral extensions of the 2nd valvula (le; Fig. 4c; sensu [86]). The apex of the 2nd valvulae of L. distinguendus features seven sawteeth that are placed laterally and staggered relative to one another (st2; Figs. 3a, f and 4i) with sensilla being found in between them. The laterally positioned sawteeth are postulated to act like a screw during the alternate rotational movements of the terebra during substrate penetration [22] and seem to be present in all chalcidoid species that undertake drilling actions [25] (Additional file 3).
Terebra (trb; Figs. 1, 2d–p, 3a and 6c–h): The acicular terebra consists of the paired 1st valvulae and the 2nd valvula and has a smooth surface. The terebra of L. distinguendus (and other chalcidoid wasps) features a single opening at the basal end, where the common oviduct (co; Fig. 5) seamlessly merges with the base of the egg canal (cf. [19–23, 25]). In chalcidoid wasps (such as L. distinguendus and Microterys flavus (Howard, 1881) (Encyrtidae) (data not yet published)), both the orifice of the venom gland reservoir (ovr; Fig. 5b–d; Additional file 4, min. 30–0:31) and the dorsolaterally situated Dufour’s gland duct (Dgd; Fig. 5) empty into the common oviduct (cf. [73, 77]) before the latter fuses with the egg canal (unlike in ichneumonoid wasps; cf. [16, 87]). The junction lies directly anterior to the basal articulation (ba; Figs. 3h and 6f) and is indicated by the furcula (Fig. 3g; Additional file 2, min. 21–0:36). The complete length of the egg canal thus functions as a conduit not only for the egg itself, but also for the expulsion of venom or other fluids during oviposition. The diameter of the terebra is even along its length (Fig. 4c; Additional file 3) between the broad basal bulbs (Figs. 3g, h and 4a,b) and the distally tapering apex (Figs. 3a and 4d, i, k). The rhachises (rh; Figs. 3c and 4g) on the ventral side of the 2nd valvula are interlocked with the aulaces (au; Figs. 3a, b and 4g, i, k) on the dorsal side of the opposing 1st valvulae via the olistheter system (oth; Fig. 4c); this enables the 1st valvulae to move along the 2nd valvulae while they are still connected to each other. The olistheters of L. distinguendus does not extend along the entire length of the terebra but end around 50 µm before its apex (Fig. 3a). The distally directed scale-like structures on the contact surfaces of both the rhachises and the aulaces (sc; Fig. 3b, c) presumably reduce frictional forces by minimizing the contact area of the olistheter elements [88]. However, these scale-likes structures potentially also forward a liquid lubricant from the colleterial glands (= accessory glands) to the apex of the olistheter system further to reduce friction in between the moving valvulae (cf. [89]). This arrangement might also enable particles to be continuously flushed out the olistheter system during drilling or venom injection. The scale-like structures might additionally create anisotropic conditions in the olistheter and thus prevent the 1st valvulae from randomly sliding back during drilling and piercing (cf. [31]). The longitudinally split and asymmetrically overlapping halves of the 2nd valvula presumably allow lateral sliding to occur towards or away from each other. Moreover, the rachises of L. distinguendus are suspended from lamellar structures of the ventral wall of the 2nd valvula (vw2; Fig. 4b, c) over their entire length, except for the apex (Fig. 4d). Thus, both the 1st and 2nd valvulae, which are connected via the olistheter system, are presumably movable in their position and may diverge tangentially. Moreover, the dorsally thickened walls of the 1st valvulae can be bent away from the midline and, in doing so, can take up the ventral membranous slack, further increasing the volume inside. This is thought to be an adaptation in several chalcidoid taxa to facilitate deformation of the terebra and temporarily to enlargement of the egg canal (ec; Fig. 4c), which is mainly formed by the two paired 1st valvulae, in order to accommodate the passing egg [39]. The olistheter system thereby must sustain the forces exerted by the egg [62, 71]. However, the maximal diameter of the apical half of the terebra is limited by the diameter of the puncture site in the substrate during oviposition. The areas of the rhachises at the basal bulbous part of the 2nd valvula presumably are also flexible (purple areas of the cuticle in Fig. 4a, b presumably indicating a higher resilin content). The internal microsculpture of the medial wall of the egg canal consists of distally orientated leaf-like ctenidia (ct; Fig. 3c, h) that contain large amounts of resilin (ct; Fig. 3h; Additional file 2, min. 05–0:20) and are found from the proximal basis to the region before the apex. The ctenidia help to push the deformable egg along the egg canal by alternate movements of the 1st valvulae, prevent regression [71, 88] and are also hypothesized to forward a liquid lubricant for the moving valvulae and thus to reduce friction [88, 90] and/or to produce a feeding tube. Both the 1st and 2nd valvulae have tapered apices. The terebra apex in many hymenopteran taxa is heavily sclerotized and hardened with metal atoms, such as calcium (Ca), manganese (Mn) and zinc (Zn). This enables the piercing of hard substrates, reduces wear and tear and prevents buckling [15, 62, 81, 91–94].
