JHR 83: 73-99 (202 1) ore JOURNAL OF = *20ereewee openasces journal

doi: 10.3897/jhr.83.64018 RESEARCH ARTICLE () Hymenopter a
e

https://jhr.pensoft.net The international Society of Hymenopteriss. REGEARCH

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

Michael Csader', Karin Mayer', Oliver Betz', Stefan Fischer'?, Benjamin Eggs'

| Evolutionary Biology of Invertebrates, Institute of Evolution and Ecology, University of Tubingen, Auf der
Morgenstelle 28, 72076, Tiibingen, Germany 2. Tiibingen Structural Microscopy Core Facility (TSM), Center
for Applied Geosciences, University of Tiibingen, Schnarrenberestrasse 94-96, 72076, Tubingen, Germany

Corresponding author: Michael Csader (m.csader@web.de)

Academic editor: J. Fernandez-Triana | Received 5 February 2021 | Accepted 22 April 2021 | Published 28 June 2021
Attp://z00 bank. org/FEA676AD-6473-4EA3-8F74-E7F83A5 72234

Citation: Csader M, Mayer K, Betz O, Fischer S, Eggs B (2021) Ovipositor of the braconid wasp Habrobracon hebetor:
structural and functional aspects. Journal of Hymenoptera Research 83: 73-99. https://doi.org/10.3897/jhr.83.64018

Abstract

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

braconids and of parasitoid wasps in general.

Keywords
3D reconstruction, Braconidae, functional morphology, Hymenoptera, parasitoid, SEM, serial section-

ing, terebra

Copyright Michael Csader et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC
BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

74 Michael Csader et al. / Journal of Hymenoptera Research 83: 73-99 (2021)

Introduction

Most hymenopteran species belong to the guild of parasitoids of other insects (Quicke
1997). The astonishing radiation of the most diverse parasitoid wasp lineages (i.e. Cer-
aphronoidea, Ichneumonoidea and Proctotrupomorpha; = Parasitoida sensu Peters et
al. 2017) has been estimated to have occurred 266-195 million years ago. This pro-
cess was presumably triggered by continuous adaptations of the parasitoid lifestyle
including features such as endoparasitism, miniaturization, and/or modifications of
the ovipositor (Peters et al. 2017). Adaptations in oviposition behavior and the mor-
phological structure of the ovipositor might not only have increased the reproductive
success of the wasps, but have potentially also enabled them to oviposit in a multitude
of different substrates, facilitating the acquisition of new hosts and host ranges (Gauld
and Bolton 1988; Quicke 1997). The ovipositor of parasitoid wasps serves a set of
functions: penetration of the substrate (if the host is concealed) or of the targeted egg/
puparium, the location, assessment, and piercing of the host, the injection of venom,
the killing of the competitors’ eggs or larvae, the finding of a suitable place for egg lay-
ing, and oviposition (Quicke et al. 1999).

The hymenopteran ovipositor is an anatomical and functional cluster that consists
of the following elements: the paired 1“ valvulae (8" gonapophyses), the 2" valvula
(fused 9° gonapophyses), the paired 3“ valvulae (9 gonostyli), the paired 1* valvifers
(fusion of the 8° gonocoxites and gonangula (Vilhelmsen 2000)), the paired 2”¢ valv-
ifers (9 gonocoxites), and the female T9 (9° abdominal tergum) (Snodgrass 1933;
Oeser 1961). All the morphological terms are applied according to the Hymenoptera
Anatomy Ontology (HAO; Yoder et al. 2010; Hymenoptera Anatomy Consortium
2021; a table of the terms used and of their definitions is given in Table Al in the Ap-
pendix 1). The 1* valvifer is connected to the 2™ valvifer via the intervalvifer articula-
tion and with the female T9 via the tergo-valvifer articulation. Each of the 1“ valvulae
is attached to the corresponding 1* valvifer via the dorsal ramus of the 1* valvula,
whereas the 2" valvula is connected to the 2" valvifer via the basal articulation and fine
ventral rami of the 2"4 valvula (cf. Bender 1943). Both the 1* and the 2" valvulae are
firmly interlocked along almost their entire length via a tongue and groove-like system
called the olistheter. They form the terebra (= ovipositor shaft) and accommodate the
ege canal (Oeser 1961; Quicke et al. 1994).

Despite many comparative studies on the terebra of hymenopterans (Snodgrass
1933; Oeser 1961; Quicke et al. 1994; LeRalec et al. 1996), the number of publica-
tions concerning the entire ovipositor is limited for the vast number of hymenopteran
(super-)families. Studies that describe all the inherent cuticular elements and muscles
of the ovipositor and (in part) also consider functional aspects include several basal
‘symphytan’ families (Smith 1970, 1972; Vilhelmsen 2000; Vilhelmsen et al. 2001),
some aculeate species (i.e. Apis melifera Linnaeus, 1758 (Apidae) (Snodgrass 1933),
species belonging to Chrysidoidea (Barbosa et al. 2021), Sceliphron destillatoruim (Il-
liger, 1807) (Sphecidae), Ampulex compressa (Fabricius, 1781) (Ampulicidae) (Graf et
al. 2021), Vespa crabro Linnaeus, 1758 (Vespidae) (Stetsun and Matushkina 2020),

Ovipositor of Habrobracon hebetor 75

and species belonging to the Crabronidae (Matushkina 2011; Matushkina and Stetsun
2016; Stetsun et al. 2019) and Pompilidae (Kumpanenko and Gladun 2018)), and a
few parasitoid species belonging to the Cynipoidea (Frihauf 1924; Fergusson 1988),
Platygastroidea (Field and Austin 1994), Chalcidoidea (Hanna 1934; King 1962; King
and Copland 1969; Copland and King 1972a, 1972b, 1972c, 1973; Copland 1976),
and Ceraphronoidea (Ernst et al. 2013). The musculoskeletal ovipositor system and the
actuation mechanisms of ichneumonoid wasps have been described recently in the ich-
neumonid Venturia canescens (Gravenhorst, 1829) (Eggs et al. 2018) and the braconid
Diachasmimorpha longicaudata (Ashmead, 1905) (van Meer et al. 2020), respectively.
Furthermore, drawings of the braconid ovipositor including all inherent cuticular ele-
ments exist for Atanycolus rugosiventris (Ashmead, 1889) (Snodgrass 1933), Apanteles
congestus (Nees, 1834) (Oeser 1961), and Stenobracon deesae (Cameron, 1902) (Alam
1953; Venkatraman and Subba Rao 1954).

However, knowledge about structural and functional aspects of the ovipositor sys-
tem of the ecologically and morphologically extremely diverse and species-rich Braco-
nidae remains limited.

Habrobracon hebetor (Say, 1836) (Fig. 1a, b) is a gregarious, idiobiont, larval ec-
toparasitoid of pyralid moths (Lepidoptera) (Paust et al. 2006) and is well known for
its use in biological pest control (Paust et al. 2006; Mbata and Shapiro-ilan 2010;
Sanower et al. 2018). Dweck et al. (2008) provided the first morphological descrip-
tions of the ovipositor of this species, with a strong focus on the terebra and its sensillar
equipment. In the present study, however, we aim to describe thoroughly all the inher-
ent cuticular elements of the ovipositor of H. /ebetor in order further to discuss their
structural, mechanical, and functional aspects. We have therefore combined scanning
electron microscopic (SEM) and light-microscopic studies on both complete cuticu-
lar structures and histological serial sections. Serial sections of the terebra have been
used to provide a 3D reconstruction that has helped us to understand its morphology
especially with regard to all its functionally clustered inherent structures. Finally, we
present a structural model of the braconid ovipositor and discuss its mode of function.

