RESEARCH ARTICLE Open Access

Structure and function of the musculoskeletalovipositor system of an ichneumonid waspBenjamin Eggs1*† , Annette I. Birkhold2† , Oliver Röhrle2 and Oliver Betz1

Abstract

Background: Modifications of the ovipositor appear to have played a prominent role in defining the host range ofparasitoid hymenopterans, highlighting an important contributing factor in shaping their oviposition strategies, lifehistories and diversification. Despite many comparative studies on the structure of the hymenopteran terebra, littleis known about functional aspects of the musculoskeletal ovipositor system. Therefore, we examined all inherentcuticular elements and muscles of the ovipositor of the ichneumonid wasp Venturia canescens (Gravenhorst, 1829),investigated the mechanics of the ovipositor system and determined its mode of function.

Results: We found that the movements of the ichneumonid ovipositor, which consists of the female T9 (9th abdominaltergum), two pairs of valvifers and three pairs of valvulae, are actuated by a set of six paired muscles. The posterior andthe anterior 2nd valvifer-2nd valvula muscles flex and extend the terebra from its resting towards an active probingposition and back. The dorsal T9-2nd valvifer muscle is modified in V. canescens and forms distinct bundles that, togetherwith the antagonistically acting ventral T9-2nd valvifer muscle, change the relative position of the 2nd valvifer to thefemale T9. Thereby, they indirectly tilt the 1st valvifer because it is linked with both of them via intervalvifer andtergo-valvifer articulation, respectively. The 1st valvifer acts as a lever arm that transfers movements to the 1stvalvula. The posterior T9-2nd valvifer muscle and the small 1st-valvifer-genital membrane muscle stabilize thesystem during oviposition.

Conclusions: From our examination of the elements of the musculoskeletal ovipositor system of ichneumonids,we discussed leverages and muscle forces and developed a functional model of the underlying working mechanismsadding to our understanding of a key feature that has largely determined the evolutionary success of the megadiverseIchneumonidae with more than 24,000 hitherto described species.

Keywords: Hymenoptera, Ichneumonidae, Kinematics, Muscles, Ovipositor, Parasitoid, SEM, SR-μCT

BackgroundThe vast majority of hymenopterans are parasitoids ofother insects. Apart from oviposition, their ovipositorserves several tasks in the parasitoid lifestyle, i.e. navi-gating or penetrating the substrate (if the host is con-cealed) or the targeted egg/puparium, assessing thehost, discriminating between suitable and previouslyparasitized hosts, piercing the host, injecting venom,oviciding the competitors’ eggs and finding a suitableplace for egg laying [1]. In some species, the ovipositoris also used to form a feeding tube for host feeding or

defensive stinging [2]. Undoubtedly, modifications ofthe ovipositor apparatus have been one of the key fac-tors in the evolution of the parasitoids’ ovipositionstrategies, the life histories and the enormous diversifi-cation of this large and ecologically important insectorder [2–4].The hymenopteran ovipositor consists of the female

T9 (9th abdominal tergum), two pairs of valvifers andthree pairs of valvulae (cf. Figs. 1a, c, 5a) derived fromthe 8th and 9th abdominal segments (7th and 8th meta-somal segments) (morphological terms are appliedaccording to the Hymenoptera Anatomy Ontology(HAO) [5–7]; a table of the terms used, their definitionsand synonyms is given in Table 2 in the Appendix). Thebasally situated valvifers accommodate the operating mus-culature, whereas all the valvulae are devoid of intrinsic

* Correspondence: benjamin.eggs@uni-tuebingen.de†Benjamin Eggs and Annette I. Birkhold contributed equally to this work.1Evolutionary Biology of Invertebrates, Institute of Evolution and Ecology,University of Tübingen, Auf der Morgenstelle 28, 72076 Tübingen, GermanyFull list of author information is available at the end of the article

BMC Zoology

© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Eggs et al. BMC Zoology (2018) 3:12 https://doi.org/10.1186/s40850-018-0037-2

http://crossmark.crossref.org/dialog/?doi=10.1186/s40850-018-0037-2&domain=pdfhttp://orcid.org/0000-0001-7618-4326http://orcid.org/0000-0002-6375-4745http://orcid.org/0000-0002-1934-6525http://orcid.org/0000-0002-5012-4808mailto:benjamin.eggs@uni-tuebingen.dehttp://creativecommons.org/licenses/by/4.0/http://creativecommons.org/publicdomain/zero/1.0/

musculature [8–10]. The 1st valvifers (fusion of the 8thgonocoxites with the gonangula [10]; = gonangulum,gonangula sensu [1]) anterordorsally are continuous withthe rami of the 1st valvulae (8th gonapophyses; = lowervalves sensu [1]). Their posterior angles articulate dorsallywith the female T9 via the tergo-valvifer articulation andventrally with the 2nd valvifers via the intervalvifer ar-ticulation. The 2nd valvifers (9th gonocoxites) extendin the form of the 3rd valvulae (9th gonostyli; = ovi-positor sheaths sensu [1]) and are anteroventrallyarticulated with the 2nd valvula (fusion of the 9thgonapophyses; = upper valve sensu [1]) [8, 9], which issecondarily re-separated except at the apex in someparasitoid taxa [11]. The interlocked 1st and 2nd val-vulae enclose the egg canal and form the terebra (=ovipositor (shaft) sensu [1]), which is embraced by the3rd valvulae when not in use. The ventral surface ofthe 2nd valvula is interlocked with both of the 1st val-vulae by a sublateral longitudinal tongue called therhachis, which runs within a corresponding groovecalled the aulax along the dorsal surface of each of the1st valvulae. This so-called olistheter system allows thethree parts of the terebra to slide longitudinally relativeto each other [9, 11]. The sensillar equipment of the1st and 2nd valvulae is highly variable among parasit-oid hymenopterans [2].Despite many descriptive studies on the comparative

morphology of the hymenopteran terebra [8, 9, 11, 12],the mode of function of the musculoskeletal ovipositorsystem has only been described in some “symphytan”families [10, 13–15], in the aculeate Apis mellifera Lin-naeus, 1758 (Apidae) [8] and Cryptocheilus versicolor(Scopoli, 1763) (Pompilidae) [16], in some species ofCynipoidea [17, 18], and in a few parasitoid species ofCeraphronoidea [19] and Chalcidoidea [20–27]. How-ever, the underlying working mechanisms of the mus-culoskeletal ovipositor system of the extremely diverseand species-rich superfamily of Ichneumonoidea hasremained largely unexplored so far and little is knownabout the actuation of the various ovipositor move-ments that are executed during oviposition. In thisstudy, we investigated structural, mechanical and func-tional aspects of the ovipositor of Venturia canescens(Gravenhorst, 1829) (Hymenoptera: Ichneumonidae:Campopleginae), a cosmopolitan, synovigenic [28],non-host feeding [29], solitary, koinobiont larval endo-parasitoid of several moth species (Lepidoptera) [30,31]. The oviposition behaviour (Additional file 1) is de-scribed by Rogers [32]. These parasitoid wasps coattheir eggs with virus-like particles (VLPs) to circum-vent their host’s immune system [33–37] and exhibitboth arrhenotokous and obligate thelytokousreproduction modes [38–41]. We aimed to (1) describethe ovipositor of V. canescens, including all inherent

cuticular elements and muscles, (2) examine the me-chanics of this musculoskeletal system, (3) determineits mode of function and (4) discuss the process ofoviposition.

Results and discussionWe combined light microscopy (LM), scanning electronmicroscopy (SEM), synchrotron X-ray phase-contrastmicrotomography (SR-μCT) and subsequent 3D imageprocessing with muscle and leverage analyses. Based onthese microscopical and microtomographical studies,we present a thorough morphological, mechanical andfunctional analysis of the musculoskeletal ovipositorsystem (Additional file 2) that steers the various move-ments executed by the female ichneumonid wasp dur-ing oviposition.

Cuticular elements of the ovipositorThe paired 1st valvulae (1vv, Figs. 1a, c, e, 2a, b, e, f,g, 4d) of V. canescens are terminally differentiated infive apically directed sawteeth (st; Fig. 2b) of decreasingsize that are used to penetrate the substrate and the host’sskin [42, 43]. Each of the 1st valvulae has a medioventralpart formed into a thickened longitudinal flap that pro-jects inwards into the egg canal (lf1; Fig. 3a; =medio-ventral seal sensu [16]). These thin chitinuous flapsare considered to effectively seal the crack between the 1stvalvulae and prevent the loss of venom and/or ovipositionfluid during oviposition [11, 44–46]. The pressure of thevenom squeezes the two membranes together and thuscloses the seal. A transverse flap called the valvillus (vlv;Fig. 2e) protrudes from their medial walls and projectsinto the central egg/venom canal (cf. [32]). Segregate val-villi are typical for taxa of Ichneumonoidea but vary inshape and number between subfamilies [11, 46]. Innon-aculeate Hymenoptera, they potentially serve as astop and release mechanism for the egg by maintainingthe egg in position within the terebra and blocking the eggcanal [32, 43, 46] or by pushing fluids into the ovipositor,thereby creating a hydrostatic pressure that forces the eggout of the terminal portion of the egg canal [43]. Theinternal microsculpture of the medial walls of the eggcanal consists of distally oriented scale-like structures;leaf-like ctenidia (ct; Fig. 2f ) occur from the proximalbasis of the valvulae to the further distally positionedregion of the valvillus, where they are replaced byspine-like subctenidial setae (scts; Fig. 2g). The cten-idia help to push the deformable egg along the eggcanal by alternate movements of the 1st valvulae andprevent it from moving backwards [43, 46, 47]. Theyare also hypothesized to deliver forward a liquid lu-bricant for the moving valvulae and thus reduce