3rd valvula (3vv; Figs. 3a, 4d and 6a–d): The relatively short semi-tubular 3rd valvula of L. distinguendus emerges at the posterior end of the 2nd valvifer (Fig. 6a–d) and ensheaths and protects the distal part of the terebra when at rest (Fig. 4d). The distally directed microsetae on the medial surface of the 3rd valvula (Fig. 3a) are thought to be involved in the cleaning of the terebra between oviposition episodes [16, 83]. The 3rd valvula might also have a sensory function [27].
1st valvifer (1vf; Figs. 3d and 6a–d, i, j): The 1st valvifer of L. distinguendus and other chalcidoids is bow-shaped [17, 19–25, 35–37]. The anteroventral angle of the 1st valvifer features a horizontal ridge, which has a medial–lateral orientation (Fig. 6i) and which is part of the tergo-valvifer articulation (tva; Figs. 3d and 6b, i, j). The posteroventral corner of the 1st valvifer is bifurcated (Fig. 6i) and is part of the intervalvifer articulation (iva; Figs. 3d, g and 6b, i, j). The interarticular ridge (iar; Fig. 6i) lies between the two articulations and might serve mechanically to stabilize the 1st valvifer. The anterodorsal angle of the 1st valvifer is continuous with the dorsal ramus of the 1st valvula (dr1; Figs. 3d, e, g, 5a, c, d, and 6c, j), which is interlocked with the dorsal projection of the 2nd valvifer (dp2; Fig. 5c, d; cf. [31]) by a system analogous to the olistheter. This tight interlocking guides the dorsal ramus and prevents it from buckling when pushing forces are applied during the protraction of the 1st valvula. Since the dorsal ramus constantly slides around the proximal bulbous end of the 2nd valvula during pro- and retraction, the ramus needs to be flexible in the sagittal plane and thus presumably contains high proportions of the elastic rubber-like protein resilin in its cuticle (cf. [95–98]).
2nd valvifer (2vf; Figs. 3d, 4a–c, 5a, c, d and 6a–d, f): The 2nd valvifer is elongated and its posterior part is placed medially of the female T9 (Fig. 6b). A conjunctive, called the genital membrane (not shown), connects the ventral margins of the paired 2nd valvifers arching above the 2nd valvula. The anterior part of the 2nd valvifer of L. distinguendus extends dorsally in a semi-circular shape and dorsally bears the dorsal projection of the 2nd valvifer (dp2; Fig. 5c, d), which is interlocked with the dorsal ramus of the 1st valvula via an interlocking system similar to the olistheter (cf. [31]). At its posterodorsal end and posterior to its medial ridge (mr2; Fig. 6f), the anterior part of the 2nd valvifer features the post-ramus flap (prf; Figs. 3d and 6b; sensu [22]), on which the dorsal projection continues, thus allowing a greater arc of movement of the 1st valvifer and therefore a greater retraction of the 1st valvula. The 2nd valvifer features two sensillar (1) the sensillar patch (sp; Fig. 3d) located anteroventrally to the intervalvifer articulation (iva; Figs. 3d, g and 6b, i, j) and (2) the row of sensilla (sr; Fig. 3e) on the dorsal margin of the 2nd valvifer. These two sensillar patches are in contact with the ventromedial side of the 1st valvifer and the dorsal ramus of the 1st valvula, respectively, and probably monitor the movements of the 1st valvula indirectly. The dorsal margins and the dorsal flanges are strengthened by cuticular ridges that putatively have a stabilizing function and prevent deformation (i.a. at the intervalvifer articulation). The posterodorsal ends of the 2nd valvifers are connected by the median bridge (mb2; Fig. 6c). The venom gland reservoir (vr; Fig. 5a, b; Additional file 2, min. 37–0:52; Additional file 4, min. 24–0:31; = acid gland reservoir sensu [19–25, 73]) is situated in between the 2nd valvifers with its proximal end lying near the base of the terebra. The Dufour’s gland (Dg; Fig. 5a; Additional file 4, min. 21–0:31; = alkaline gland sensu [19–25, 73]) is situated dorsolaterally to the venom gland reservoir (cf. [77, 79]).