Materials and methods

Study animals

The H. hebetor (Fig. 1a, b) specimens used in this study originated from Sauter &
Stepper GmbH (Ammerbuch, Germany), where they were bred on larvae of Ephestia
kuehniella Zeller, 1879 (Lepidoptera: Pyralidae).

Sample preparation and light microscopy

For whole mount samples, female H. hebetor were killed in 70 % ethanol at 45 °C.
The metasoma was cut off and macerated in 10% aqueous potassium hydroxide for up

76 Michael Csader et al. / Journal of Hymenoptera Research 83: 73-99 (2021)

a b

1vf tva

mb2 vv 2VV

2vt sp iva

Figure |. Habitus images of female Habrobracon hebetor: \ateral (a) and ventral (b) ascpect. Schematic
drawing of the ovipositor of H. hebetor (lateral view) based on the light microscopic and SEM images (C).
Abbreviations: lvf = 1* valvifer; lvv = 1* valvula; 2vf = 2"4 valvifer; 2vv = 2"! valvula; 3vv = 3" valvula; ar9
= anterior ridge of T9; ba = basal articulation; blb = bulb; dr1 = dorsal ramus of the 1* valvula; hsl = hook-
shaped lobe of the 2"! valvifer; iva = intervalvifer articulation; mb2 = median bridge of 2 valvifers;
sr = sensillar row of the 2" valvifer, sp = sensillar patch of the 2"! valvifer; T9 = female T9; tva = tergo-

valvifer articulation.

to 24 h at room temperature to remove tissues, cleaned in distilled water on a mini-
shaker, and dehydrated stepwise in ethanol. We then dissected the ovipositor out of the
metasoma by using thin tungsten needles, mounted the specimen onto a microscope
slide, and embedded it in Entellan® (Merck KGaA, Darmstadt, Deutschland).

For semithin serial sections, we anaesthetized female H. hebetor with carbon diox-
ide. The metasomas were removed and, in order to avoid autolysis, immediately sub-
mersed in a primary fixative containing 2.5% glutaraldehyde and 5% sucrose, buffered
with 0.1 M cacodylate to pH 7.4. During this fixation, the samples were held in an ice
bath at approximately 4 °C for 4 h. Samples were rinsed three times for 10 min in pre-
chilled 0.1 M cacodylate buffer (pH 7.4) and post-fixed by using a solution of 1% os-
mium tetroxide in 0.1 M cacodylate buffer at 4 °C for 4 h. After being further rinsed in
the same buffer, the samples were dehydrated through a graded series of ethanol with
steps of 30% for 15 min and 50% for 10 min at 4 °C, three times per step, and of 70%

Ovipositor of Habrobracon hebetor ras

for 10 min, three times at room temperature. The following steps were performed at
room temperature. Ev bloc staining was conducted using a saturated solution of 70%
ethanolic uranyl acetate for 12 h on a rotatory shaker. Afterwards, dehydration was
continued in 5% steps, three times for 10 min each. ‘The fully dehydrated samples were
washed in 100% propylene oxide twice for 1 h , and subsequently infiltrated in Spurr
low-viscosity embedding resin (Polysciences Inc., Warrington, PA, USA) via a propyl-
ene oxide:resin mixture at ratios of 1:1, 1:3, and 1:7 for 1 h per step and then in pure
resin for 17 hona rotatory shaker. As a last incubation step, the samples were placed in
fresh pure resin, embedded in silicon molds, and polymerized at 70 °C for 8 h.

Semithin sections of 600 nm were cut perpendicularly to the terebra of H. hebetor
by using an ultramicrotome Leica Ultracut UTC (Leica Microsystems GmbH, Wetzlar,
Germany) equipped with a diamond knife (45° knife angle; DuPont Instruments, Wilm-
ington, DE, USA); a series was obtained with 1920 sections. Semithin sections were then
mounted on a microscopic slide by using a ‘Perfect Loop for Light Microscopy’ (Electron
Microscopy Sciences, Hatfield, PA, USA), stained with Stevenel’s blue (del Cerro et al.
1980) for 40 min at 60 °C, and subsequently washed in distilled water twice for 5 min
each. After being dried, the stained sections were embedded in ‘Xylolfreies Eindeckmittel’
(Engelbrecht Medizin- und Labortechnik GmbH, Edermiide, Germany).

The image stack for the 3D reconstruction was generated using a Zeiss Axioplan
(Carl Zeiss Microscopy GmbH, Jena, Deutschland) light microscope, equipped with
a Nikon D7100 single-lens reflex digital camera (Nikon K.K., Tokio, Japan) and Heli-
con Remote software version 3.6.2.w (Helicon Soft Ltd, Kharkiv, Ukraine). Flawed
images (missing or folded structures and staining problems) were replaced by a copy
of the previous or the following image (this was the case for fewer than 3% of the im-
ages) for reconstruction purposes. Adobe Photoshop Lightroom version 6.0 (Adobe
Systems, San José, CA, USA) was used for initial image processing (white balancing,
color contrasting, black and white conversion), whereas Fiji (Schindelin et al. 2012;
available online at https://imagej.net/Fiji) was employed for cropping, CLAHE filter-
ing, and image stack calibration by using the plugin TrakEM2 (Cardona et al. 2012). A
preliminary least square rigid alignment followed by an elastic alignment of the image
stack was performed using the “Elastic Stack Alignment’ plugin (Saalfeld et al. 2012).
The aligned image stack was then imported into Amira version 6.0 (FEI Company,
Hillsboro, OR, USA). We pre-segmented the 1 and 2°¢ valvulae, the duct of the
venom gland and the ligaments that connect the terebra and the 2" valvifer in the soft-
ware’s segmentation editor by manually labeling approximately every 15" virtual slice
along the terebra and every 4" virtual slice in the proximal bulbous region and assigned
them to different ‘materials’. The labels served as an input for automated segmenta-
tion by using the Biomedical Image Segmentation App Biomedisa (available online at
https://biomedisa.de) (Lésel et al. 2020). The output of Biomedisa was then partially
corrected manually in Amira, and a final surface model was generated.

Schematic drawings of the cross-sections of the terebra were generated in Inkscape
version 0.92.4 (Inkscape Community; available online at http://www.inkscape.org/)
based on the original light-microscopic images of the semithin sections.

78 Michael Csader et al. / Journal of Hymenoptera Research 83: 73-99 (2021)

For lateral and ventral habitus images, female wasps were imaged with a Key-
ence VHX-7000 Digital Microscope (Keyence Corporation, Osaka, Japan) using

focus stacking.

Scanning electron microscopy (SEM)

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

Results and discussion

As in all hymenopterans, the ovipositor of H. hebetor consists of three pairs of valvulae,
two pairs of valvifers, and the female T9 (Fig. 1c).