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Fig. 1 SEM images of Venturia canescens. a The posterior part of the metasoma (lateral view) with the exhibited ovipositor that consists of thefemale T9, two pairs of valvifers and three pairs of valvulae. Because of the storage in ethanol and the drying procedure, the 3rd valvulae are coiledand do not embrace the terebra (formed by the interlocked 1st and 2nd valvulae) as in living animals (left is anterior). b Habitus image of V. canescens(lateral aspect). c–e Ovipositor excised from the genital chamber (left is anterior; c, lateral view; d, dorsolateral view; e, ventral view), so that thearticulations of the 1st valvifer and the female T9 (tergo-valvifer articulation) and of the 1st valvifer with the 2nd valvifer (intervalvifer articulation)become visible. The dorsal rami of the 1st valvulae are continuous with the 1st valvifers. The fat arrows represent the direction of view of the otherSEM images. f–g Detailed images of the tergo-valvifer and the intervalvifer articulation (lateral view, left is anterior) and the sensillar patch of the 2ndvalvifer (in g). Abbreviations: 1vf, 1st valvifer; 1vv, 1st valvula; 2vf, 2nd valvifer; 2vv, 2nd valvula; 3vv, 3rd valvula; dr1, Dorsal ramus of the 1st valvula; iar,Interarticular ridge of the 1st valvifer; iva, Intervalvifer articulation; sp, Sensillar patch of the 2nd valvifer; T6, 6th abdimonal tergum; T7, 7th abdominaltergum; T8, 8th abdominal tergum; T9, Female T9; T10, 10th abdominal tergum; tva, Tergo-valvifer articulation

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friction between the valvulae during oviposition [42,45, 46, 48].The 2nd valvula (2vv; Figs. 1a, c, 2a, b, c, d, 4d) is

bulbous at its proximal end and basally articulatedwith the 2nd valvifers via the basal articulation (ba;Fig. 4i; blue region in Fig. 3). There are openings oneach of the dorsolateral sides of the bulbs that presum-ably enable the passage of eggs, venom and other

fluids. The dorsal ramus of the 2nd valvula extendsalong its dorsal margin and bears the processus articu-laris (pra; Fig. 5h) laterally at its proximal part (anter-ior) and the processus musculares (prm; Fig. 5h)dorsally. On its ventral side, the 2nd valvula bears therhachises (rh; Fig. 2b, c, d), which are interlocked withboth the aulaces (au; Fig. 2e, f, g) on the dorsal side ofthe opposing paired 1st valvulae via the olistheter

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Fig. 2 SEM images of Venturia canescens (left is anterior). a, b The apex of the terebra (a, lateral view; b, ventral view; for a transverse section seeFig. 3) showing the notch and the rhachis, which ends at the very apex of the 2nd valvula, and five sawteeth directed apically and decreasing insize apically on each of the 1st valvulae. The valvulae bear various types of sensilla with the campaniform sensilla being numerous at the apicesof both the 1st and the 2nd valvulae. c Upon removal of the 1st valvulae, the rhachises at the ventral side of the 2nd valvula become visible(ventrolateral view). d The rhachises show distally directed scales/serrations. e The inner surface of the apex of the right 1st valvula shows a singlevalvillus and the aulax. f, g The egg canal formed by the 1st and 2nd valvifers bears a microsculpture consisting of distally oriented ctenidia (f),which become further distally replaced by spine-like subctenidial setae (g) at the apex of the terebra. The aulaces of the 1st valvulae, similar tothe rhachis, show distally oriented scales. The fat arrow in a represents the direction of view of the image in b. Abbreviations: 1vv, 1st valvula; 2vv,2nd valvula; au, Aulax; cs, Campaniform sensilla; ct, Ctenidium; no, Notch; rh, Rhachis; sc, Scales; scts, Subctenidial setae; st, Sawtooth; vlv, Valvillus

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system (oth; Fig. 4h2), which extends all the way to theapex. The 2nd valvula of V. canescens and other ich-neumonids (e.g. taxa belonging to the subfamilies ofCampopleginae, Cremastinae, Ctenopelmatinae, Neor-hacodinae and Tryphoninae) consists of two halves thatare joined together for the most of their length by adorsal notal membrane (nm; Fig. 3a; cf. [32, 45]) butare fused at the apex [11], so that the 2nd valvula pos-sesses a lumen that is undivided at the apex of the tere-bra (arrows in red region of Fig. 3b) but that splits intotwo lumina for a substantial proportion of its proximalpart. The blunt tip of the 2nd valvula dorsally possessesa distal notch (no; Fig. 2a, c), which is assumed to beassociated with moderating penetration of the host cu-ticle [42] or to maintain a grip on the inner surface ofthe host cuticle and thereby providing a momentaryclasping mechanism in the host’s skin to ensure con-tinuous engagement with the host during oviposition [43].Almost all ichneumonid species with a pre-apical notchare larval endoparasitoids of holometabolous insects [43].At their external surface, both the 1st and the 2nd valvulaeof V. canescens exhibit canpaniform sensilla (cs; Fig. 2b),

which are concentrated at the apices of the valvulae, espe-cially distally of the distal notch of the 2nd valvula andposteriorly of the sawteeth of the 1st valvulae (cf. [45]).However, the sensillar equipment of the terebra was notfurther investigated in this study (but see [49]).The terebra (trb; Fig. 1b, 3) consists of the paired

1st valvulae and the 2nd valvula, which are tightlyinterlocked by the olistheter (oth; Fig. 4h2). The dis-tally directed scales/serrations on the surfaces of boththe rhachises and the walls of the aulaces (sc; Fig. 2d, f,g) potentially reduce friction forces by minimizing thecontact area of the olistheter elements [46]. However,we hypothesize that these scales might also serve otherfunctions: (1) they, analogous to the ctenidia, mightforward a liquid lubricant from the metasoma to theapex of the olistheter system to reduce friction be-tween the moving valvulae (cf. [48]), and/or (2) theymight create anisotropic conditions in the olistheter byincreasing frictional forces whenever a valvula ispushed in proximal direction, thereby preventing the1st valvulae from randomly sliding back during pier-cing/drilling. The terebra extends far beyond the tip of

Fig. 3 SR-μCT images of the terebra of Venturia canescens. a 3D visualization of the whole terebra in the metasoma. b Virtual cross sectionsthrough the terebra from proximal to distal. Proximal (blue); every 65 μm, a cross section is displayed because of strong morphological changessuch as the bulbous proximal end of the 2nd valvula. According to the limited morphological changes along the longitudinal axis, for the nextpart (green), a cross section is shown only every 260 μm over the next 3380 μm. The most distal 900 μm (red) shows, once again, large morphologicalvariations such as the spindle-shaped cavity formed by all three valvulae; therefore, a cross section is shown every 65 μm. The arrows indicate theundivided distal parts of the 2nd valvula. Abbreviations: 1vv, 1st valvula; 2vf, 2nd valvifer; 2vv, 2nd valvula; 3vv, 3rd valvulae; blb, Bulb; ec, Egg canal; lf1,Longitudinal flap of the 1st valvula; nm, Notal membrane; ssc, Spindle-shaped cavity; trb, Terebra

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the metasoma. The diameter of the terebra decreasesfrom the proximal to its distal end, although the partin between remains similar in diameter throughout.The cross sections of both the 1st and the 2nd valvulaeare notably different across the length of the terebra

(Fig. 3b). The egg canal is largely defined by the 1stvalvulae but its dorsal side is formed by the 2nd val-vula (ec; Fig. 3a). At the apex of the terebra, the 1stvalvulae are enlarged and form an approximatelyspindle-shaped cavity (ssc; red region in Fig. 3) that is

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Fig. 4 Segmented 3D model of the structures involved in the ovipositior movements in Venturia canescens. a, b Cuticular elements and musclesof the ovipositor (a, medial view, left is anterior; b lateral view, left is posterior). c Muscles involved (the cuticular structures are semi-transparent):1st valvifer-genital membrane muscle (grey); anterior 2nd valvifer-2nd valvula muscle (pink); posterior 2nd valvifer-2nd valvula muscle (dark green);dorsal T9-2nd valvifer muscle part a (light green); dorsal T9-2nd valvifer muscle part b (olive); ventral T9-2nd valvifer muscle (blue); posterior T9-2ndvalvifer muscle (cyan). d Selected cuticular elements involved (the ovipositor muscles are semi-transparent): 1st valvifer (orange); 2nd valvifer (yellow);1st valvulae (pink); 2nd valvula (purple). The 3rd valvulae are not shown here. e–j Joints involved with their degrees of freedom depicted as dashedarrows. e Cuticular elements of the ovipositor and their inherent structures. f Enlarged view of rotation joints between the 1st valvifer and the 2ndvalvifer (intervalvifer articulation) and between the 1st valvifer and female T9 (tergo-valvifer articulation). g Joints with assumed rotation and translationdegree of freedom between the 2nd valvifer and the female T9 (assumed movements indicated by white dashed arrows, assumed rotation angle bywhite dashed lines). h Translational joints with tongue and groove connection between the dorsal rami of the 1st valvula and the dorsal projection ofthe 2nd valvifer (h1; image of the SR-μCT data stack; location of the virtual cross section is indicated in e by small number 1), and between the 1st andthe 2nd valvulae via the olistheter system: the tonge-like rhachises on the ventral surface of the 2nd valvula and the corresponding grooves calledaulaces along the dorsal surface of each on each of the 1st valvulae (h2; image of the SR-μCT data stack; location of the virtual cross section isindicated in e by small number 2). i Rotational joint between the 2nd valvifer and the 2nd valvula called the basal articulation (the valvifersand the female T9 are semi-transparent). j Joints and movements enabled by the 1st valvifer, which acts as a lever. Abbreviations: 1vf, 1st valvifer; 1vv, 1stvalvula; 2vf, 2nd valvifer; 2vv, 2nd valvula; af9, Anterior flange of T9; asdf, Anterior section of the dorsal flange of the 2nd valvifer; ba, Basal articulation;bl, Basal line; blb, Bulb; ca, Cordate apodeme; dp2, Dorsal projection of the 2nd valvifer; dr1, Dorsal ramus of the 1st valvula; ec, Egg canal; hsl, Hook-shaped lobe of the 2nd valvifer; iar, Interarticular ridge of the 1st valvifer; iva, Intervalvifer articulation; m1, 1st valvifer-genital membrane muscle; m2,Anterior 2nd valvifer-2nd valvula muscle; m3, Posterior 2nd valvifer-2nd valvula muscle; m4a, Dorsal T9-2nd valvifer muscle part a; m4b, Dorsal T9-2ndvalvifer muscle part b; m5, Ventral T9-2nd valvifer muscle; m6, Posterior T9-2nd valvifer muscle; mb2, Median bridge of the 2nd valvifers; oth, Olistheter;psdf, Posterior section of the dorsal flange of the 2nd valvifer; T9, Female T9; tm4b, Tendon of the dorsal T9-2nd valvifer muscle part b; tva,Tergo-valvifer articulation