Female T9 (T9; Figs. 3d and 6a–d): The female T9 of L. distinguendus is U-shaped and situated lateral to the posterior part of the 2nd valvifers (Fig. 6b). Its elongated anteriorly projecting arms articulate with the 1st valvifers via the tergo-valvifer articulations (tva; Figs. 3d and 6b, i, j). The cordate apodeme (not shown) on the anterior margin of the female T9 is located posterior to the articulation. The dorsal margins are strengthened by the anterior flange of T9, which presumably mechanically stabilizes the female T9 during oviposition. Medially, the anterior flange of T9 bifurcates and forms a dorsomedial crest-like ridge that runs almost the entire length of the female T9. This ridge serves as a muscle attachment area both medially and laterally and presumably increases the mechanical stability of the female T9.
Basal articulation (ba; Figs. 3h and 6f): The two articular surfaces of this ball-and-socket-like articulation are located on the socket-like pars articularis of the anteroventral part of the 2nd valvifer and the ball-like processus articulated laterally on the bulb of the 2nd valvula. This rotational joint presumably also allows some pivotal and rotational movements of the 2nd valvula and thus of the whole terebra.
Intervalvifer articulation (iva; Figs. 3d, g and 6b, i, j): The 1st and 2nd valvifer are connected via the intervalvifer articulation, a rotational joint that allows a rotation of the 1st valvifer in the sagittal plane only [32]. This articulation consists of the bifurcated posteroventral corner of the 1st valvifer (iva; Fig. 6i), which encloses the articulation site at the 2nd valvifer. Thereby, one furcal structure of the 1st valvifer is placed medially and one laterally to the 2nd valvifer.
Tergo-valvifer articulation (tva; Figs. 3d and 6b, i, j): The 1st valvifer lies adjacent to the female T9 via the tergo-valvifer articulation, which is situated dorsally to the intervalvifer articulation. It is a rotational joint that allows the 1st valvifer to rotate in the sagittal plane only [32]. This articulation consists of a horizontal ridge at the 1st valvifer (tva; Fig. 6i) and a corresponding counterpart at the female T9 situated near the cordate apodeme.
In total, nine paired ovipositor muscles have been identified that drive and actuate the associated skeletal apparatus (Table 1). Three of these muscles (i.e. the 1st valvifer-genital membrane muscle, the ventral 2nd valvifer-venom gland reservoir muscle and the T9-genital membrane muscle) are described here for the first time in chalcidoids.Table 1Ovipositor muscles of Lariophagus distinguendus (abbreviations in brackets) and their origin, insertion (cf. Additional file 4) and presumed function as verified in the present contributionMuscle name (abbreviation)OriginInsertionPresumed functions1st valvifer-genital membrane muscle (m-1vf-gm)*Medial surface of the posteroventral part of the 1st valvifer, at the centre between the tergo-valvifer and the intervalvifer articulation (Fig. 6c, d, f)Anteriorly at the genital membrane (Fig. 4c)Tensor muscle for stabilization of the 1st valvifer during ovipositor movementsDorsal 2nd valvifer-venom gland reservoir muscle (m-d-2vf-vr)Medial surface of the most anterior part of the 2nd valvifer (Fig. 6c–f)Dorsally at the anterior part of the venom gland reservoir (Fig. 5a, b)Supporting the discharge of venom gland reservoir secretion and probably of Dufour’s gland secretion, tensor muscle for stabilization of the 2nd valvifer during ovipositor movementsVentral 2nd valvifer-venom gland reservoir muscle part a (m-v-2vf-vr-a)*Medial surface of the most anterior part of the 2nd valvifer, ventrally to the origin of m-d-2vf-vr (Fig. 6d–f)Laterally at the orifice of the venom gland reservoir (Fig. 5c, d)Increasing the diameter of the orifice of the venom gland