Overall structure of the terebra

The 1* and 2™ valvulae form the terebra and enclose the egg canal (cf. Figs 2c—g, 3;
Suppl. material 1). The terebra of H. hebetor extends far beyond the posterior tip of the
metasoma. They are interconnected by a tongue and groove-like system, called the olis-
theter (oth; Fig. 2a—h). The olistheter comprises two longitudinal ridges that are called
the rhachises (rh; Figs 2a, 5c, d) on both sides at the ventral surface of the 2" valvula and
that fit into corresponding T-shaped grooves termed the aulaxes (au; Figs 2a, 4b, f, h)

Figure 2. (next page) Cross sections through the terebra of Habrobracon hebetor (from proximal to distal);
schematic drawings of the 1 and 2" valvula (ai) based on the light microscopic images of the presented
semithin sections (a’=i’ 600 nm; stained with Stevenel’s blue). The drawings are of the same size ratio. The
2™! valvulae possesses, in the proximal region, two lumina that merge into one in the most distal region
(h=i). The bulbs (b) and the valvilli (g) are visible. The orange lines (in e, f) mark the position of the di-
stally pointing ctenoid structures on the dorsal surfaces of the 1‘ valvulae, which are in close contact with
the ventral surface of the 2"¢ valvula. The genital membrane connects the dorsal margins of the 2" valvifers
(b’-h’) c fine cuticular structures arise from the dorsal and ventral parts of the 2" valvula and define the
lumina of the bulbs (arrow) hI olistheter-like interlocking system connecting the medial surfaces of the
apices of the paired 1* valvulae (arrow). Final segmented 3D reconstruction based on a semithin section
series (600 nm thickness) a*=i* position of each single section marked on the final 3D reconstruction of
the terebra. Abbreviations: lvv = 1* valvula; 2vv = 2" valvula; 3vv = 3" valvula; au = aulax; blb = bulb; cr
= longitudinal crack of 2" valvula; ec = egg canal; fll = longitudinal flap of the 1* valvula; gm = genital
membrane; igs = internal guiding structure; 11 = lumen of 1* valvula; 12 = lumen of 2"¢ valvula; lb = lumen
of the bulb; lg = ligament; oth = olistheter; rh = rhachis; vd = duct of the venom gland; vl = valvillus.

Ovipositor of Habrobracon hebetor

79

100 um

80 Michael Csader et al. / Journal of Hymenoptera Research 83: 73-99 (2021)

Ig

Figure 3. 3D reconstruction of the basal part of the terebra of Habrobracon hebetor composed of the 2"
valvulae and the paired 1* valvulae, based on a semithin section series (600 nm thickness a lateroventral
aspect b lateral aspect € dorsal aspect d ventral aspect). The duct of the venom gland enters dorsoproximally
on the left side only. The two ligaments connect the 2" valvula to the anterior parts of the 2™ valvifers (not
visible in these images). The black circle (b) indicates the rotation axis of the basal articulation. The lines
of the processus articularis and processus musculares (b) point to their presumed position according to the
results of van Meer et al. (2020). The peak-like structure at the anterior end of the 2! valvula (red arrow
in a, b) is part of the processus musculares. There are two openings (black arrows in a, C) at the proximal
side of the bulbs. The duct of the venom gland enters on the left side only. Abbreviations: lvv = 1* valvula;
2vv = 2™ valvula; blb = bulb; ec = egg canal; lg = ligament; pa = processus articularis; pm = processus

musculares; vd = duct of the venom gland.

along the dorsal surface of each of the 1* valvulae. This system allows the 1* valvulae to
slide longitudinally relative to each other when actuated by the corresponding operat-
ing muscles (Oeser 1961; Quicke et al. 1994). Distally pointing scale-like structures are
found on both the olistheter elements and might reduce the friction forces by reducing
the contact surface between the 1* valvulae and the 2™ valvula (sc; Fig. 4f) (Rahman
et al. 1998).

In H. hebetor, the cross sections of the terebra differ notably along its length
(Fig. 2a—i). A common oviduct enters the proximal bulbous part of the terebra (Bender

Ovipositor of Habrobracon hebetor 81

Figure 4. SEM images of the 1 valvulae of Habrobracon hebetor a-c apex of 1* valvula with sawteeth
(a lateral aspect b—c dorsal aspect). The aulaces are visible (b) d-e valvillus of 1* valvula (distal is right)

f distally oriented scale-like structures on the lateral walls of the aulax g leaf-like ctenidia on the medial
side of 1“ valvula h 1* valvula with distally oriented ctenoid structures on its dorsal side (contact surface
with the 2"¢ valvula) and ctenidia on its medial side (contact surface with the opposing 1* valvula building
the egg canal in the distal part of the terebra). Abbreviations: lvv = 1* valvula; au = aulax; ct = ctenidium;

rcs = row of ctenoid structures; sc = scales; st = sawtooth; vl = valvillus.

1943; Pampel 1914), where it ends at the base of the egg canal (Quicke 1997). Dis-
tally, the egg canal is largely defined by the 1* valvulae, but with the dorsal side being
formed by the 2™4 valvula. The diameter of the terebra decreases from proximal to
distal, whereas the diameter of the egg canal remains constant for a long distance from
proximal until the valvillus (see subsection ‘1 valvulae’).

Is* valvulae

The paired 1* valvulae of H. hebetor form the ventral half of the terebra (1vv; Figs Ic,
2a-i, 3, 7a). The proximal end of each 1* valvula is continuous with its dorsal ramus

82 Michael Csader et al. / Journal of Hymenoptera Research 83: 73-99 (2021)

(dr1; Figs lc, 7c, h), which is fused with the anterodorsal corner of the 1* valvifer
(Figs 7a, c, e, 8).

At their apices, the 1“ valvulae of H. hebetor possess several sawteeth, which de-
crease in size apically (st; Fig. 4a, b, c) (cf. Dweck et al. 2008). They probably serve
to penetrate the substrate and the host’s skin and tissue. Distally pointing ctenoid
structures (rcs; Fig. 4h) arranged in rows can be found on the dorsal surfaces of the 1“
valvulae, which are in close contact with the ventral surface of the 2™ valvula (orange
line; Fig. 2e, f). These ctenoid structures potentially reduce friction forces by minimiz-
ing the contact surface between the 1“ and the 2"¢ valvula. The aulaces do not extend
all the way to the apex of the 1* valvulae but end just before the lateral sawteeth occur
(au; Fig. 4b). Both 1* valvulae are separated for the most of their length. However,
mediodorsally at their very apex, the two 1“ valvulae become interlocked dorsally by
a mechanism similar to that of the olistheter (Fig. 2h1; also see fig. 4a of Dweck et al.
2008). Such a mechanism has previously been observed in other braconids (Zaglypto-
gastra (Quicke 1991), Aleiodes, Ligulibracon and Odontobracon (Quicke et al. 1994),
and D. longicaudata (van Meer et al. 2020)) and is suggested to be an adaptation to the
injection of venom into the host while laying the egg externally (Dweck et al. 2008).
In addition, this mechanism might also increase the stability of the apex of the terebra
when the host cuticle is pierced (Quicke 2015).

A single valvillus situated on the inner surface of each 1* valvulae protrudes inside
the egg canal (vl; Figs 2g, 4d, e; cf. Dweck et al. 2008). The valvillus is a bilaterally con-
cave structure lying in the distal third of the terebra and occupies the whole diameter
of the egg canal. Valvilli can be found in the Ichneumonoidea and in various families
of the Apocrita (Snodgrass 1933; Quicke et al. 1992; Rahman et al. 1998). They are
postulated to serve as a stop and release mechanism for the egg by maintaining the egg
in position within the terebra and blocking the egg canal in Ichneumonoidea (Rogers
1972; Rahman et al. 1998; Boring et al. 2009), or for venom pumping in Apocrita
(Quicke et al. 1992). However, in the ectoparasitoid H. hebetor, the eggs are observed
to advance and even partially emerge ventrally at the base of the terebra, i.e. in between
the 1* valvulae and near the genital opening (Prozell et al. 2006; Wiihrer et al. 2009,
see also Shaw 2017). Further distally, the valvilli seem to divert the egg ventrally be-
tween the 1* valvulae and to press it out completely, since the egg does not emerge at
the tip of the terebra but rather ventrally in between the 1* valvulae approximately at
the region at which the valvilli are located (Prozell et al. 2006; Witihrer et al. 2009). We
therefore suggest that the valvilli guide the relatively large egg ventrally out in between
the 1* valvulae. The latter are capable of being widely spread in this region because of
the olistheter mechanism (Shaw 2017). In cross sections further apically to the valvilli,
an egg canal is rarely visible or is absent (Fig. 2h, i), which suggests that at that point
the egg has already left the terebra. In addition, the apical interlocking in between the
two 1* valvulae (red arrow; Fig. 2h1), which is similar to that of the olistheter, prevents
the canal from expanding at the very apex. Proximal to the valvillus, the walls of the
ege canal carry leaf-like ctenidia (ct; Fig. 4g, h), which are arranged in rows and are
directed towards the distal end of the terebra. These rows of ctenidia point distally in

