-
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: [email protected]†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:[email protected]://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
Eggs et al. BMC Zoology (2018) 3:12 Page 2 of 25
-
a
e
d
c
b
200 m
1000 m
200 m 200 m
200 m
2vv
3vv
2vf
1vf
T9
2vf
3vv
T9
1vf
2vf
1vv
dr1
dr1
iva
tva
T9 T10
T8 2vf 1vf
T6
T7
1vv
e
1vv
3vv
2vv
1vv
d
trb
1vf
50 m
10 m
T9
T9 1vf
2vf
dr1
2vf
iar
tva
iva
iva
sp
f g
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
Eggs et al. BMC Zoology (2018) 3:12 Page 3 of 25
-
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
cs
2vv
1vv
no
b
c
a
d
g 5 m 5 m
2vv
1vv
1vv rh
au au
no
ct
scts sc
sc
e 20 m
vlv au
rh
20 m
10 m
50 m
10 m rh sc
f
b
st
2vv 2vv
1vv
1vv 1vv
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
Eggs et al. BMC Zoology (2018) 3:12 Page 4 of 25
-
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
Eggs et al. BMC Zoology (2018) 3:12 Page 5 of 25
-
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
1
a
c
e
b
d
f h
2vf
2
2
tva
iva
1
translational joint
rotation joint (tva)
rotation joint (iva)
j i
g
ca
1vf
T9
T9 1vf 2vf
m1
m2
m3
m4a m4b
m5
m6
2vv
1vv
tm4b
iar
1vf
dr1
dp2
oth
2vv
1vv ec
dr1
dp2 hsl
ca blb
asdf bl psdf
mb2
af9
blb
ba
2vf
dr1
1vf
T9
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
Eggs et al. BMC Zoology (2018) 3:12 Page 6 of 25
-
a 2vf 1vf T9
2vv
b
m1
f m5
F5
d
m6
m4a m4b
F4
l F2
F3 ba
i
ba 2vf
1vf dr1
iva
tva
T9 blb
2vv
1vv
2
1
F4 3
6
4 7
6
3
m4a/b
m5
m2
m3
F5
F2
F3
c 1vv
3
4
5
3 4
e 6
7
8
6
7
m2
m3
1vv 2vv
g
1
2
F2
F3
m2
m3
h
pra
prm
vd
ba
a'
b'
a .
a = 66 m b = 84 m
F3
F2 b
1vv
2vv
m
ba n trb 3vv
c'
F4x
F5x
d'
c
.
.
k .
c = 103 m
F1vv4
tva
iva
dr1
F1vv5
F1vv5 F1vv4
1vf
j
F5x F4x
F4
F5 iva
tva
Fig. 5 (See legend on next page.)
Eggs et al. BMC Zoology (2018) 3:12 Page 7 of 25
-
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