reservoir, thus controlling the venom dischargeVentral 2nd valvifer-venom gland reservoir muscle part b (m-v-2vf-vr-b)*Medial surface of the most anterior part of the 2nd valvifer, posteroventrally to the origin of m-v-2vf-vr-a (Fig. 6d–f)Laterally at the orifice of the venom gland reservoir, ventrally to the insertion of m-v-2vf-vr-a, shortly before the orifice of the venom gland reservoir enters the common oviduct (Fig. 5c, d)Anterior 2nd valvifer-2nd valvula muscle (m-a-2vf-2vv)Medial region along the anterodorsal arch of the 2nd valvifer (Fig. 6c, d)At the processus articularis on the 2nd valvula, laterally at the bulbs of the 2nd valvula (Fig. 6f–h)Pulling of the corresponding bulb of the 2nd valvula dorsad (thus inducing lateral bending movements of the terebra) in the active probing position, assistance in the rotation of the terebra during oviposition process, elevator of the terebra back into its resting position (once withdrawn from the substrate), holding the terebra in resting positionPosterior 2nd valvifer-2nd valvula muscle (m-p-2vf-2vv)Medial region along the ventral part of the 2nd valvifer (Fig. 6c, d)At the processus musculares on the 2nd valvula, dorsally at the anteriorly directed horn-like processes of the bulbs of the 2nd valvula (Fig. 6f–h)Rotation of the terebra during oviposition process, inducing partial deformation of the 2nd valvula by moving its two halves tangentially towards each other, holding of the terebra in the active probing positionDorsal T9-2nd valvifer muscle part a (m-d-T9-2vf-a)Lateral region along the posterodorsal part of the female T9, laterally along its dorsomedial ridge (Fig. 6a–d)Anterior section of the dorsal flange of the 2nd valvifer, posterior to its medial ridge (Fig. 6c, d, f)Protractor of the 1st moves the 2nd posteriorly and the female T9 anteriorly towards each other, causing the 1st valvifer to tilt anteriorly and thus the 1st valvula to slide distally relative to the 2nd valvulaDorsal T9-2nd valvifer muscle part b (m-d-T9-2vf-b)Medial region along the posterodorsal part of the female T9, ventromedially to its dorsomedial ridge (Fig. 6c, d)Anterior section of the dorsal flange of the 2nd valvifer via a tendon, ventrally to the insertion of m-d-T9-2vf-a (Fig. 6c, d)Ventral T9-2nd valvifer muscle (m-v-T9-2vf)At the cordate apodeme at the anterior margin of the female T9, posteriorly to the tergo-valvifer articulation (Fig. 6c, d)Medial surface along the posterior section of the dorsal flange of the 2nd valvifer (Fig. 6c, d)Retractor of the 1st moves the 2nd anteriorly and the female T9 posteriorly apart from each other, causing the 1st valvifer to tilt posteriorly and thus the 1st valvula to slide proximally relative to the 2nd valvulaPosterior T9-2nd valvifer muscle (m-p-T9-2vf)Medial surface of the posterodorsal part of the female T9 (Fig. 6c, d)Median bridge of the 2nd valvifersTensor muscle for stabilization by holding the posterior part of the 2nd valvifer in position during ovipositor movementsT9-genital membrane muscle (m-T9-gm)*Medial surface of the posterodorsal part of the female T9, dorsally of the origin of m-p-T9-2vf (Fig. 6c, d)Posteriorly at the genital membraneTensor muscle for stabilizationThe muscles marked with * are described here for the first time in chalcidoids
1st valvifer-genital membrane muscle (m-1vf-gm; Figs. 4c and 6d, e, f): This muscle is the only muscle of the 1st valvifer. It originates at the medial surface of the posteroventral part of the 1st valvifer, i.e. at the centre between the tergo-valvifer and the intervalvifer articulation (Fig. 6c, d, f), and inserts anteriorly on the genital membrane (Fig. 4c). We here describe the m-1vf-gm for the first time in Chalcidoidea. Previous authors (e.g. [17, 19–25, 36]) might have overlooked its presence because of to its minute size.