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partly occluded by the valvilli of each of the 1st valvu-lae (cf. [32]).The paired 3rd valvulae (3vv; Figs. 1a, c, e, 3) emerge

at the posterior end of the 2nd valvifer and ensheathand protect the terebra when at rest. The lateral wallsof the 3rd valvulae of V. canescens and other parasitoidwasps with long external terebrae are annulated by finetransversal narrow furrows (cf. [50]), which makes themflexible and allow their extensive deformation duringoviposition. Since the valvulae lack intrinsic muscles,deformation must arise as a passive response to exter-nal pressures. The ability to bend the 3rd valvulae facil-itates oviposition [50], however, it is not yet clear if V.canescens is able to support the flexion of the terebratowards an active probing position and its steering dur-ing the search for a potential host with their 3rd valvu-lae or if they simply follow the movements of theterebra (Fig. 5n; Additional file 1; cf. [32]). The distallydirected dense microsetae on the inner surface of the3rd valvulae (cf. [45]) are thought to be involved incleaning the ovipositor sensilla between oviposition

episodes [2, 12, 50]. The 3rd valvulae potentially alsohave a sensory function [1].The paired 1st valvifers (1vf; Figs. 1a, c, d, f, g, 4b, d, j) of

V. canescens and other ichneumonid species are shortand show an almost oblong shape (with roundededges) [8], unlike the bow-shaped 1st valvifers of spe-cies of Chalcidoidea [21, 23–26] or the triangularlyshaped 1st valvifers of species of Apoidea [8, 9, 51, 52].The posterior angles of the 1st valvifer are doublymovably articulated with the modified female T9 viathe tergo-valvifer articulation and via its posteroventralcorner with the 2nd valfiver by means of the intervalvi-fer articulation (tva/iva; Figs. 1c, f, g, 4f, j). A strength-ened ridge called the interarticular ridge (iar; Figs. 1f,4f ) occurs between the two articulations and mightmechanically stabilize the 1st valvifer during ovipos-ition. The anterodorsal angle of the 1st valvifer is con-tinuous with the dorsal ramus of the 1st valvula (dr1;Figs. 1c, d, f, 4h1, i, j), which is interlocked with thedorsal projection of the 2nd valvifer (dp2; Fig. 4e, h1)by a system analogous to the olistheter. This tight

(See figure on previous page.)Fig. 5 Mechanics of the musculoskeletal ovipositor system of Ventuia canescens. a–g, i Kinematics of the musculoskeletal ovipositor system; acting(input) muscle forces are visualized by solid red arrows (b, d, f, g, i) and resulting (output) movements by solid black arrows (c, e, g, i). a–g, j–m 3Dmodel of the ovipositor system (medial view, left is anterior). b m1 potentially serves as a tensor muscle for stabilization of the ovipositor systemduring oviposition. c, d, i Contraction of both m4a and m4b (F4 in d, i) moves the 2nd valvifer posteriorly and the female T9 anteriorly towards eachother (small number 3 in c, i), thus indirectly causing the 1st valvifer to tilt anteriorly (small number 4 in c, i). This is possible because the 1st valvifer isarticulated with both the 2nd valvifer and the female T9 via the intervalvifer and tergo-valvifer articulations that act as rotational joints. The 1st valviferthereby functions as a lever arm that transfers the movement to the dorsal ramus of the 1st valvula and consequently causes the 1st valvula to slidedistally relative to the 2nd valvula (small number 5 in c). These movements might also facilitate the extension of the terebra back towards its restingposition (c). m6 thereby stabilizes the ovipositor system by holding the 2nd valvifer and the female T9 in position and preventing them to rotatearound the articulations (d). e, f, i Contraction of m5 (F5 in f, i) moves the 2nd valvifer anteriorly and the female T9 posteriorly apart from each other(small number 6 in e, i), thus causing the 1st valvifer to tilt posteriorly (small number 7 in e,i) and consequently causing the 1st valvula to slideproximally relative to the 2nd valvula (small number 8 in e). These movements might also facilitate the flexion of the terebra (e). g, i Contraction of m3(F3 in g, i) causes the bulbs to pivot anteriorly at the basal articulation, thus flexing the 2nd valvula and, therefore, the whole terebra (small number 2in g, i). Contraction of m2 (F2 in g, i) extends the terebra back towards its resting position (small number 1 in g, i). h Light microscopical image of theinsertion regions of m2 and m3 at the processus articularis and the processus musculares, respectively (lateral view, left is anterior). The duct of thevenom gland reservoir of the 2nd valvifer ends at the lateral openings of the bulbous region of the 2nd valvula. i Resulting schematic drawing of themechanism of the tilting movements of the 1st valvifer and of the flexion/extension of the terebra (lateral view, left is anterior, not to scale). Only thetwo pairs of antagonistically acting muscles that are mainly responsible for these movements are represented in simplified terms (m2/m3 and m4/m5).The muscles stabilizing the system (m1 and m6) are not depicted here. j–m Simplified mechanical scheme of the leverages of the ovipositor in theresting position; acting (input) muscle forces are visualized by solid red arrows, their horizontal force vector components and the resulting (output)forces by thin red arrows (j, k), the anatomical (in)levers by solid black lines and the effective (= mechanical) levers by thin black lines, and the jointangles (α, β, ε) are given (k, m). j, l Major direction of the acting muscle forces (F2, F3, F4 and F5) from a muscle’s insertion point to the centre point ofits origin. j, k Under the simplified assumption that the 2nd valvifer, which acts as the frame of reference, and the female T9 are guided and cannottwist but only move towards or apart from each other along the horizontal anterior–posterior axis, the input force vectors F4x and F5x act horizontallyat the 1st valvifer at the tergo-valvifer-articulation. The distance between the tergo-valvifer articulation (where the force is applied) and the intervalviferarticulation (joint axis/pivot point) is the anatomical inlever c; for torques see eqs. 4, 5. The 1st valvifer acts as a lever with the effective outlever d’,resulting in pro- or retraction forces at the dorsal ramus of the 1st valvula F1vv4 and F1vv5; see eqs. 6, 7. l, m Input force vectors F2 and F3 acting at theproximal end of the 2nd valvula with the basal articulation as joint axis and the anatomical inlevers a and b; for torques see eqs. 2, 3. n Schema of afemale wasp flexing its terebra to an active position for oviposition (after [32]) (Additional file 1), which might be supported by the flexible 3rd valvulae(not shown in a–m). Abbreviations: 1vf, 1st valvifer; 1vv, 1st valvula; 2vf, 2nd valvifer; 2vv, 2nd valvula; 3vv, 3rd valvua; ba, Basal articulation; blb, Bulb;dr1, Dorsal ramus of the 1st valvifer; F, Force; Fx, Horizontal vector components of a force; iva, Intervalvifer articulation; m1, 1st valvifer-genitalmembrane muscle; m2, Anterior 2nd valvifer-2nd valvula muscle; m3, Posterior 2nd valvifer-2nd valvula muscle; m4a, Dorsal T9-2nd valvifermuscle part a; m4b, Dorsal T9-2nd valvifer muscle part b; m5, Ventral T9-2nd valvifer muscle; m6, Posterior T9-2nd valvifer muscle; pra,Processus articularis; prm, Processus musculares; T9, Female T9; trb, Terebra; tva, Tergo-valvifer articulation; vd, Duct of the venom glandreservoir of the 2nd valfiver

Eggs et al. BMC Zoology (2018) 3:12 Page 8 of 25

interlocking guides the dorsal rami and prevents themfrom buckling when pushing forces are applied duringthe protraction of the 1st valvulae. The rami makeacute angles around the proximal bulbous end of the2nd valvula. The cuticle in the part of the dorsal ramithat slides around the angle during pro- or retractionof the 1st valvulae needs to be flexible in the sagittalplane and might contain high proportions of the veryelastic rubber-like protein resilin (cf. [53–55]).The paired 2nd valvifers (2vf; Fig. 1a, c, e, f, g, 4b, d)

are elongated and their posterior parts are placed medi-ally of the female T9. A conjunctiva, called the genitalmembrane (not shown), connects the ventral marginsof both the 2nd valvifers arching above the 2nd valvula.The 2nd valvifer bears the dorsal flange, which extendsupon its dorsal margin and which is divided by asharply defined ridge called the basal line (bl; Fig. 4e)into an anterior and a posterior section. The anteriorsection of the dorsal flange of the 2nd valvifer (asdf;Fig. 4e) dorsally bears the dorsal projection of the 2ndvalvifer (dp2; Fig. 4e, h1) and extends upwards in ahook-shaped lobe (hsl; Fig. 4e; sensu [8]) at its postero-dorsal end, which might allow a greater arc of move-ment of the 1st valvifer and therefore a greaterprotraction of the 1st valvulae. The dorsal margins andthe dorsal flanges are strengthened by cuticular ridgesthat might have a stabilizing function to prevent de-formation. Sensillar patches (sp; Fig. 1g) can be seen onthe 2nd valvifer near the intervalvifer and the basal ar-ticulation (cf. [56]), monitoring the movements of the1st vlavifer and therefore the connected 1st valvula orthe position of the bulbs of the 2nd valvula. The poster-ior section of the dorsal flange of the 2nd valvifer (psdf;Fig. 4e) is elongated and oriented almost vertically. Attheir posterodorsal ends, the 2nd valvifers are con-nected by the median bridge (mb2; Fig. 4e). The duct ofthe venom gland reservoir (vd; Fig. 5h) is situated inbetween the paired 2nd valvifers.The female T9 (T9; Figs. 1a, c, e, f, g, 4b, d) is

elongated and anterodorsally bears a hook-shapedstructure. Medially at its anterior end, the T9 formsa funnel-like structure at the cordate apodeme (ca;Fig. 4e, f, g), situated posteriorly to the tergo-valviferarticulation. This structure has not yet been de-scribed in parasitoid hymenopterans. The anterodor-sal and dorsal margins of the female T9 isstrengthened by the anterior flange of T9 (af9; Fig. 4e) thatmight mechanically stabilize the female T9 duringoviposition.

Joints of the musculoskeletal ovipositor systemThe musculoskeletal ovipositor system possesses threemain joints.

The basal articulation (ba; Fig. 4i) connects the lat-erally placed bulbs of the 2nd valvula with the thickenedanteroventral parts of the 2nd valvifers via a rotationaljoint. This joint might also allow some limited pivotingmovements of the 2nd valvula and therefore of thewhole terebra.Both the 2nd valvifer and the female T9 are con-

nected with the 1st valvifer by the intervalvifer articu-lation and the tergo-valvifer articulation (iva/tva;Figs. 1c, f, g, 4f, j), respectively, forming a double joint.The tergo-valvifer articulation is situated dorsal to theintervalvifer articulation. Both of these articulations actas rotational joints; thus, the 1st valvifer is movable inthe sagittal plane only.