Ovipositor of Habrobracon hebetor 83

Figure 5. SEM images of the 2" valvula of Habrobracon hebetor a overview of the 2"¢ valvula (ventral

aspect with the interlocked 1% valvulae removed) b proximal part of 2"¢ valvula featuring the bulbs ¢ apex
of 2™ valvula with the ending of the rhachises (arrow) and campaniform sensillae (medioventral aspect)
d-e middle part of the 2™! valvula showing the rhachises and distally oriented ctenidia f sensilla at the
apex of the 2™ valvula (detail image of ¢). Abbreviations: 2vv = 2"¢ valvula; blb = bulb; cs = campaniform

sensilla; ct = ctenidia; rh = rhachis.

the direction of egg movement and presumably prevent the regression of the egg dur-
ing the oviposition process (Austin and Browning 1981). Setiform structures (= sub-
ctenidial setae sensu Rahman et al. 1998) are also found at the inner walls of the 1%
valvulae lying distally to the valvilli. They are arranged in distinct rows.

Each 1* valvula contains a lumen (11, Fig. 2a—i) whose cuticular walls differ along
its length. Proximally, the cuticle is thin but becomes thicker towards the middle and
diminishes again apical to the valvillus (Fig. 2a—i). In cross section, the shape of the 1“
valvula differs between the basal region and the rest of the terebra. In the basal part,
it is triangular in shape (lvv; Fig. 2a), whereas further distally, it appears more oval
(1vv; Fig. 2b-i). A longitudinal flap extends along the mediodorsal edge for most of the
length of the 1* valvulae and is clearly recognizable in cross sections (f1; Fig. 2a—f). This
flap is highly prominent in the proximal part of the terebra but vanishes further apically
(fll; Fig. 2g—i). It might seal the egg canal to prevent the leaking of venom, since the
pressure of the venom has been suggested to squeeze the two flaps together and therefore
to seal the gap (Quicke et al. 1994; Shaw 2017). It has been observed in almost all the
examined braconid species (Quicke and van Achterberg 1990; Quicke et al. 1994).

2" valvula

In H. hebetor, the 24 valvula (2vv; Figs 1c, 2a-i, 3, 5a, 7a) form the dorsal half of
the terebra, and its proximal bulbous end (blb; Figs 1c, 2b, 3, 5b, 7e, h; called the

84 Michael Csader et al. / Journal of Hymenoptera Research 83: 73-99 (2021)

bulbs in the following) is connected with the 2" valvifer via the basal articulation
(ba; Figs 1c, 7h).

The apex of the 2°¢ valvula is not serrated but is slightly enlarged before it narrows
towards the tip (Figs 5a, c, 7a). In contrast to many ichneumonid and other braconid
species (cf. Boring et al. 2009; Shah et al. 2012; Eggs et al. 2018), the 2™ valvula of H.
hebetor does not feature a prominent apical notch. Campaniform sensilla can be found
in this area (cs; Fig. 5f) (for a discussion of the sensillary equipment of the terebra
of H. hebetor, see Dweck et al. 2008). Similar to the aulaces on the 1° valvulae, the
thachises (rh; Fig. 5c, d) do not extend all the way to the apex but end at about the
same distance away from the apex as seen for the aulaces (arrow; Fig. 5c). The apical
half of the ventral side of the 2"¢ valvula forms the dorsal wall of the egg canal and is,
similar to the 1 valvulae, covered by rows of ctenidia directed distally (ct; Fig. 5e). As
previously discussed for the 1* valvulae, these structures might prevent the regression
of the egg during oviposition (cf. Rahman et al. 1998). Medioproximally, the bulbs
feature ligaments (lg; Figs 2a, 3a, b, d) that connect the 2! valvula with the anterior
section of the 2™! valvifer. The ligament marks the region at which parts of the 24
valvifer merge into the anterior part of the 2™ valvula. The bulbs also contain a lumen
(Ib; Fig. 2b). The proximal end of the 2 valvula bears the processus articularis (pa;
Figs 3b, 7h) laterally and the processus musculares (pm; Figs 3b, 7h) at the anterior
peak-like structure of the 2™ valvula (red arrow; Fig. 3a, b). However, the medial 24
valvifer-2"* valvula muscle (M-2vfl-2vlv) that might stabilize the 2"! valvifer and that
was newly described in the braconid D. longicaudata by Meer et al. (2020) was absent
in our serial sections. There are two openings (black arrows; Fig. 3a, c) at the proximal
side of the bulbs. The duct of the venom gland enters the dorsoproximal area of the
bulbs on the left side only (vd; Figs 2a, b, 3, Suppl. material 2) (cf. Bender 1943, who
investigated the anatomy and histology of the female reproductive organs of the closely
related Habrobracon juglandis (Ashmead, 1889)). Further distally, the closed duct of
the venom gland seems to disappear and to merge with the egg canal formed by the
valvulae (Suppl. material 2). In this area, the venom presumably flows into the egg
channel that is formed by both the 1* and 2Ӣ valvulae with the longitudinal flaps of
the 1* valvulae acting as a seal (fll; Fig. 2a—f).

Proximally, the 2™ valvula features a distinct longitudinal crack at the ventral side
along the middle, which is clearly visible in cross-section (cr; Fig. 2c—g), presumably
indicating the paired origin of the 2" valvulae. At the basal part of the 2™ valvula, fine
cuticular structures (arrow; Fig. 2c) arise from it dorsal and ventral parts and define
the two lumina (12; Fig. 2c—-g) that run almost the entire length of the 2"¢ valvula and
that fuse at the apex (Fig. 2h, i). Proximally, the ventral part of the 2° valvula gradu-
ally changes shape and forms a U-shaped structure that extends distally into the egg
canal (Suppl. material 2). This internal structure (igs; Fig. 2c-e) presumably helps
in guiding the egg by forming a temporary egg canal. Without this internal guid-
ing structure, the diameter of the egg canal would be large in this proximal region;
this might lead to a lowered internal pressure and thus to problems when the egg is

pushed further distally.

Ovipositor of Habrobracon hebetor 85

valvulae b inner proximal surface of the 3“ valvulae with fine trichomes distally ¢ outer surface of the 3

valvulae, proximally exhibiting annulation caused by fine transverse furrows (arrow) d outer surface of the
3 valvulae at the transition zone (arrow) between the distal longitudinal ridge to the proximal vertically
folded surface at the region in which the 3“ valvulae expand e inner surface of the apex of a 3“ valvula

covered by trichomes. Abbreviations: 3vv = 3" valvula; trb = terebra; t = trichome.

3 valvulae

The paired 3 valvulae of H. hebetor originate at the distal end of the 2™ valvifers and
extend far beyond the posterior tip of the metasoma towards the tip of the terebra
(Figs 1b,c, 7a, e). Each is U-shaped in cross-section (3vv; Figs 2g¢’—i’, 6a) and they
completely ensheath and protect the terebra in the resting position (3vv, trb; Figs
2¢’-i’, 6a) (cf. Bender 1943; Dweck et al. 2008). The distal third of the 3 valvulae is
enlarged (Figs 6a, 7a), and their lateral surfaces differ over the course of their length:
proximally, the 3“ valvulae are annulated by fine transverse furrows (arrow; Fig. 6c;
cf. Vilhelmsen 2003; Eggs et al. 2018), whereas the enlarged distal part lacks these
structures (arrow; Fig. 6d). Trichomes, which Dweck et al. (2008) have described as
trichoid sensilla, cover most of the external surface of the 3“ valvulae (Fig. 6a). The
density of the trichomes varies along the length of the 3 valvulae and is highest at
the apex (Fig. 6a).