Dorsal 2nd valvifer-venom gland reservoir muscle (m-d-2vf-vr; Figs. 5a–c and 6d, e, f): This muscle originates at the medial surface of the most anterior part of the 2nd valvifer (Fig. 6c–f) and inserts dorsally at the anterior part of the venom gland reservoir (Fig. 5a, b), which is located ventrally of the common oviduct. Most previous authors (e.g. [17, 19–25]) have overlooked the presence of this muscle; it was only mentioned by [73].
Ventral 2nd valvifer-venom gland reservoir muscle (m-v-2vf-vr-a/b; Figs. 5a, c, d and 6d–f): This muscle forms two distinct bundles. Its anterodorsal part (m-v-2vf-vr-a) originates at the medial surface of the most anterior part of the 2nd valvifer, ventrally to the origin region of the dorsal 2nd valvifer-venom gland reservoir muscle (Fig. 6d–f), and inserts laterally at the orifice the venom gland reservoir (Fig. 5c, d). The other part (m-v-2vf-vr-b) originates at the medial surface of the anterior part of the 2nd valvifer, posteroventrally to the origin region of part a (Fig. 6d–f), and inserts laterally at the orifice of the venom gland reservoir, ventrally to the insertion of part a and shortly before the orifice of the venom gland reservoir enters the common oviduct (Fig. 5c, d). To our knowledge, this muscle has also not yet been described in chalcidoids (but see [99–101] for the description of a similar set of muscles in ants).
Anterior 2nd valvifer-2nd valvula muscle (m-a-2vf-2vv; Fig. 6d, f, g, h): This muscle originates at the medial region along the anterodorsal arch of the 2nd valvifer (Fig. 6c, d) and inserts at the processus articularis, located laterally on the bulbs of the 2nd valvula (Fig. 6f–h).
Posterior 2nd valvifer-2nd valvula muscle (m-p-2vf-2vv; Fig. 6d, f, g, h): This muscle originates at the medial region along the ventral part of the 2nd valvifer (Fig. 6c, d) and inserts at the processus musculares, located dorsally on the anteriorly directed horn-like processes of the bulbs of the 2nd valvula (Fig. 6f–h).
Dorsal T9-2nd valvifer muscle (m-d-T9-2vf-a/b; Fig. 6d): This muscle is modified in its insertion and forms two distinct muscle bundles. One part of this muscle (m-d-T9-2vf-a) originates at the lateral region along the posterodorsal part of the female T9, i.e. laterally along its dorsomedial ridge (Fig. 6a–d), and inserts at the anterior section of the dorsal flange of the 2nd valvifer, posterior to its medial ridge (Fig. 6c, d, f). The other part (m-d-T9-2vf-b) originates at the medial region along the posterodorsal part of the female T9, i.e. ventromedially to its dorsomedial ridge (Fig. 6c, d), and inserts at the anterior section of the dorsal flange of the 2nd valvifer via a tendon (t-m-d-T9-2vf-a; Fig. 3g), located ventrally to the insertion region of m-d-T9-2vf-a (Fig. 6c, d).
Ventral T9-2nd valvifer muscle (m-v-T9-2vf; Fig. 6d): This muscle originates at the cordate apodeme, which is located at the anterior margin of the female T9, posteriorly to the tergo-valvifer articulation (Fig. 6c, d), and inserts at the medial surface along the posterior section of the dorsal flange of the 2nd valvifer (Fig. 6c, d).
Posterior T9-2nd valvifer muscle (m-p-T9-2vf; Fig. 6d): This muscle originates at the medial surface of the posterodorsal part of the female T9 (Fig. 6c, d) and inserts at the median bridge of the 2nd valvifers. Previous studies on the chalcidoid ovipositor [17, 19–25] report only one muscle originating in the posterior region of the female T9. The authors presumably were unable to distinguish this muscle from the T9-genital membrane muscle described below.
T9-genital membrane muscle (m-T9-gm; Fig. 6d): This muscle originates at the medial surface of the posterodorsal part of the female T9, dorsally of the origin region of the posterior T9-2nd valvifer muscle (Fig. 6c, d), and inserts posteriorly at the genital membrane. We here describe the m-T9-gm for the first time in Chalcidoidea.