Ovipositor musclesThe maximum tensions at constant muscle length (iso-metric tension) that individual insect muscles can exertgreatly vary between species, ranging from 19 to700 kPa [57, 58] (e.g. approximately 38 kPa exerted bythe asynchronous dorso–ventral flight muscle in Bombusterrestris (Linnaeus, 1758) at 30 °C [59]). In case of par-allel muscle fibres, the maximum force (F) created by amuscle can be estimated by using the specific tension (f )and the mean cross section area (CSA; Table 1) accord-ing to the equation:F = CSA · f (eq. 1)However, there are, to the best of our knowledge,

no studies hitherto that measured tensions of abdom-inal muscles of hymenopterans we could refer to.The ovipositor of V. canescens possesses a set of six

paired muscles (Fig. 4c; Table 1), one of them (m4)forming two distinct bundles.The paired 1st valvifer-genital membrane muscles

(m1) are the only muscles of the 1st valvifer. Theyoriginate at the medial surface of the posteroventralpart of the 1st valvifer, i.e. between the tergo-valviferand the intervalvifer articulation, and insert anteriorlyon the genital membrane. They are the smallest mus-cles of the ovipositor with a CSA of 0.0008 mm2 each(Table 1).The paired fan-shaped anterior 2nd valvifer-2nd

valvula muscles (m2) arise at the medial region alongthe anterodorsal part of the 2nd valvifer, largely at theanterior section of the dorsal flange (asdf; Fig. 4e), andinsert at the processus articularis (pra; Fig. 5h), aprocess that extends laterally from the proximal partof the 2nd valvula to form the medial part of thebasal articulation. These muscles have a CSA of0.0032 mm2 each (Table 1).The paired posterior 2nd valvifer-2nd valvula

muscles (m3) originate at the medial region along theventral part of the 2nd valvifer and insert at the

Eggs et al. BMC Zoology (2018) 3:12 Page 9 of 25

Table

1Ovipo

sitormuscles

ofVenturia

canescens.Themuscles

(abb

reviations

inbrackets),theirorigin,insertio

nandassumed

functio

narede

scrib

ed.Inadditio

n,themeasured

volume,meanleng

thandthemeancrosssectionarea

(CSA

)of

thesing

lemuscles

arelisted

musclename(labe

l)origin

insertion

assumed

functio

nvolume

[mm

3 ]mean

leng

th[m

m]

meancross

sectionarea

(CSA

)[mm

2 ]

1stvalvifer-g

enitalm

embranemuscle(m

1)medialsurface

ofthepo

steroventralpart

ofthe1stvalvifer,inthecentre

between

thetergo-valviferand

theintervalvifer

articulation

anteriorly

onthegenitalm

embrane

tensor

muscleforstabilizatio

nof

the1st

valvifersdu

ringoviposito

rmovem

ents

0.0001

0.175

0.0008

anterior2nd

valvifer-2nd

valvulamuscle

(m2)

medialregionalon

gtheanterodo

rsalpart

ofthe2ndvalvifer

attheprocessusarticularis

extensor

oftheterebra(tow

ards

the

restingpo

sitio

n)0.0015

0.455

0.0032

posterior2

ndvalvifer-2ndvalvulamuscle

(m3)

med

ialreg

ionalon

gtheventralp

artof

the2n

dvalvifer

attheprocessusmusculares

flexorof

theterebra(tow

ards

theactive

prob

ingpo

sitio

n):causesthebu

lbto

pivotanterio

rlyat

thebasalarticulation

0.0029

0.760

0.0039

dorsalT9-2nd

valviferm

uscleparta(m

4a)

lateralreg

ionalon

gthepo

sterod

orsal

partof

theanterio

rmarginof

femaleT9

anteriorsectionof

thedo

rsalflang

eof

the2ndvalvifer,partlyatthe

dorsalho

ok-shapedlobe

protractor

ofthe1stvalvulae:m

oves

the2n

dvalviferpo

steriorly

andthe

femaleT9

anterio

rlytowards

each

othe

r,causingthe1stvalviferto

tiltanterio

rlyandthus

the1stvalvulato

slidedistally

relativeto

the2n

dvalvula

0.0047

0.950

0.0050

dorsalT9-2nd

valviferm

usclepartb(m

4b)

med

ialreg

ionalon

gthepo

sterod

orsal

partof

theanterio

rmarginof

femaleT9

anteriorsectionof

thedo

rsalflang

eof

the2ndvalviferviatend

on,

ventrally

tom4a

0.0029

0.740

0.0039

ventralT9-2n

dvalvifermuscle(m

5)medialregionof

theanterodo

rsalpartof

femaleT9,partly

onthecordateapod

eme

alon

gthepo

steriorsectionof

the

dorsalflang

eof

the2n

dvalvifer

retractorof

the1stvalvulae:m

oves

the

2ndvalviferanterio

rlyandthefemale

T9po

steriorly

apartfro

meach

othe

r,causingthe1stvalviferto

tiltpo

steriorly

andthus

the1stvalvulato

slide

proxim

allyrelativeto

the2n

dvalvula

0.0062

0.805

0.0077

posteriorT9-2nd

valvifermuscle(m

6)med

ially

from

thepo

sterod

orsalp

artof

femaleT9

med

ianbridge

ofthe2n

dvalvifers

muscleforstabilizatio

nby

holdingthe

posteriorpartsof

the2n

dvalvifersin

positio

ndu

ringoviposito

rmovem

ents

0.0004

0.280

0.0015

Allmeasuremen

tswerede

term

ined

directly

from

the3D

musclemasks

oftheSR

-μCTda

taset.Th

esevalues

potentially

arelower

than

inlivingan

imalsdu

eto

shrin

king

artefacts.Th

etotalm

uscleleng

thwas

determ

ined

asthedistan

cebe

tweenthecentre

points

ofthemuscleattachmen

ts.C

SAwas

determ

ined

asmusclevo

lume/muscleleng

th

Eggs et al. BMC Zoology (2018) 3:12 Page 10 of 25

processus musculares (prm; Fig. 5h), namely the apo-deme that extends dorsally from the proximal part ofthe 2nd valvula to the genital membrane. These mus-cles have a CSA of 0.0039 mm2, which is similar tothat of m2 (Table 1).The paired dorsal T9-2nd valvifer muscles (m4a/

b) are modified in their insertion and form two dis-tinct muscle bundles, as it is also known to occur inthe ichneumonid genus Megarhyssa Ashmead, 1858[8, 60]. One part of these muscles (m4a) arises at thelateral region along the posterodorsal part of the an-terior margin of female T9 and inserts at the anteriorsection of the dorsal flange of the 2nd valvifer (asdf;Fig. 4e) and partly on the hook-shaped lobe of the2nd valvifer (hsl; Fig. 4e). The other part (m4b) isfan-shaped and originates at the medial region alongthe posterodorsal part of the anterior margin of fe-male T9. The muscle tendons (tm4b; Fig. 4f, g) alsoinsert at the anterior section of the dorsal flange ofthe 2nd valvifer, ventrally to the insertion region ofm4a. The tendon of m4b thereby traverses thefunnel-like structure at the cordate apodeme (ca; Fig.4f, g) of the female T9. Muscles m4a and m4b arelong thick muscles with a CSA of 0.0050 mm2 and0.0039 mm2, respectively (Table 1).The paired ventral T9-2nd valvifer muscles (m5)

arise from the medial region of the anterodorsal part ofthe female T9, partly at the funnel-like structure at thecordate apodeme (ca; Fig. 4f, g), and insert along theposterior section of the dorsal flange of the 2nd valvifer(psdf; Fig. 4e). These are the largest ovipositor muscleswith a CSA of 0.0077 mm2.The paired posterior T9-2nd valvifer muscles (m6)

arise medially at the posterodorsal part of the femaleT9 and insert at the median bridge of the 2ndvalvifers (mb2; Fig. 4e). They are the second smallestmuscles of the ovipositor with a CSA of 0.0015 mm2

(Table 1).The literature concerning the musculoskeletal ovi-

positor system of ichneumonoid wasps is limited andsome inconsistent statements have been made aboutcertain ovipositor muscles. We describe the 1stvalvifer-genital membrane muscle for the first time inan ichneumonoid species. Either this small muscle isnot present in all ichneumonoid species or, morelikely, previous authors (e.g. [8, 60]) might have over-looked its presence. In Megarhyssa macrurus lunator(Fabricius, 1781) (Hymenoptera: Ichneumonidae), Ab-bott [60] described the 1st valvifer-2nd valvifer muscleas ‘a small muscle connecting the “runner” plate [=2nd valvifer] with the dorsal margin of the “kidney”plate [= 1st valvifer]’. However, this muscle has nei-ther been found in Megarhyssa atrata (Fabricius,1781) (Hymenoptera: Ichneumonidae) by Snodgrass

[8] nor in V. canescens in the present study andmight have been mistaken for the anterior 2ndvalvifer-2nd valvula (m2) muscle by this author.In general, the musculoskeletal ovipositor system of

ichneumonoid wasps is similar to that of the parasit-oid hymenopteran species belonging to Ceraphronoi-dea [19], a superfamily that is closely related toIchneumonoidea [61]. However, the ceraphronoidslack the anterior 2nd valvifer-2nd valvula muscle [19]that is present in V. canescens and other ichneumo-nids. All chalcidoid species investigated to date withregard to the ovipositor muscles (Agaonidae [26],Aphelinidae [27], Chalcididae [20], Eurytomidae [23],Pteromalidae [21, 25] and Torymidae [24]) comprisethe same set of muscles as ichneumonids but lack the1st valvifer-genital membrane muscle. All the taxa ofChalcidoidea, Ceraphronoidea and Ichneumonoideainvestigated hitherto (including our study of V. canes-cens) lack the 1st valvifer-2nd valvifer muscle, lateralT9-2nd valvifer muscle, 2nd valvifer-genital membranemuscle and T9-genital membrane muscle, which havebeen described in other hymenopteran taxa [7].