The inner surface of the 3 valvulae facing the terebra is densely covered by
trichomes (t; Fig. 6b, e), particularly at the distal enlarged part (Fig. 6a, e). These
structures might be involved in cleaning the terebra sensilla between oviposition epi-
sodes (Quicke et al. 1999; Vilhelmsen 2003). Observations have shown that the 3"
valvulae also play a role in stabilizing the terebra during oviposition (Prozell et al.
2006; Withrer et al. 2009; Vilhelmsen 2003; Cerkvenik et al. 2017; Eggs et al. 2018;
van Meer et al. 2020).

86 Michael Csader et al. / Journal of Hymenoptera Research 83: 73-99 (2021)

Ovipositor of Habrobracon hebetor 87

| valvifer

In lateral view, the paired 1* valvifers of H. hebetor have a compact triangular shape
with rounded edges (Ivf; Figs 1c, 7a, c-e, 8). The intervalvifer articulation (iva; Figs Ic,
7a, c-f), a rotational joint, is located at the rounded posteroventral side and connects
the 1* valvifer to the 2"¢ valvifer. The ventral edge of the 1* valvifer is placed laterally
of the 2" valvifer and seems to be in contact with a sensillar patch (sp; Figs Ic, 7b,
d_f) that extends dorsally at the anterior beginning of the the dorsal flange of the 24
valvifer (df2; Fig. 7d, f). The tergo-valvifer articulation (tva; Fig. 7a, ce, g) connects
the 1* valvifer to the female T9. A ridge, called the interarticular ridge of the 1* valvi-
fer (iar; Figs 1c, 7c—e, 8), extends in between the two articulations. This ridge might
mechanically stabilize the 1* valvifer and prevent it from extensive deformation. At its
anterodorsal corner (arrow; Fig. 8), the 1“ valvifer is fused with the dorsal ramus of the
1* valvula (dr1; Figs 1c, 7c, h, 8), which is continuous with the 1* valvula.

24 valvifer

The paired 2Ӣ valvifers of H. hebetor are elongated in the longitudinal axis (2vf Figs 1c,
7a, e). The anteromedial socket-like part of the 2°4 valvifer is connected to the laterally
placed bulbs of the 2° valvula (blb; Figs 1c, 3, 7e, h) via the ball-and-socket-like basal
articulation (ba; Figs 1c, 7h). The posterior ends of both the 2" valvifers are connected to
the 3“ valvulae (3vv; Figs 1c, 7a, e). At their posterodorsal ends, the two 2™ valvifers are
connected by a median bridge (mb2; suggested position indicated in Fig. 1c). A massive
dorsal spike (ds; Fig. 7e), a structure that has not as yet been described in other parasitoid
wasps, is present at the posterior end of the 2" valvifer and potentially serves as an apode-
me. In addition, a flexible cuticular area, a conjunctiva called the genital membrane (gm;
Fig. 2d’), connects the ventral margins of the 2"¢ valvifers arching above the 2"¢ valvula.

Figure 7. (previous page) SEM (a=d) and light microscopic (e=i) images of the ovipositor of Habrobra-
con hebetor a overview of the ovipositor (lateral aspect; visible pore-like structures are presumably artefacts
of detached trichomes) € 1* valvifer exhibiting the interarticular ridge and the hook-shaped lobe of the 2"
valvifer. The 1* valvifer is continuous with the dorsal rami of the 1* valvula and is articulated with the 2™
valvifer and the female T9 via the intervalvifer articulation and the tergo-valvifer articulation, respectively
d sensillar patch of the 2" valvifer (made visible by partly removal of the 1* valvifer) b, f sensillar patch
of the 2" valvifer € overview of the 2" valvifer and female T9. The arrow shows the dorsal hump of the
T9 g tergo-valfiver articulation between the 1“ valvifer and female T9 h detail image of e. The laterally
placed bulbs of the most proximal part of the 24 valvula are articulated with the paired 2" valvifers via the
basal articulation i sensilla in a row at the dorsal margin of the 2"¢ valvifer. Abbreviations: Ivf =1* valvifer;
lw = 1* valvula; 2vf = 2" valvifer; 2vv = 2" valvula; 3vv = 3“ valvula; ar9 = anterior ridge of T9; ba = basal
articulation; blb = bulb; df2 = dorsal flange of 2" valvifer; dm2 = dorsal margin of the 24 valvifer; dr1
= dorsal ramus of the 1 valvula; ds = dorsal spike of the 24 valvifer; hsl = hook-shaped lobe of the 2"¢
valvifer; iar = interarticular ridge of the 1* valvifer; iva = intervalvifer articulation; sp = sensillar patch of
the 2" valvifer; sr = sensillar row of the 2"¢ valvifer; pa = processus articularis; pm = processus musculares;

T9 = female T9; trb = terebra; tva = tergo-valvifer articulation.

88 Michael Csader et al. / Journal of Hymenoptera Research 83: 73-99 (2021)

tva
lar

|

\ F (in retraction)
Ps F (in protraction)
Cc
|

iva

Figure 8. Functional lever model of 1* valvifer with both articulations and the beginning of the dorsal
ramus of the 1* valvula (arrow) with the intervalvifer articulation acting as pivot point. c = anatomical
inlever ; C = effective (= mechanical) inlever; d = anatomic outlever; d‘ = effective (= mechanical) outlever;

= Force input at the 1* valvifer; F = Force output at the 1*

(in protraction)? ~ (in retraction) (out protraction)? ~ (out retraction)
P P

valvifer that is transferred to the dorsal ramus of the 1* valvula. c¢ - ay =d. a: Abbreviations: Ivf = 1*
valvifer; dr1 = dorsal ramus of the 1* valvula; iar = interarticular ridge of the 1* valvifer; iva = intervalvifer

articulationtva = tergo-valvifer articulation.

At its anterodorsal corner, the 2" valvifer extends upwards in a hook-shaped lobe
(hsl; Fig. 7a, c, e; sensu Snodgrass 1933), and features the elongated anterodorsal ridge
of the 2" valvifer, the so called dorsal margin of the 2" valvifer (dm2; Fig. 7c, d, h).
The dorsal projection of the 2"¢ valvifer, a tongue-like structure situated on the dorsal
margin of the 2™ valvifer, is continuous with the olistheter. The corresponding groove
is located on the dorsal side of the dorsal ramus of the 1* valvula (dr1; Fig. 7c, h; cf. fig.
4h1 of Eggs et al. 2018) and enables its back and forth movement. This hook-shaped
lobe might guide and stabilize the 1 valvifer during its posterior pivoting but might
also allow for a larger arc of movement of the 1* valvifer and therefore a greater retrac-
tion distance of the 1“ valvulae (cf. Eggs et al. 2018).

Two main ridges are found on the 2™ valvifer, i.e. (1) the dorsal flange of the 2"!
valvifer (df2; Fig. 7d, f), which expands from the sensillar patch in the direction of
the hook-shaped lobe and posteriorly from the sensillar patch to the origin of the 3“
valvulae (Fig. 7e), and (2) the dorsal margin of the 2" valvifer (dm2; Fig. 7c, d, h). The
two cuticular ridges might have a stabilizing function to prevent deformation. The 24
valvifer of H. hebetor does not feature a basal line (e.g. in contrast to the ichneumonid
Venturia canescens (Gravenhorst, 1829), see fig. 4e of Eggs et al. 2018), a ridge that
extends from the pars articularis to the dorsal flange of the 24 valvifer.