The set of nine paired ovipositor muscles in L. distinguendus comprises two pairs of two antagonistically working muscles that are mainly responsible for the various ovipositor movements, three muscles stabilizing the musculoskeletal system, and two muscles related to the function of the venom gland reservoir (Table 1).
Depression and elevation of the terebra: The 2nd valvula is connected with the 2nd valvifer by a rotational joint called the basal articulation (ba; Figs. 3h, 6f and 7a). Two muscles (m-a-2vf-2vv, m-p-2vf-2vv) insert at the bulbous region around this articulation. The insertion region of the posterior 2nd valvifer-2nd valvula muscle (m-p-2vf-2vv; Fig. 6f–h) at the 2nd valvula is located dorsal of the basal articulation, whereas its region of origin at the 2nd valvifer is located posteroventral to it (Fig. 6c, d). Taxa from other superfamilies use the m-p-2vf-2vv to depress their terebra towards an active probing position (e.g. Ichneumonoidea [31, 32]). However, female L. distinguendus have never been observed to depress their terebra in such a manner. Instead, these wasps bend their whole metasoma downwards to bring their terebra into the drilling position. Once the apex of the terebra is engaged in the substrate, the metasoma is lifted upwards again, while the terebra remains in its depressed position (Fig. 2b–d; Additional file 1, min. 06–0:11; cf. [45, 47]). This behaviour has also been reported for other pteromalids [40, 46] and species of Torymidae [23], Eurytomidae [21], Encyrtidae (data not yet published) and Eulophidae [19]. Therefore, in pteromalids (and possibly also in other chalcidoid taxa), we assume that the m-p-2vf-2vv is adapted in its function (see paragraph ‘Rotation of the terebra’ of the subchapter ‘Mechanisms of terebra bending and rotation’ below). During this indirect depression of the terebra, the bulbs of the 2nd valvula might be pulled out of the socket-like anterior ends of the 2nd valvifer ventrally by pushing them slightly apart, resulting in a slight translation of the pivot point (= joint axis or fulcrum) of the basal articulation (cf. [32]). The insertion region of the anterior 2nd valvifer-2nd valvula muscle (m-a-2vf-2vv; Fig. 6f–h) at the 2nd valvula is situated posteroventrally of both the basal articulation and the insertion region of m-p-2vf-2vv, whereas its origin at the 2nd valvifer is located posterodorsally of this articulation (Fig. 6c, d). After an oviposition attempt, the terebra is withdrawn from the substrate. Since slender structures such as the terebra can support much higher tensile than compressive stresses, the withdrawal does not damage it [62]. A contraction of the anterior 2nd valvifer-2nd valvula muscle (Fm-a-2vf-2vv; Fig. 7a) presumably initiates the elevation of the terebra (arrow 9; Fig. 7a; Table 1). The passive rebound of the bulbs of the 2nd valvula into the socket-like anterior ends of the 2nd valvifer presumably further supports the elevation of the terebra passively and helps to stabilize it in its resting position (cf. elevation of the terebra in ceraphronoids, which completely lack the m-a-2vf-2vv [102]). The anatomical cluster comprising the 2nd valvifer, the 2nd valvula and the two muscles connecting them is a simple mechanical system in which the 2nd valvula is a two-armed class 1 lever, whereby the effective (= mechanical) inlever arm and the joint angle (attachment angle) of m-a-2vf-2vv change over the range of motion (cf. [31]).Fig. 7Mechanisms driving the various ovipositor movements of Lariophagus distinguendus, and the importance of the terebra movements during the oviposition process. a–c Functional model of the mechanisms driving the various ovipositor movements in the resting and the active probing position (only the left side of the paired ovipositor elements are depicted; not to scale). Acting (input) muscle forces are visualized by solid red arrows and resulting (output) movements by solid black arrows. a Mechanism of the tilting movement of the 1st valvifer and the resulting pro- and retraction of the 1st valvulae (lateral view, left is anterior). Only the two