Mechanics and mode of function of the musculoskeletalovipositor systemThe set of six paired ovipositor muscles in V. canes-cens (Fig. 4c; Table 1) comprises two pairs of two an-tagonistically working muscles that are mainlyresponsible for the various ovipositor movements, andtwo muscles stabilizing the musculoskeletal system.Based on the following functional model, we assumethat the anterior (m2) and the antagonistically actingposterior 2nd valvifer-2nd valvula muscles (m3) ex-tend or flex the terebra, whereas the two parts of thedorsal T9-2nd valvifer (m4a/b) and the antagonistic-ally acting ventral T9-2nd valvifer muscle (m5) indir-ectly protract or retract the 1st valvulae. Therelatively small 1st valvifer-genital membrane muscle(m1) and the posterior T9-2nd valvifer muscle (m6)might predominantly serve for the stabilization of theovipositor system during oviposition.

Flexion and extension of the terebraThe 2nd valvula of V. canescens is connected withthe 2nd valvifers by a rotational joint called the basalarticulation (ba; Figs. 4i, 5h, i, l, m). Two antagonis-tic muscles (m2, m3) insert at the bulbous regionaround this articulation (Fig. 5h). The insertion re-gion of the posterior 2nd valvifer-2nd valvula muscle(m3) at the 2nd valvula is located dorsally of thebasal articulation, whereas its region of origin at the2nd valvifer is located posteroventrally to it. There-fore, a contraction of m3 (F3; Fig. 5g, i) causes the

Eggs et al. BMC Zoology (2018) 3:12 Page 11 of 25

bulbs (blb; Fig. 4e, i) to pivot anteriorly at the basalarticulation. This leads to a flexion of the 2nd valvulaand the interlocked 1st valvulae from its resting pos-ition between the paired 3rd valvulae towards an ac-tive probing position (small number 2; Fig. 5g, i;Table 1). An alternate contraction of m3 on eitherside might also cause the terebra to rotate to a cer-tain degree. The insertion region of the anterior 2ndvalvifer-2nd valvula muscle (m2) at the 2nd valvula issituated posteroventrally of both the basal articulationand the insertion region of m3, whereas its origin atthe 2nd valvifer is located posterodorsally of the ar-ticulation. Hence, when m2 (F2; Fig. 5g, i) contracts,the terebra is extended towards its resting position(small number 1; Fig. 5g, i; Table 1).The anatomical cluster comprising the 2nd valvifer,

the 2nd valvula and the two muscles connecting them(Fig. 5l) is a simple mechanical system in which the2nd valvula is a two-armed class 1 lever. The ratio ofthe anatomical inlevers (a = 66 μm and b = 84 μm; Fig.5m) is 1:1.27. The torques (M) of the muscle forces ofthe anterior and posterior 2nd valvifer-2nd valvulamuscle (F2 and F3) on the basal articulation in the rest-ing position can be estimated by using the maximumforce of the muscle (F; cf. eq. 1), the lengths of theanatomical inlever arms and the attachment angles ofthe muscles at the 2nd valvula (α = 154° and β = 96°;Fig. 5m) according to the equations:M2 = F2 · a · sin(α) (eq. 2)M3 = F3 · b · sin(β) (eq. 3)However, the lengths of the effective (= mechanical)

inlever arms (a’ and b’; Fig. 5m) vary greatly with at-tachment angle (joint angle), i.e. during the flexion orextension of the terebra. The attachment angle of m3in the resting position is almost 90°; thus, the effect-ive inlever arm is almost optimal, so that the force ofm3 can be optimally transmitted to the 2nd valvula,which leads to a high torque. By contrast, the attach-ment angle of m2 in the resting position is far below90° but increases when the wasp flexes its terebra to-wards the active probing position. This results in anincrease in length of the effective inlever arm, an op-timal force transmission of m2 at the basal articula-tion and consequently a high torque. High torques atthe basal articulation might be crucial to enable theextensive movements for both the flexion and exten-sion of the terebra, despite the relatively small ana-tomical inlevers.

Pro- and retraction of the 1st valvulaeThree muscles (m4–m6) connect the 2nd valviferwith the female T9, both these structures being con-nected with the 1st valvifer by the intervalvifer

articulation or the tergo-valvifer articulation (iva/tva;1c, f, g, 4f, j, 5i–k), forming a double joint. The inser-tion regions at the 2nd valvifer of both parts of thedorsal T9-2nd valvifer muscle (m4a/b) lie anterodor-sally, whereas the regions of origin at the female T9are posterodorsally located of both articulations. Acontraction of m4a and m4b (F4; Fig. 5d, i) movesthe 2nd valvifer posteriorly and the female T9 anteri-orly towards each other (small number 3; Fig. 5c, i),whereby the tension of the posterior T9-2nd valvifermuscle (m6) presumably prevents the involved cuticu-lar elements to rotate around the articulations. Thismovement causes the 1st valvifer to tilt anteriorly(small number 4; Fig. 5c, i) because it is articulatedwith both the 2nd valvifer and the female T9 via rota-tional joints (intervalvifer and tergo-valvifer articula-tion). The 1st valvifer acts as a one-armed class 3lever that transfers its tilting movement to the dorsalramus of the 1st valvula, causing the 1st valvula toslide distally relative to the 2nd valvula (small number5; Fig. 5c). Both m4a and m4b act as protractors ofthe 1st valvulae (Table 1). They might also assist inextending the terebra (Fig. 5c), as a simultaneous pro-traction of the 1st valvulae places the terebra underunilateral tension due to friction between the olisth-eter elements of the 1st and 2nd valvulae. The originof the antagonistic ventral T9-2nd valvifer muscle(m5) at the female T9 is situated posterodorsally nearthe intervalvifer articulation and posterior to thetergo-valvifer articulation, whereas its insertion regionat the 2nd valvifer is located posteroventrally of boththese articulations. Its contraction (F5; Fig. 5f, i)moves the 2nd valvifer anteriorly with respect to thefemale T9 (small number 6; Fig. 5e, i), thus indirectlycausing the 1st valvifer to tilt posteriorly (small num-ber 7; Fig. 5e, i) and the 1st valvulae, as a direct con-sequence, to slide proximally relative to the 2ndvalvula (small number 8; Fig. 5e). Therefore, m5 actsas a retractor of the 1st valvulae (Table 1). It mightalso assist in flexing the terebra (Fig. 5e), as a simul-taneous retraction of both of the 1st valvulae placesthe terebra under a unilateral tension due to frictionbetween the olistheter elements of the 1st and 2ndvalvulae. Muscles m4a and m4b act antagonisticallyagainst m5, i.e. m4a/b protract the 1st valvulae,whereas m5 retracts them. The posterior T9-2nd val-vifer muscle (m6) stabilizes the ovipositor system byholding the 2nd valvifer and the female T9 in positionand prevents them to rotate around the articulations(Fig. 5d; Table 1), although some limited movementsin dorso–ventral direction at their posterior ends arelikely to occur (cf. Fig. 4g).The following assumptions were made for a simpli-

fied estimation of the torques (M) of the muscle

Eggs et al. BMC Zoology (2018) 3:12 Page 12 of 25

forces of the dorsal and ventral T9-2nd valvifermuscle (F4 and F5): (1) The 2nd valvifer acts as theframe of reference; therefore, the intervalvifer articu-lation (iva; Figs. 1c, f, g, 4f, j, 5i, j, k) acts as thepivot point (= joint axis or fulcrum) at which the 1stvalvifer tilts; and (2) the 2nd valvifer and the femaleT9 are guided and cannot twist around the articula-tions but only move towards to or apart from eachother along the horizontal anterior–posterior axiswithout friction occurring. Under these assumptions,the horizontal force vector components of m4 andm5 (F4× = cos(γ) · F4 and F5× = cos(δ) · F5 with γ = 5°and δ = 24°; Fig. 5j, k) act at the 1st valvifer at thetergo-valvifer articulation (tva; Figs. 1c, f, 4f, j, 5i, j,k). Therefore, the torque (M) of F4× and F5× on theintervalvifer articulation in the resting position can beestimated by using the horizontal vector component(F×) of the maximum force of a muscle (cf. eq. 1),the length of the anatomical inlever arm (c = 103 μm;Fig. 5k)—which is the distance between tergo-valviferand intervalvifer articulation—and the joint angle (ε =113°; Fig. 5k) according to the equations:M4 = F4× · c · sin(ε) (eq. 4)M5 = F5× · c · sin(ε) (eq. 5)The 1st valvifer acts as a lever with the effective out-

lever (d’; Fig. 5k), which is defined as the length be-tween the intervalvifer articulation and the pointwhere the 1st valvifer continues as dorsal ramus of the1st valvula. The resulting pro- or retracting forces atthe dorsal ramus of the 1st valvula (Fvvm4 and Fvvm5;Fig. 5k) can be estimated by using the horizontal vec-tor components (F×) of the forces acting on the 1stvalvifer at the tergo-valvifer articulation, the length ofthe effective inlever arm (c’ = c · sin(ε) = 94.8 μm; Fig.5k) and the effective outlever arm according to theequations:F1vv4 = (F4× · c’) / d’ (eq. 6)F1vv5 = (F5× · c’) / d’ (eq. 7)The distance that the 1st valvifer moves is equally

transferred to the 1st valvula. Thereby, the shape ofthe 1st valvifer and the positions of the tergo-valviferand the intervalvifer articulations influence the wayhow the 1st valvula is moved, i.e. the more closelythe two articulations are situated to each other andthe further they are away from the anterior angle ofthe 1st valvifer, the further the 1st valvula will sliderelative to the 2nd valvula along the olistheter [19].An increase of the quotient of the effective outleverto the effective inlever (d’: c’ ratio) results in asmaller force output but an increase in the potentialmaximum velocity and mechanical deflection, i.e. anincrease in the speed and the movement distance ofthe dorsal rami of the 1st valvulae. Their tight inter-locking with the dorsal projection of the 2nd valvifer

prevents them from buckling and transfers the move-ments to the apex of the valvulae. The double jointsystem of the 1st valvifer enables an pro- and retrac-tion of the 1st valvulae.The 1st valvifer-genital membrane muscle (m1) po-

tentially serves as a tensor muscle that stabilizes the1st valvifers during their fast alternate movements byholding them in position laterally to the 2nd valvifers(Fig. 5a, b; Table 1).