Ovipositor of Habrobracon hebetor 89

Clusters of sensillae (“styloconic sensillae” according to Dweck et al. 2008) occur in
two regions. The first cluster, called the sensillar patch (sp; Figs 1c, 7b, d—f), is situated
ventrally of the intervalvifer articulation and is covered by the 1* valvifer laterally. The
second cluster occurs at the dorsal margin of the 2"¢ valvifer (sr; Figs 1c, 7h, i). These
sensilla are arranged in a row and are in contact with the dorsal ramus of the 1* valvula.
The two sensilla clusters presumably monitor the pro- and retraction movements of
the 1“ valvifers and the attached 1* valvulae, respectively. The density of sensilla in the
patch is much higher than that on the dorsal margin of the 2"¢ valvifer.

Female T9

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

Mode of function of the ovipositor

Functional models of the actuation and movement mechanisms based on thorough
analyses of the musculoskeletal system of an ichneumonid and a braconid wasp have
recently been described (Eggs et al. 2020, van Meer et al. 2020) and are summarized
in the following. Although, in our study, we have not considered the muscles of the
system, we have basically found the same arrangement of cuticular elements in the
ovipositor system of H. hebetor as described in both of the above-mentioned studies.
Hence, we assume analogous functional morphological conditions, although we point
out any possible H. hebetor-specific modifications.

The ovipositor movements are mainly actuated by two pairs of antagonistically
working muscles (further described below), i.e. (1) the depression (i.e. downward rota-
tion to the active position) and elevation (i.e. upward rotation back to the resting posi-
tion) of the terebra, and (2) the pro- and retraction of the 1* valvulae. Smaller muscles,
ie. the 1% valvifer-genital membrane muscle or the posterior T9-2"¢ valvifer muscle,
might predominantly serve to stabilize the ovipositor system during oviposition.

(1) Depression and elevation of the terebra: The basal articulation is composed of
the processus articularis (pa; Figs 3b, 7h) at the 2" valvulae and the pars articularis at
the 2™ valvifer. The pars articularis is a small area of anteroventral corners of the 24
valvifer, whereas the processus articularis is the respective structure of the bulb. The
posterior 2"4 valvifer-2"4 valvula muscle depresses the terebra, i.e. rotates it downwards
to the active position from its resting position between the paired 3 valvulae. The
tendon of this muscle inserts at the processus musculares (pm; Figs 3b, 7h), which is
situated at the peak-like posterior part of the 2"! valvula (arrow; Fig. 3a, b) and thus

90 Michael Csader et al. / Journal of Hymenoptera Research 83: 73-99 (2021)

increases the moment arm. However, the moment arm most probably changes over the
range of motion of the terebra. In H. hebetor, we assume that the virtual line that can
be drawn perpendicularly to the length axis of the terebra through the ligaments (lg;
Figs 2a, 3a, b, d) lying anterolaterally on the bulbs (blb; Figs 2b, 5a, b, 7e, h) most likely
forms the rotation axis (= joint axis, pivot point or fulcrum; black circle; Fig. 3b), since
the ligaments form the connections of the 2" valvula with the anterior parts of the 2"4
valvifers and can only stretch to a limited extent. Van Meer et al. (2020) postulate that,
in the braconid D. longicaudata, the rotation axis lies directly anterior to the bulbs. In
addition, these authors have observed that, during terebra depression (towards an active
probing position), the lateral bulbs are pulled out of the socket-like anterior parts of
the 2" valvifers, which are pushed slightly apart. The ball-and-socket-like connection is
therefore assumed mainly to stabilize the terebra in its resting position. The antagonisti-
cally acting anterior 2° valvifer-2"' valvula muscle inserts at the processus musculares
and elevates the terebra, i.e. rotates it back upwards towards the resting position.

(2) Pro- and retraction of the 1* valvulae: The 1* valvifer, 2"! valvifer, and the
female T9 form a mechanical cluster of functionally interconnected elements (for de-
tailed functional models see fig. 5 of Eggs et al. 2018 and fig. 8 of van Meer et al.
2020). The dorsal and the antagonistically acting ventral T9-2™ valvifer muscle change
the relative position of the 2" valvifer and the female T9. Both of these structures are
connected with the 1* valvifer via the intervalvifer and the tergo-valvifer articulation
(Fig. 7c), respectively. Moreover, both are rotational joints that allow rotation in the
sagittal plane only. The 1* valvifer acts as a lever (Fig. 8) that transfers its movements to
the dorsal ramus of the 1* valvula (dr1; Figs 7c, h, 8). Contraction of the dorsal T9-2"4
valvifer muscle leads to an anterior rotation of the 1° valvifer around the intervalvifer
articulation. The 1% valvifer acts as a lever that transfers these movements to the dorsal
ramus of the 1* valvula, thus causing the 1* valvula to slide distally relative to the 2"¢
valvula, i.e. to protract. Vice versa, contraction of the antagonistic ventral T9-2™ valvi-
fer muscle leads to a posterior rotation of the 1“ valvifer, causing the 1“ valvula to slide
proximally to the 2Ӣ valvula, i.e. to retract (Eggs et al. 2018; van Meer et al. 2020).
The hook-shaped lobe of the 2"! valvifer (hsl; Fig. 7a, c, e) might allow a larger arc of
movement of the 1* valvifer and therefore a larger retraction distance of the 1* valvu-
lae. During the retraction of the 1* valvula, the dorsal ramus of the 1* valvula (dr1; Fig.
7c, h) can slide along the dorsal projection of the 2"¢ valvifer almost until the posterior

end of the hook-shaped lobe (hsl; Fig. 7a, c, e).

In the context of the described movements, the 1* valvifer acts as a one-armed class 3
lever (force arm smaller than load arm). In our lever model (Fig. 8), we use the 2" valvifer
(2vf; Fig. 1c) as a frame of reference. However, in reality, all involved cuticular elements
can move relative to each other. The anatomical inlever (c; Fig. 8) equals the distance
between the intervalvifer parca and the tergo-valvifer articulation (where the input

force is applied; F. Fig. 8). The distance between the intervalvifer

rotraction)? oes beracionte
articulation and eae » beginning of the dorsal ramus of the 1“ valvula at the anterodorsal
end of the 1* valvifer equals the anatomical outlever (d; Fig. 8). The ratio of effective

outlever (d’; Fig. 8) and the effective inlever (c’; Fig. 8) are indicative for the potential

Ovipositor of Habrobracon hebetor 91

maximum velocity, the mechanical deflection, and the amount of force transmission to
the 1* valvula. An increase of the d’:c’ ratio results in an increase of the potential maxi-
mum velocity and mechanical deflection but entails a smaller force output (F,

out protraction)’

beset ae Fig. 8) of the 1* valvulae. In resting position, the anatomical in- and outlever
are both very similar to their respective effective levers, thereby creating high torques at
the intervalvifer articulation and ensuring an optimal force transmission when pro- or
retracting the 1“ valvulae. During oviposition, the left and the right 1“ valvulae slide back
and forth alternately at a high frequency. These valvula movements are crucial for drilling
and precise egg laying (Vilhelmsen 2000; Cerkvenik et al. 2017; van Meer et al. 2020).