pairs of antagonistically working muscles that are responsible for these movements (m-a-2vf-2vv/m-p-2vf-2vv and m-d-T9-2vf/m-v-T9-2vf) are represented in simplified terms. The muscles stabilizing the ovipositor system (m-1vf-gm; m-p-T9-2vf; m-T9-gm) and those supporting the venom and reproductive systems (m-d-2vf-vr; m-v-2vf-vr) are not shown. Contraction of (both parts of) m-d-T9-2vf (Fm-d-T9-2vf) slides the 2nd valvifer posteriorly and the female T9 anteriorly towards each other (arrow 1), thus indirectly causing the 1st valvifer to tilt anteriorly (arrow 2). This is possible because the 1st valvifer is articulated with both the 2nd valvifer and the female T9 via the intervalvifer and tergo-valvifer articulation, respectively. The 1st valvifer thereby functions as a lever arm that transmits the movement to the dorsal ramus of the 1st valvula (arrow 3) and consequently causes a protraction of the 1st valvula (arrow 4). M-p-T9-2vf and m-T9-gm thereby presumably stabilize the system by holding the 2nd valvifer and the female T9 in position and preventing them from rotating around the articulations. Contraction of m-v-T9-2vf (Fm-v-T9-2vf) slides the 2nd valvifer anteriorly and the female T9 posteriorly apart from each other (arrow 5), thus causing the 1st valvifer to tilt posteriorly (arrow 6). This movement is transmitted via the dorsal ramus of the 1st valvula (arrow 7) and consequently causes its retraction (arrow 8). When the terebra is withdrawn from the substrate, a contraction of the m-a-2vf-2vv (Fm-a-2vf-2vv) presumably causes the bulbs to pivot posteriorly around the basal articulation, thus elevating the 2nd valvula and therefore the whole terebra back into its resting position (arrow 9). b, c Mechanisms of the bending and rotational movements of the terebra (b lateral view, left is anterior; c dorsal view; schematic drawing of wasp in lateral view). During oviposition, a contraction of m-a-2vf-2vv cannot elevate the terebra back towards its resting position (as described in other hymenopteran taxa), because the terebra is anchored at the puncture site in the substrate. In this situation, a contraction of one of the paired m-a-2vf-2vv (Fm-a-2vf-2vv) in the active probing position pulls the corresponding bulb and thus one half of the longitudinally split and asymmetrically overlapping 2nd valvula dorsad along its longitudinal axis (arrow 10) because of the orientation of the muscle and the resulting direction of the force vector. Since the halves of the 2nd valvula are fused at the apex, this movement causes the distal part of the terebra (i.e. the part inside the cavity in the substrate) to bend to the left or a contraction of the left m-a-2vf-2vv causes the 2nd valvula and thus the whole terebra to bend to the left (arrow 11), a contraction of the right m-a-2vf-2vv causes a bend to the right. In addition, a contraction of one of the m-p-2vf-2vv (Fm-p-2vf-2vv) in the active probing position presumably causes the 2nd valvula and thus the whole terebra to rotate back and forth at the basal articulation along its longitudinal axis to a certain a contraction of the left m-p-2vf-2vv causes the 2nd valvula and thus the whole terebra to rotate anti-clockwise when viewed from the dorsal side (arrow 12), whereas a contraction of the right m-p-2vf-2vv results in a clockwise rotation (cf. Additional file 1). Contractions of the m-a-2vf-2vv might support these rotational movements. The rotation allows the bending movements to take effect in different directions. d Timeline of the oviposition process of an idiobiont ectoparasitoid wasp highlighting the importance of terebra movements during the various stages (stages in parenthesis do not occur in L. distinguendus; stages with * do not occur in all parasitoid lifestyles but are particularly notable in idiobiont ectoparasitoids). Abbreviations: 1st valvifer; 1st valvula; 2nd valvifer; 2nd valvula; 3rd valvula; Basal articulation; Dorsal ramus of the 1st valvula; F: Force; Intervalvifer articulation; m-a-2vf-2vv: Anterior 2nd valvifer-2nd valvula muscle; m-d-T9-2vf: Dorsal T9-2nd valvifer muscle; m-p-2vf-2vv: Posterior 2nd valvifer-2nd valvula muscle; m-v-T9-2vf: Ventral T9-2nd valvifer muscle; T9: Female T9 (9th abdominal tergum); Tergo-valvifer articulation; Terebra