Process of ovipositionAfter a female wasp has found a suitable ovipositionsite, the contraction of the posterior 2nd valvifer-2ndvalvula muscles (m3) causes the 2nd valvula and theinterlocked 1st valvulae to flex anteriorly towards theactive probing position [19]. This flexing and the gen-eral employment of the terebra of V. canescens (as inmany other ichneumonoid wasp taxa [62, 63]) mightbe assisted by the annulated and flexible 3rd valvulaeand the generally improved manoeuvrability of themetasoma of the Apocrita [64]. The 2nd valvifer isthen rotated away from the dorsal surface of themetasoma concomitantly with the terebra. During theso-called cocking behaviour (sensu [32]) of V. canes-cens, the 2nd valvifer and the terebra flex simultan-eously. In V. canescens, this characteristic behaviouris always performed prior to the actual ovipositionand is assumed to correlate with the egg being passeddown into the spindle-shaped cavity at the apex ofthe terebra in readiness for oviposition [32, 45]. Theparasitoid then performs localized probing movementswith the unsheathed terebra in the substrate (Add-itional file 1). Drilling movements of the terebra arenot needed, since the hosts of V. canescens live insoft substrates. Once a suitable host is found, stab-bing movements are conducted, whereby the terebrais quickly inserted into the host caterpillar [32, 65].Thereby, alternate contractions of the dorsal T9-2ndvalvifer muscles (m4a/b) and the ventral T9-2nd val-vifer muscles (m5) indirectly execute the penetrationmovements of the 1st valvulae (which are docu-mented in a braconid wasp [66]). In some species ofBraconidae (the sister group of Ichneumonidae), thesemovements of the 1st valvulae are known to enablethe wasps to actively steer their terebra to some ex-tent: asymmetrical apex forces at the terebra in a vis-cid medium—caused by varying its asymmetrical tipby pro- or retracting one 1st valvula with respect tothe other—result in a passive bending of the terebra[66], or restrictions in inter-element displacements(e.g. strongly swollen short regions pre-apically on therhachises) cause the terebra to bend due to tensileand compressive forces [67]. Throughout penetration,

Eggs et al. BMC Zoology (2018) 3:12 Page 13 of 25

the relative position of the valvifers and consequentlyof the 1st valvulae might be monitored via the sensil-lar patches of the 2nd valvifers situated anteriorly tothe intervalvifer articulations. In addition to penetrat-ing the substrate, the longitudinal alternate move-ments of the 1st valvulae presumably serve to passthe egg along the terebra. This is facilitated by theegg canal microsculpture consisting of distally ori-ented scales (ctenidia and subctenidial setae) thatpush the egg towards the apex of the terebra andhold it in position by preventing backward move-ments [43, 46, 47]. Shah [45] suggests that the valvilliassist in moving the egg in the terminal part of theterebra by using hydrostatic pressure for a speedy de-livery of the egg into the host. In V. canescens, thelaying of an egg into the haemocoel of the host cater-pillar takes only a fraction of a second [32, 45]. Afteroviposition and withdrawal of the terebra, the anterior2nd valvifer-2nd valvula muscles (m2) extend theterebra back towards its resting position between theinternal concave faces of the 3rd valvulae [10]. Ovi-position is commonly followed by cleaning behaviourduring which the wasp especially grooms its antennaeand terebra.

ConclusionsThe examination of the elements of the musculoskel-etal ovipositor system of V. canescens and its under-lying working mechanisms adds to our understandingof a key feature in the evolution of parasitoid hyme-nopterans, a feature that has impacted the evolutionarysuccess of ichneumonid wasps (with more than 24,000described [68] and more than 100,000 estimated spe-cies [69]) and parasitoid hymenopterans in general(with 115,000 described and 680,000 estimated species[70]). Whereas the basic organization of the ovipositoris remarkably uniform among the Hymenoptera [8],huge variations exist in its structure [9, 11, 12], whichare associated with the employment of the terebra inthe different taxa of parasitoid species (cf. [62, 63, 71,72]). Further studies that combine thorough morpho-logical analyses of a parasitoid’s musculoskeletalovipositor system with investigations of its parasitoid–host interactions are needed in order to understandhow morpho-physiological traits have influenced theevolution of behavioural, ecological and life historytraits and vice versa in the megadiverse parasitoidHymenoptera.

MethodsThe V. canescens specimens used in this study origi-nated from the thelytokous lab colony of BiologischeBeratung Ltd. (Berlin, Germany) from whom we also

received larvae of the host Ephestia kuehniella Zeller,1879 (Lepidoptera: Pyralidae). The wasps were kept ina glass box (20 · 30 · 20 cm) and reproduced after theaddition of several pyralid larvae within a mealy sub-strate to the box every third week (Additional file 1).Three times a week, the imagos were fed with wateredhoney absorbed onto paper towels. The room was keptat a constant temperature of 24°C.

Light microscopy (LM) and scanning electron microscopy(SEM)The ovipositor was excised and dissected from the geni-tal chamber of ethanol-fixed animals by using fine for-ceps, macerated in 10% aqueous potassium hydroxide(KOH) for 12–15 h at room temperature if necessary,cleaned in distilled water and dehydrated stepwise inethanol (C2H6O).For light microscopy, specimens were mounted onto

microscopic slides (76 mm · 26 mm, VWR Inter-national, Radnor, PA, USA), embedded in Euparal(Waldeck GmbH & Co. KG, Münster, Germany) and,after drying, investigated with a light microscope ofthe type Zeiss Axioplan (Carl Zeiss MicroscopyGmbH, Jena, Germany) equipped wit a Nikon D7100single-lens reflex digital camera (Nikon Corporation,Tokyo, Japan) and the software Helicon Remote ver-sion 3.6.2.w (Helicon Soft Ltd., Kharkiv, Ukraine) (forfocus stacking Helicon Focus version 6.3.7 Pro;RRID:SCR_014462).For scanning electron microscopy (SEM), specimens

were air-dried for at least one week in a desiccator. Thesamples were mounted with double-sided adhesive tapeonto stubs, sputter-coated with 19 nm pure gold (Au)by using an Emitech K550X (Quorum TechnologiesLtd., West Sussex, UK) and investigated with a scan-ning electron microscope of the type Zeiss EVO LS 10(Carl Zeiss Microscopy GmbH, Jena, Germany) and thesoftware SmartSEM version V05.04.05.00 (Carl ZeissMicroscopy GmbH, Jena, Germany).After completion of the microscopical studies, the

remaining wasps were killed by freezing them at − 20°C.

Synchrotron X-ray phase-contrast microtomography(SR-μCT)Two metasomas of ethanol-fixed female V. canescenswere dehydrated stepwise in ethanol andcritical-point-dried by using a Polaron 3100 (QuorumTechnologies Ltd., West Sussex, UK) to minimizeshrinking artefacts by water loss during the tomog-raphy procedure. The anterior ends of the metasomaswere glued onto the tips of plastic pins, so that theovipositor tip was oriented upright, and mountedonto the goniometer head of the sample stage for

Eggs et al. BMC Zoology (2018) 3:12 Page 14 of 25

tomography. Synchrotron X-ray phase-contrast micro-tomography (SR-μCT) [73] was performed at thebeamline ID19 at the European Synchrotron RadiationFacility (ESRF) (Grenoble, France) at 19 keV (wave-length 8 · 10− 11 m) and an effective detector pixelsize of 0.68 μm with a corresponding field of view of1.43 · 1.43 mm; 6000 projections were recorded overthe 180 degree rotation. The detector-to-sample dis-tance was 12 mm. As the structures of interest werelarger than the field of view, four separate imagestacks were acquired. Therefore, the sample was repo-sitioned in between the imaging procedure, resultingin a certain overlap of two consecutive images. The3D voxel datasets were reconstructed from the 2D ra-diographs by using the filtered back-projection algo-rithm [74, 75] developed for absorption contrasttomography.

Registration and segmentation of SR-μCT imagesTo obtain a high-resolution 3D image of the ovipositorand the inherent muscles, two consecutive images fromthe stack were geometrically aligned in an iterative 3Drigid registration procedure (Additional file 3). Astepwise strategy was applied for the registration. Thetwo data sets were aligned according to the transla-tion of the sample stage in between imaging. The im-ages were then rigidly registered by using normalizedmutual information of the grey value images as asimilarity measure, with a line search algorithm forthe optimization approach. A hierarchical strategy wasapplied to reduce the risk of finding local minima,starting at a coarse resampling of the datasets andproceeding to finer resolutions. Finally, an affinetransformation by using a Lanczos interpolation (cf.[76]) was performed that interpolated both imagesinto the same coordinate system. As a result, all fourimages were matched in a common coordinate sys-tem. An edge-preserving smoothing filter was appliedfor the segmentation of the individual structures. Seg-mentation was based on local differences in densities,as chitinous structures have higher densities thanmuscles. Therefore, grey value images were binarizedby using a dual threshold approach that allowed theextraction and separation of regions with differentdensities.

Image processing and extraction of individual morphologicalstructuresThe obtained two masks of muscles and denser struc-tures were further processed to differentiate theminto their various morphological components. There-fore, a semi-automatic extraction of biological struc-tural features was applied by using geometric

information. First, small islands were removed withan opening filter and, subsequently, the connectedcomponents were automatically labelled. Second, theresulting chitinous structures were manually split atthe connection points between the female T9 and thevalvifers and at the olistheter mechanism of the tere-bra, as these fine structures could not be segmentedautomatically because of the limited resolution of theimages. For each muscle bundle, insertion regions(apodemes) were identified on the cuticular elementsat both muscle ends, with the whole muscle betweenthe apodemes being determined in a semi-automatedinterpolation process. This resulted in individual la-bels for the six muscles involved in ovipositor actu-ation mechanics. A Gaussian filter was applied forsmoothing the 3D masks of the individual chitinousand muscular structures and 3D morphological volu-metric models of the biological structures weregenerated.Image processing was performed by using the software

Amira version 6.0 (FEI, Hillsboro, OR, USA;RRID:SCR_014305) and the custom MATLAB scriptsversion R2016a (The MathWorks, Inc., Natick, MA,USA; RRID:SCR_001622).

Muscle and leverage analysesMuscle volume, mean length and mean cross sectionarea were determined from the 3D data sets. The ob-tained muscle volume values potentially are lowerthan in living animals due to shrinking artefacts. Thetotal muscle length and the major direction of themuscle force was determined as the distance betweenthe centre points of the attachments of the musclesand the direction of the line in between, respectively.The exact locations of the muscles’ origins and inser-tions were verified with light microscopy. The meancross section area (CSA) was determined as themuscle volume / muscle length. However, the orien-tation of the single muscle fibre might deviate fromthe direction of the main muscle force (cf. [77]),which potentially results in an underestimation ofthe estimated CSA of an individual muscle and thusits maximum muscle force but also an overesti-mation of its maximum contraction distance. Theanatomical inlevers were measured from the 3D dataset and the joint angles were determined. The ana-tomical lever was defined as the length of the linebetween the joint axis and the point where themuscle force is applied, i.e. the tendon attachmentpoint. The effective lever arm, which is pivotal forthe efficiency of the force transmission, is defined asthe perpendicular distance between the projection ofthe line of action of the tendon attachment pointand the joint axis.