The shape of the 1* valvifer varies between the various hymenopteran superfamilies
(Oeser 1961). Ichneumonoid species such as the braconid H. /ebetor in the present
study possess a 1“ valvifer with a rounded compact shape (Snodgrass 1933; Eggs et
al. 2018), in contrast to the elongated and bow-shaped 1* valvifers of members of the
superfamily Chalcidoidea (Copland and King 1972a, 1972b, 1972c, 1973), the trian-
gularly shaped 1“ valvifers of Apis mellifera (Linnaeus, 1758) and other aculeate species
(Snodgrass 1933; Oeser 1961; Matushkina 2011; Matushkina and Stetsun 2016; Stet-
sun and Matushkina 2020; Graf et al. 2021), and the highly diverse 1* valvifers of ba-
sal hymenopterans (e.g. the robust-appearing 1* valvifers of Tentredinidae (Snodgrass
1933; Vilhelmsen 2000) or the triangular 1“ valvifers in some Xyelidae (Vilhelmsen
2000). The ecomorphological consequences of these morphological differences remain
to be explored in future systematic comparative analyses with respect to the parasitiza-
tion of other hosts in different substrates and habitats.

The two sensilla clusters found on the 2" valvifer of H. hebetor (sp, sr; Fig. 7b,
d—f, i). probably play an important role in monitoring the pro- and retraction of the
1* valvulae, since their accurate actuation is of major importance for successful egg
deposition (van Meer et al. 2020). Unlike in H. hebetor or V. canescens (Eggs et al.
2018), the sensilla patch at the intervalvifer articulation of other parasitoid wasps can
be extremely reduced, e.g. in Pteromalidae (Chalcidoidea) with only three single sen-
silla (Copland and King 1972b). The question remains as to whether both the density
and number of sensilla are linked to the importance of the control of the movements
involved in oviposition, and whether this correlates with the shape of the 1* valvifer.

Conclusion

All the cuticular elements of the ovipositor of Habrobracon hebetor play a crucial role for
successful oviposition. The 2" valvifer and the female T9 exhibit many muscle insertions,
the 1* valvifer acts as a lever that transmits movements to the 1“ valvulae, and the terebra
serves as a device for precise venom injection, host assessment, and accurate egg laying. All
the cuticular elements feature many distinct structures in addition to the microsculpture
that is crucial for the performance of these tasks. Our work also has shown that a 3D re-
construction based on a histological section series preserves useful information about the
exact morphology and position of inherent structures thereby enabling us to draw con-
clusions about their function. Future comparative examination of the inherent ovipositor

92 Michael Csader et al. / Journal of Hymenoptera Research 83: 73-99 (2021)

elements, their morphological structure, and the underlying mechanical and functional
aspects has the potential to increase our understanding of a putative key feature in the
evolution of parasitoid hymenopterans, a feature that probably has significantly impacted
the evolutionary success of braconid wasps (more than 18,000 described (Quicke 2015)
and about 43,000 estimated species (Jones et al. 2009)) and of parasitoid hymenopterans
in general (115,000 described and 680,000 estimated species (Heraty 2009)).

Acknowledgements

We thank Paavo Bergmann for valuable hints regarding histological techniques, Moni-
ka Meinert for assistance with SEM, Verena Pietzsch and James H. Nebelsick with the
Keyence digital microscope, Theresa Jones for linguistic corrections of the manuscript,
and Lars Vilhelmsen and Donald L. J. Quicke for their useful comments.

This work was funded by the German Research Foundation (DFG) as part of the
Transregional Collaborative Research Centre (SFB-TRR) 141 “Biological design and
integrative structures’ (project A03 ‘Inspired by plants and animals: actively actuated
rod-shaped structures exhibiting adaptive stiffness and joint-free kinematics’). The ar-
ticle processing charge was covered by the DFG and the Open Access Publishing Fund
of the University of Tiibingen.

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Appendix |

Ovipositor of Habrobracon hebetor

OF

Table Al. Morphological terms relevant to the hymenopteran ovipositor system. The terms (abbrevia-

tions used in this article in brackets) are used and defined according to the Hymenoptera Anatomy On-

tology Portal (HAO) (Yoder et al. 2010; Hymenoptera Anatomy Consortium 2021) and the according
Uniform Resource Identifiers (URJ) are listed.

Anatomical term definition / concept URI
(abbreviation)

1* valvifer (1vf) The area of the 1* valvifer-1* valvulae complex that is proximal to the aulax, bears the 9" | http://purl.obolibrary.

tergal condyle of the 1* valvifer and the 2™ valviferal condyle of the 1* valvifer and is | org/obo/HAO_0000338
connected to the genital membrane by muscle.

1* valvifer-genital The ovipositor muscle that arises from the posterior part of the 1* valvifer and inserts http://purl.obolibrary.
membrane muscle anteriorly on the genital membrane anterior to the T9-genital membrane muscle. org/obo/HAO_0001746

1* valvula, 1" The area of the 1* valvifer-1* valvulae complex that is delimited by the proximal margin | http://purl.obolibrary.
valvulae (1vv) of the aulax. org/obo/HAO_0000339

2™ valvifer (2vf) The area of the 2™ valvifer-2"¢ valvulae-3" valvulae complex that is proximal to the basal | http://purl.obolibrary.
articulation and to the processus musculares and articulates with the female T9. org/obo/HAO_0000927

2™ valvula (2vv) The area of the 2™ valvifer-2™ valvulae-3" valvulae complex that is distal to the basal http://purl.obolibrary.

articulation and to the processus musculares and is limited medially by the median body

org/obo/HAO_0000928

axis.
3" valvula, 3"! The area of the 2"¢ valvifer-3"valvulae complex that is posterior to the distal vertical http://purl.obolibrary.
valvulae (3vv) conjunctiva of the 2" valvifer-3" valvulae complex. org/obo/HAO_0001012
anterior 2" valvifer- | The ovipositor muscle that arises from the anterodorsal part of the 2™ valvifer and inserts | http://purl-obolibrary.
2™ valvula muscle subapically on the processus articularis. org/obo/HAO_0001166
anterior ridge of T9 | ‘The ridge that extends along the anterior margin of female T9 and receives the site of | http://purl.obolibrary.
(ar9) origin of the ventral and the dorsal T9-2" valvifer muscles. org/obo/HAO_0002182
anterior section of | The area of the dorsal flange of the 2 valvifer that is anterior to the site of origin of the | http://purl.obolibrary.
dorsal flange of 2" basal line. org/obo/HAO_0002173
valvifer
apodeme The process that is internal. http://purl.obolibrary.
org/obo/HAO_0000142
aulax (au) The impression that is on the 1* valvifer-1* valvula complex accommodates the rhachis. | http://purl.obolibrary.
org/obo/HAO_0000152
basal articulation The articulation that is part of the 2™ valvifer-2™ valvula-3" valvula complex and http://purl.obolibrary.
(ba) adjacent to the rhachis. org/obo/HAO_0001177
basal line of the 2"* | The line on the 2" valvifer that extends between the pars articularis and the dorsal flange | http://purl.obolibrary.
valvifer of 2"4 valvifer. org/obo/HAO_0002171
bulb (blb) The anterior area of the dorsal valve [composite structure of the fused 2"4 valvulae] that is} http://purl-obolibrary.
bulbous. org/obo/HAO_0002177
conjunctiva ‘The area of the cuticle that is more flexible than adjacent sclerites. http://purl.obolibrary.
org/obo/HAO_0000221
distal notch of the The notch that is distal on the dorsal valve [composite structure of the fused 2"4 http://purl.obolibrary.
dorsal valve (no) valvulae]. org/obo/HAO_0002179
dorsal flange of the | The flange that extends on the dorsal margin of the 2" valvifer. Part of the ventral T9-2"* | hetp://purl.obolibrary.
2"4 valvifer (df2) valvifer muscle attaches to the flange. org/obo/HAO_0001577
dorsal projection of | The projection that is located on the 2"¢ valvifer and corresponds to the proximal end of | http://purl.obolibrary.
the 2"¢ valvifer (dp2) the rhachis. org/obo/HAO_0002172
dorsal ramus of the | ‘The region that extends along the dorsal margin of the 1“ valvula and bears the aulax. | http://purl.obolibrary.
1* valvula (dr1) org/obo/HAO_0001579
dorsal T9-2" valvifer| The ovipositor muscle that arises along the posterodorsal part of the anterior margin of | http://purl.obolibrary.
muscle female T9 and inserts on the anterior section of the dorsal flanges of the 2"! valvifer. _ | org/obo/HAO_0001569
egg canal (ec) The anatomical space that is between the left and right olistheters. http://purl.obolibrary.
org/obo/HAO_0002191
female T9 (T9) The tergite that is articulated with the 1* valvifer and is connected to the 2" valvifer via | _http://purl.obolibrary.
muscles. org/obo/HAO_0000075
flange The projection that is lamella-like and is located on a rim, carina, apodeme or edge. http://purl.obolibrary.
org/obo/HAO_0000344
genital membrane ‘The conjunctiva that connects the ventral margins of the 2° valvifers arching above the | http://purl.obolibrary.