Pro- and retraction of the 1st valvulae: Three muscles (m-d-T9-2vf, m-v-T9-2vf, m-p-T9-2vf) connect the 2nd valvifer with the female T9. Both of these cuticular structures are connected with the 1st valvifer via the intervalvifer articulation and the tergo-valvifer articulation (iva/tva; Figs. 3d, 6b, i, j and 7a), respectively. The insertion region of both parts of the dorsal T9-2nd valvifer muscle (m-d-T9-2vf-a/b; Fig. 6c, d) at the 2nd valvifer are situated anterodorsally, whereas their regions of origin at the female T9 are located posterodorsally of both articulations (Fig. 6c, d). A simultaneous contraction of m-d-T9-2vf-a and m-d-T9-2vf-b (summarized as Fm-d-T9-2vf; Fig. 7a) slides the 2nd valvifer posteriorly with respect to the female T9 (arrow 1; Fig. 7a). This causes the 1st valvifer to tilt anteriorly (arrow 2; Fig. 7a), because it is articulated with both the 2nd valvifer and the female T9 via rotational joints. The 1st valvifer acts as a lever that transforms its tilting movement to the dorsal ramus of the 1st valvula (arrow 3; Fig. 7a). Its tight interlocking with the dorsal projection of the 2nd valvifer prevents it from buckling and transmits the movements to the apex of the 1st valvula, causing it to slide distally relative to the 2nd valvula, i.e. to protract (arrow 4; Fig. 7a; Table 1). In the active probing position, the dorsal ramus is less curved, which presumably reduces friction [32]. The region of origin of the antagonistically acting ventral T9-2nd valvifer muscle at the female T9 (m-v-T9-2vf; Fig. 6c, d) is situated posterodorsally near the intervalvifer articulation and posterior to the tergo-valvifer articulation, whereas its insertion region at the 2nd valvifer is located posteroventrally of both these articulations (Fig. 6c, d). Its contraction (Fm-v-T9-2vf; Fig. 7a) slides the 2nd valvifer anteriorly with respect to the female T9 (arrow 5; Fig. 7a), thus indirectly causing the 1st valvifer to tilt posteriorly (arrow 6; Fig. 7a) and the 1st valvula to slide proximally relative to the 2nd valvula, i.e. to retract (arrows 7, 8; Fig. 7a; Table 1). The vibration-like rapid reciprocal alternate pro- and retracting movements of the 1st valvulae are crucial for drilling and precise egg laying (Fig. 2m–p; Additional file 1, min. 11–1:35, 56–2:22, 36–4:00; cf. [32, 44, 47]). The following assumptions have been made for a simplified estimation of the torques (M) exerted by the forces of the dorsal and ventral T9-2nd valvifer muscles (Fm-d-T9-2vf/Fm-v-T9-2vf; Fig. 7a): (1) The 2nd valvifer acts as the frame of reference; therefore, the intervalvifer articulation (iva; Figs. 6i, j and 7a) acts as a pivot point around which the 1st valvifer tilts; (2) the movements of 2nd valvifer and the female T9 are constrained by the musculoskeletal system and cannot twist around the articulations but only slide telescopically towards or against each other along the anterior–posterior axis; and (3) frictional forces in the system can be neglected. In reality, all cuticular elements can move relatively to each other. However, under these assumptions, the horizontal force vector components acting in the anterior–posterior axis (Fm-d-T9-2vf(x)-in/Fm-v-T9-2vf(x)-in; Fig. 6j) act at the 1st valvifer at the tergo-valvifer articulation (tva; Figs. 6i, j and 7a). Therefore, the torques (M) of Fm-d-T9-2vf and Fm-v-T9-2vf that act at the intervalvifer articulation in the resting position can be estimated by using the horizontal vector components (Fm-d-T9-2vf(x)-in/Fm-v-T9-2vf(x)-in; Fig. 6j) of the maximum force of a muscle, the length of the anatomical inlever arm (a; Fig. 6j), i.e. the distance between the intervalvifer and the tergo-valvifer articulation, and the joint angle (α; Fig. 6j) according to the 1\documentclass[12pt]{minimal}
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M_{{\text{m-d-T9-2vf}}} = F_{{{\text{m-d-T9-2vf}}\left( {\text{x}} \right){\text{-in}}}} \cdot {\text{a}} \cdot \sin(\alpha)