Eggs et al. BMC Zoology (2018) 3:12 Page 15 of 25

Appen

dix

Table

2Morph

olog

icalterm

srelevant

tothehymen

opteranoviposito

rsystem

.The

term

s(abb

reviations

used

inthisarticlein

brackets)areused

andde

fined

accordingto

the

Hym

enop

tera

AnatomyOntolog

y(HAO)[5–7];therespectiveUniform

Resource

Iden

tifiers(URI)a

ndthesyno

nymsfoun

din

thecitedliteraturearelisted

anatom

icalterm

(abb

reviation)

definition

/con

cept

URI

syno

nymscommon

lyfoun

din

literature

1stvalvifer(1vf)

Thearea

ofthe1stvalvifer-1stvalvulacomplex

that

isproxim

alto

theaulax,be

arsthe9thtergalcond

yleof

the1stvalviferandthe2n

dvalviferalcon

dyleof

the1st

valviferandisconn

ectedto

thege

nitalm

embraneby

muscle.

http://pu

rl.ob

olibrary.org/obo

/HAO_0000338

1.Valvifer[9];fulcralp

late

[20–27];go

nang

ulum

,go

nang

ula[1];go

nocoxite

8[18];g

onocoxite

XIII[13,

14];kidn

eyplate[60];triang

ular

plate[8];vorderer

Valvifer[9];Winkelplatte[17]

1stvalvifer-1stvalvulacomplex

Theanatom

icalclusterthat

iscompo

sedof

thesclerites

that

articulates

with

the9thabdo

minaltergite

andthe

2ndvalvifer.

http://pu

rl.ob

olibrary.org/obo

/HAO_0002158

1stvalvifer-2ndvalviferm

uscle

Theoviposito

rmusclethat

arises

from

theinterarticular

ridge

ofthe1stvalviferandinsertson

the2n

dvalvifer

http://pu

rl.ob

olibrary.org/obo

/HAO_0002189

1stvalvifer-ge

nitalm

embrane

muscle(m

1)Theoviposito

rmusclethat

arises

from

thepo

sterior

partof

the1stvalviferandinsertsanterio

rlyon

the

genitalm

embraneanterio

rto

theT9-gen

italm

embrane

muscle.

http://pu

rl.ob

olibrary.org/obo

/HAO_0001746

anterio

rtergosternalstrictormuscle[14]

1stvalvula(1vv)

Thearea

ofthe1stvalvifer-1stvalvulacomplex

that

isde

limiteddistallyby

theproxim

almarginof

theaulax.

http://pu

rl.ob

olibrary.org/obo

/HAO_0000339

1.Valvula[9];go

napo

physis8[18];g

onapop

hysisVIII

[13,14];lancet

[8,60];low

ervalve[1,2,11,42,44,

46,71,72];Stechb

orste[9,17];stylet[20–27];ventral

stylet

[45,49];ventralvalve

[43,47,66,67];ventral

valvula[45,49]

2ndvalvifer(2vf)

Thearea

ofthe2n

dvalvifer-2n

dvalvula-3rdvalvula

complex

that

isproxim

alto

thebasalarticulationand

totheprocessusmuscularesandarticulates

with

thefe-

maleT9.

http://pu

rl.ob

olibrary.org/obo

/HAO_0000927

2.Valvifer[9];go

nocoxite

9[1,18];g

onocoxite

IX[13,

14];hinterer

Valvifer[9];inne

rplate[20];~

inne

roviposito

rplate[21,22,24,26,27];o

blon

gplate[8];

oblong

ePlatte

[9,17];run

nerplate[60]:~

semicuricular

sheet[23,25]

2ndvalvifer-2n

dvalvula-3rd

valvulacomplex

Thearea

that

isconn

ectedto

the9thtergite

andthe

1stvalviferviaconjun

ctiva,isarticulated

tothe1st

tergite,and

bearstheaulax.

http://pu

rl.ob

olibrary.org/obo

/HAO_0002175

2ndvalvifer-3rd

valvulacomplex

Thearea

ofthe2n

dvalvifer-2n

dvalvula-3rdvalvula

complex

that

isproxim

alto

thebasalarticulation.

http://pu

rl.ob

olibrary.org/obo

/HAO_0002181

2ndvalvifer-g

enitalm

embrane

muscle

Theoviposito

rmusclethat

arises

anterio

rlyfro

mthe

dorsalflang

eof

the2n

dvalviferandinsertsanterio

rlyon

thedo

rsalpartof

thege

nitalm

embrane.

http://pu

rl.ob

olibrary.org/obo

/HAO_0001672

2ndvalviferalcon

dyleof

the

1stvalvifer

Thecond

ylethat

islocatedon

the1stvalviferand

articulates

with

the1stvalviferalfossa

ofthe2n

dvalvifer.

http://pu

rl.ob

olibrary.org/obo

/HAO_0002167

2ndvalvula(2vv)

Thearea

ofthe2n

dvalvifer-2n

dvalvula-3rdvalvula

complex

that

isdistalto

thebasalarticulationandto

theprocessusmuscularesandislim

itedmed

ially

bythe

med

ianbo

dyaxis.

http://pu

rl.ob

olibrary.org/obo

/HAO_0000928

2.Valvula[9];do

rsalvalve[43,47,66,67];do

rsal

stylet

[45,49];do

rsalvalvula[45,49];go

napo

physis9

[18];g

onapop

hysisIX

[13,14];Schien

enrin

ne[9,17];

sheath

[21,22,27];~

stylet

[=slen

derdistalpartof

theun

ited2n

dvalvulae][8]

stylet

sheath

[20,23–26];

uppe

rvlave[1,2,11,42,44,46,71,72];(fu

sed)

ventralvalves[60]

Eggs et al. BMC Zoology (2018) 3:12 Page 16 of 25

http://purl.obolibrary.org/obo/HAO_0000338http://purl.obolibrary.org/obo/HAO_0002158http://purl.obolibrary.org/obo/HAO_0002189http://purl.obolibrary.org/obo/HAO_0001746http://purl.obolibrary.org/obo/HAO_0000339http://purl.obolibrary.org/obo/HAO_0000927http://purl.obolibrary.org/obo/HAO_0002175http://purl.obolibrary.org/obo/HAO_0002181http://purl.obolibrary.org/obo/HAO_0001672http://purl.obolibrary.org/obo/HAO_0002167http://purl.obolibrary.org/obo/HAO_0000928

Table

2Morph

olog

icalterm

srelevant

tothehymen

opteranoviposito

rsystem

.The

term

s(abb

reviations

used

inthisarticlein

brackets)areused

andde

fined

accordingto

the

Hym

enop

tera

AnatomyOntolog

y(HAO)[5–7];therespectiveUniform

Resource

Iden

tifiers(URI)a

ndthesyno

nymsfoun

din

thecitedliteraturearelisted(Con

tinued)

anatom

icalterm

(abb

reviation)

definition

/con

cept

URI

syno

nymscommon

lyfoun

din

literature

3rdvalvula(3vv)

Thearea

ofthe2n

dvalvifer-3rdvalvulacomplex

that

ispo

steriorto

thedistalverticalconjun

ctivaof

the2n

dvalvifer-3rdvalvulacomplex.

http://pu

rl.ob

olibrary.org/obo

/HAO_0001012

3.Valvula[9];articulatingpalps[25];d

orsalvalve

[60];

gono

stylus

[18];g

onostylusIX

[13,14];~inne

roviposito

rplate[23];o

vipo

sitorsheath

[1,2,11,43–

45,49

];palp

[20];she

ath[67];she

athlobe

[8];

Stache

lscheide

[9,17];terminalpalp

[24]

9thtergalcond

yleof

the1st

valvifer

Thecond

ylethat

islocatedon

the1stvalviferand

articulates

with

the1stvalviferalfossa

ofT9.

http://pu

rl.ob

olibrary.org/obo

/HAO_0002160

annu

lus

Thecarin

athat

istransverse

andextend

sacross

the

lateralw

allo

ftheterebra.

http://pu

rl.ob

olibrary.org/obo

/HAO_0001173

anterio

r2n

dvalvifer-2n

dvalvulamuscle(m

2)Theoviposito

rmusclethat

arises

from

theanterodo

rsal

partof

the2n

dvalviferandinsertssubapically

onthe

processusarticularis.

http://pu

rl.ob

olibrary.org/obo

/HAO_0001166

anterio

rgo

nocoxapo

physealm

uscle[13,14];

gonapo

physis9levator[18];ram

usmuscleof

the

2ndvalvula[8];shaftelevator

muscle[27]

anterio

rangleof

the1st

valvifer

Thecorner

onthe1stvalviferthat

marks

thepo

sterior

endof

the1stvalvula.

http://pu

rl.ob

olibrary.org/obo

/HAO_0002168

anterio

rarea

ofthe2n

dvalvifer

Thearea

ofthe2n

dvalviferwhich

isanterio

rto

the

anatom

icallinethat

istheshortestdistance

from

the

1stvalviferalfossa

ofthe2n

dvalviferandtheventral

marginof

the2n

dvalvifer.

http://pu

rl.ob

olibrary.org/obo

/HAO_0002169

anterio

rflang

eof

the1st

valvifer

Theflang

ethat

extend

santerio

rlyon

the1stvalvifer

andoverlaps

with

thepo

steriormarginof

theanterio

rarea

ofthe2n

dvalvifer.

http://pu

rl.ob

olibrary.org/obo

/HAO_0002166

anterio

rflang

eof

abdo

minal

tergum

9(af9)

Theflang

ethat

extend

salon

gtheanterolateralm

argin

offemaleT9.

http://pu

rl.ob

olibrary.org/obo

/HAO_0001171

Apo

dem

[9]

anterio

rrid

geof

T9Therid

gethat

extend

salon

gtheanterio

rmarginof

femaleT9

andreceives

thesite

oforigin

oftheventral

andthedo

rsalT9-2nd

valvifermuscles.

http://pu

rl.ob

olibrary.org/obo

/HAO_0002182

anterio

rsectionof

dorsal

flang

eof

2ndvalvifer(asdf)

Thearea

ofthedo

rsalflang

eof

the2n

dvalviferthat

isanterio

rto

thesite

oforigin

ofthebasalline.