(gm)

2" valvula.

org/obo/HAO_0001757

98 Michael Csader et al. / Journal of Hymenoptera Research 83: 73-99 (2021)

Anatomical term
(abbreviation)
interarticular ridge
of the 1* valvifer

definition / concept

The ridge that extends along the posterior margin of the 1“ valvifer between the
intervalvifer and tergo-valvifer articulations.

URI

http://purl.obolibrary.
org/obo/HAO_0001562

(iar)

intervalvifer The articulation between the 1* valvifer and 2" valvifer. http://purl.obolibrary.
articulation (iva) org/obo/HAO_0001558

median bridge of the} The area that connects posterodorsally the 2" valvifers and is the site of attachment for | http://purl.obolibrary.
2"4 valvifers (mb2) the posterior T9-2™ valvifer muscle. org/obo/HAO_0001780

notal membrane ‘The conjunctiva that connects the medial margins of the 2"¢ valvulae. http://purl.obolibrary.
org/obo/HAO_0001733

notch ‘The part of the margin of a sclerite that is concave. http://purl.obolibrary.
org/obo/HAO_0000648

olistheter (oth) The anatomical cluster that is composed of the rhachis of the 2°‘ valvula and the aulax of | http://purl.obolibrary.
the 1* valvula. org/obo/HAO_0001103

ovipositor The anatomical cluster that is composed of the 1* valvulae, 2" valvulae, 3“ valvulae, 1* | hetp://purl.obolibrary.
valvifers, 2"! valvifers and female T9. org/obo/HAO_0000679

ovipositor apparatus ‘The anatomical cluster that is composed of the ovipositor, abdominal terga 8-10, http://purl.obolibrary.
abdominal sternum 7 and muscles connecting them. org/obo/HAO_0001600

ovipositor muscle The abdominal muscle that inserts on the ovipositor. http://purl.obolibrary.
org/obo/HAO_0001290

pars articularis / pars | The articular surface that is situated anteriorly on the ventral margin of the 2° valvifer_ | http://purl.obolibrary.
articulares and forms the lateral part of the basal articulation. org/obo/HAO_0001606

posterior 2" valvifer-| The ovipositor muscle that arises posteroventrally from the 2™ valvifer and inserts on the | http://purl.obolibrary.
2" valvula muscle processus musculares of the 2*¢ valvula. org/obo/HAO_0001815

processus articularis | The process that extends laterally from the proximal part of the 2"! valvula and forms the | http://purl.obolibrary.

/ processus
articulares
processus musculares
/ processus

muscularis

rhachis (rh)

ridge

sawtooth (st)

sclerite

sensillar patch of the
2" valvifer (sp)
sensillum

terebra (trb)

tergite

tergo-valvifer
articulation (tva)

median part of the basal articulation, and corresponds to the site of attachment for the
anterior 2" valvifer-2"¢ valvula muscle. The processus articularis is part of the sclerite.
The apodeme that extends dorsally from the proximal part of the 2°‘ valvula to the
genital membrane and receives the site of attachment of the posterior 2" valvifer-2"*

valvula muscle.

‘The ridge that extends along the ventral surface of the 2° valvula that is partially enclosed

by the aulax.

‘The apodeme that is elongate.

The process that is located along the ventral margin of the 1“ valvula of the dorsal margin

of the 2"¢ valvula.

‘The area of the cuticle that is less flexible than adjacent conjunctivae.

The patch that is composed of placoid sensilla adjacent to the intervalvifer articulation.
A sense organ embedded in the integument and consisting of one or a cluster of sensory
neurons and associated sensory structures, support cells and glial cells forming a single
organized unit with a largely bona fide boundary.

The anatomical cluster that is composed of the 1* and 2” valulae.

‘The sclerite that is located on the tergum.

The articulation that is located between the female T9 and the 1* valvifer and is
composed of the 9" tergal condyle of the 1* valvifer and the 1* valviferal fossa of the 9

org/obo/HAO_0001704

http://purl.obolibrary.
org/obo/HAO_0001703

http://purl.obolibrary.
org/obo/HAO_0000898
http://purl.obolibrary.
org/obo/HAO_0000899
http://purl.obolibrary.
org/obo/HAO_0001681
http://purl.obolibrary.
org/obo/HAO_0000909
http://purl.obolibrary.
org/obo/HAO_0001671
http://purl.obolibrary.
org/obo/HAO_0000933

http://purl.obolibrary.
org/obo/HAO_0001004

http://purl.obolibrary.
org/obo/HAO_0001005

http://purl.obolibrary.
org/obo/HAO_0001636

tergite.
valvillus (vlv) ‘The sclerite that articulates on the 1* valvula and projects into the egg/poison canal. http://purl.obolibrary.
org/obo/HAO_0001619
venom gland The gland reservoir that is between the 2" valvifers. http://purl.obolibrary.

reservoir of the 2"4

valvifer (vd)

org/obo/HAO_0002176

ventral ramus of the The area of the 2" valvifer-2"¢ valvula-3" valvula complex that bears the rhachis. http://purl.obolibrary.
2” valvula org/obo/HAO_0001107
ventral T9-24 The ovipositor muscle that arises from the lateral region of female T9 and inserts along | http://purl.obolibrary.

valvifer muscle

the posterior part of the dorsal flange of the 2"¢ valvifer.

org/obo/HAO_0001616

Ovipositor of Habrobracon hebetor 99

Supplementary material |

Video S1

Authors: Michael Csader, Karin Mayer, Oliver Betz, Stefan Fischer, Benjamin Eggs

Data type: Video file (mp4)

Explanation note: Animation of the rotated segmented 3D reconstruction of the
terebra of Habrobracon hebetor.

Copyright notice: This dataset is made available under the Open Database License
(http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License
(ODDbL) is a license agreement intended to allow users to freely share, modify, and
use this Dataset while maintaining this same freedom for others, provided that the
original source and author(s) are credited.

Link: https://doi.org/10.3897/jhr.83.64018.suppl1

Supplementary material 2

Video S2

Authors: Michael Csader, Karin Mayer, Oliver Betz, Stefan Fischer, Benjamin Eggs

Data type: Video file (mp4)

Explanation note: Animation of the rotated segmented 3D reconstruction of the prox-
imal region of the terebra of Habrobracon hebetor (cf. Fig. 3), highlighting the 1“
and 2Ӣ valvulae, the ligaments, and the duct of the venom gland.

Copyright notice: This dataset is made available under the Open Database License
(http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License
(ODDbL) is a license agreement intended to allow users to freely share, modify, and
use this Dataset while maintaining this same freedom for others, provided that the
original source and author(s) are credited.

Link: https://doi.org/10.3897/jhr.83.64018.suppl2