http://pu

rl.ob

olibrary.org/obo

/HAO_0002173

~semicircular

sheet[23–27]

apod

eme

Theprocessthat

isinternal.

http://pu

rl.ob

olibrary.org/obo

/HAO_0000142

articular

surface

Thearea

that

islocatedon

thescleriteandthat

makes

movabledirect

contactwith

anothe

rsclerite.

http://pu

rl.ob

olibrary.org/obo

/HAO_0001485

aulax(au)

Theim

pression

that

ison

the1stvalvifer-1stvalvula

complex

accommod

ates

therhachis.

http://pu

rl.ob

olibrary.org/obo

/HAO_0000152

Falzede

rStechb

orste[17];g

roove[20,21,25]

basalarticulation(ba)

Thearticulationthat

ispartof

the2n

dvalvifer-2n

dvalvula-3rdvalvulacomplex

andadjacent

tothe

rhachis.

http://pu

rl.ob

olibrary.org/obo

/HAO_0001177

Basalgelen

k[9];bu

lbou

sarticulation[18,21,25–27]

basallineof

the2n

dvalvifer

Thelineon

the2n

dvalviferthat

extend

sbe

tweenthe

parsarticularisandthedo

rsalflang

eof

2ndvalvifer.

http://pu

rl.ob

olibrary.org/obo

/HAO_0002171

Eggs et al. BMC Zoology (2018) 3:12 Page 17 of 25

http://purl.obolibrary.org/obo/HAO_0001012http://purl.obolibrary.org/obo/HAO_0002160http://purl.obolibrary.org/obo/HAO_0001173http://purl.obolibrary.org/obo/HAO_0001166http://purl.obolibrary.org/obo/HAO_0002168http://purl.obolibrary.org/obo/HAO_0002169http://purl.obolibrary.org/obo/HAO_0002166http://purl.obolibrary.org/obo/HAO_0001171http://purl.obolibrary.org/obo/HAO_0002182http://purl.obolibrary.org/obo/HAO_0002173http://purl.obolibrary.org/obo/HAO_0000142http://purl.obolibrary.org/obo/HAO_0001485http://purl.obolibrary.org/obo/HAO_0000152http://purl.obolibrary.org/obo/HAO_0001177http://purl.obolibrary.org/obo/HAO_0002171

Table

2Morph

olog

icalterm

srelevant

tothehymen

opteranoviposito

rsystem

.The

term

s(abb

reviations

used

inthisarticlein

brackets)areused

andde

fined

accordingto

the

Hym

enop

tera

AnatomyOntolog

y(HAO)[5–7];therespectiveUniform

Resource

Iden

tifiers(URI)a

ndthesyno

nymsfoun

din

thecitedliteraturearelisted(Con

tinued)

anatom

icalterm

(abb

reviation)

definition

/con

cept

URI

syno

nymscommon

lyfoun

din

literature

bulb

(blb)

Theanterio

rarea

ofthedo

rsalvalve[com

posite

structureof

thefused2n

dvalvulae]that

isbu

lbou

s.http://pu

rl.ob

olibrary.org/obo

/HAO_0002177

Backen

[17];b

ulbo

usbasalp

artof

theun

ited2n

dvalvulae

[8];bo

ulbo

ussockets[25,26];pivotin

gprocess[20];sockets[27]

carin

aTheprocessthat

iselon

gate

andexternal.

http://pu

rl.ob

olibrary.org/obo

/HAO_0000188

cond

yle

Thearticular

surface

that

isconvex

andisinserted

into

thefossaof

anadjacent

sclerite.

http://pu

rl.ob

olibrary.org/obo

/HAO_0000220

conjun

ctiva

Thearea

ofthecuticlethat

isweaklysclerotized

,with

thin

exocuticle.

http://pu

rl.ob

olibrary.org/obo

/HAO_0000221

cordateapod

eme(ca)

Theapod

emeon

theanterio

rmarginof

femaleT9.

TheventralT9-2n

dvalvifermuscleattaches

partlyon

theapod

eme.

http://pu

rl.ob

olibrary.org/obo

/HAO_0001585

Apo

physe[9]

distalno

tchof

thedo

rsal

valve(no)

Theno

tchthat

isdistalon

thedo

rsalvalve[com

posite

structureof

thefused2n

dvalvulae].

http://pu

rl.ob

olibrary.org/obo

/HAO_0002179

distalverticalconjun

ctivaof

the2n

dvalvifer-3rdvalvula

complex

Theconjun

ctivathat

traversesthe2n

dvalvifer-3rd

valvulacomplex

andislocateddistalto

themed

ian

bridge

ofthe2n

dvalvifers.

http://pu

rl.ob

olibrary.org/obo

/HAO_0002180

dorsalflang

eof

the2n

dvalvifer

Theflang

ethat

extend

son

thedo

rsalmarginof

the

2ndvalvifer.Partof

theventralT9-2n

dvalvifermuscle

attaches

totheflang

e.

http://pu

rl.ob

olibrary.org/obo

/HAO_0001577

dorsaleVerdickung

sleiste[9]

dorsalprojectio

nof

the2n

dvalvifer(dp2

)Theprojectio

nthat

islocatedon

the2n

dvalviferand

correspo

ndsto

theproxim

alen

dof

therhachis.

http://pu

rl.ob

olibrary.org/obo

/HAO_0002172

~ramus

edge

[23,25,27]

dorsalramus

ofthe1st

valvula(dr1)

Theregion

that

extend

salon

gthedo

rsalmarginof

the

1stvalvulaandbe

arstheaulax.

http://pu

rl.ob

olibrary.org/obo

/HAO_0001579

1stramus

[16];Ram

usde

r1.Valvula[9];ramus

ofthe1stvalvula[8,51];Stechbo

rstenb

ogen

[9]

dorsalramus

ofthe2n

dvalvula

Thearea

that

extend

salon

gthedo

rsalmarginof

the

2ndvalvula,be

arstheprocessusarticularisanterio

rlyandtheprocessusmusculareson

theantero-dorsal

region

andarticulates

with

the2n

dvalviferviathe

basalarticulation.

http://pu

rl.ob

olibrary.org/obo

/HAO_0002190

2ndramus

[16];Ram

usde

r2.Valvula[9];

Schien

enbö

gen[9,17]

dorsalscleriteof

the1st

valvifer

Thescleriteof

the1stvalviferthat

islocateddo

rsallyof

thetransvalviferalcon

junctiva.

http://pu

rl.ob

olibrary.org/obo

/HAO_0002163

dorsalT9-2nd

valvifermuscle

(m4a/b)

Theoviposito

rmusclethat

arises

alon

gthe

posterod

orsalp

artof

theanterio

rmarginof

femaleT9

andinsertson

theanterio

rsectionof

thedo

rsalflang

esof

the2n

dvalvifer.

http://pu

rl.ob

olibrary.org/obo

/HAO_0001569

anterio

rtergog

onocoxalmuscle[13,14];do

rsal/

ventral[=parta/partb]

anterio

rtergalmuscleof

the

2ndvalvifer[8];extensor

muscles

oflancet

[60];

uppe

r/lower

[=parta/partb]

protractor

ofgo

napo

physis8[18];upp

er/lo

wer

[=parta/partb]

stylet

protractor

muscle[27]

dorsalvalve

Thearea

that

isarticulated

with

therig

htandleft2n

dvalvifersat

thebasalarticulationandbe

arsthe

rhachises.[Term

sometim

esused

forthecompo

site

structureof

thefused2n

dvalvulae.]

http://pu

rl.ob

olibrary.org/obo

/HAO_0001658

[cf.2n

dvalvula]

Eggs et al. BMC Zoology (2018) 3:12 Page 18 of 25

http://purl.obolibrary.org/obo/HAO_0002177http://purl.obolibrary.org/obo/HAO_0000188http://purl.obolibrary.org/obo/HAO_0000220http://purl.obolibrary.org/obo/HAO_0000221http://purl.obolibrary.org/obo/HAO_0001585http://purl.obolibrary.org/obo/HAO_0002179http://purl.obolibrary.org/obo/HAO_0002180http://purl.obolibrary.org/obo/HAO_0001577http://purl.obolibrary.org/obo/HAO_0002172http://purl.obolibrary.org/obo/HAO_0001579http://purl.obolibrary.org/obo/HAO_0002190http://purl.obolibrary.org/obo/HAO_0002163http://purl.obolibrary.org/obo/HAO_0001569http://purl.obolibrary.org/obo/HAO_0001658

Table

2Morph

olog

icalterm

srelevant

tothehymen

opteranoviposito

rsystem

.The

term

s(abb

reviations

used

inthisarticlein

brackets)areused

andde

fined

accordingto

the

Hym

enop

tera

AnatomyOntolog

y(HAO)[5–7];therespectiveUniform

Resource

Iden

tifiers(URI)a

ndthesyno

nymsfoun

din

thecitedliteraturearelisted(Con

tinued)

anatom

icalterm

(abb

reviation)

definition

/con

cept

URI

syno

nymscommon

lyfoun

din

literature

eggcanal(ec)

Theanatom

icalspacethat

isbe

tweentheleftandrig

htolistheters.

http://pu

rl.ob

olibrary.org/obo

/HAO_0002191

Eikanal[9];Inn

enkanal[9,17]

femaleT9

(T9)

Thetergite

that

isarticulated

with

the1stvalviferandis

conn

ectedto

the2n

dvalviferviamuscles.

http://pu

rl.ob

olibrary.org/obo

/HAO_0000075

9.Tergit[9];9thtergum

[8];ou

teroviposito

rplate

[21–27];ou

terplate[20];q

uadrateplate[8];

quadratischePlatte

[9,17];sledplate[60];T9[1,10,

15,19,51,52];terga

9[45,49];tergite

9[12,18];

tergite

IX[13,14];tergum

9[10,15,51,52];tergum

IX[16]

flang

eTheprojectio

nthat

islamella-like

andislocatedon

arim

,carina,apod

emeor

edge

.http://pu

rl.ob

olibrary.org/obo

/HAO_0000344

fossa

Thearticular

surface

that

isconcaveand

accommod

ates

thecond

yleof

anothe

rsclerite.

http://pu

rl.ob

olibrary.org/obo

/HAO_0000353

furcula

Thescleritethatisproximalto

the2ndvalviferand

receives

thesiteof

originof

thepo

sterior2nd

valvifer-2nd

valvula

muscle.

http://pu

rl.o