Arthropod Structure & Development 50 (2019) 15e23

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Arthropod Structure & Development

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The sperm pump and genital coupling of Panorpodes kuandianensis

(Mecoptera: Panorpodidae)
Xin Tong, Bao-Zhen Hua*
Key Laboratory of Plant Protection Resources and Pest Management, Ministry of Education, College of Plant Protection, Northwest A&F University, Yangling,
Shaanxi, 712100, China

a r t i c l e i n f o a b s t r a c t

Article history: Males of Panorpodidae possess a special sperm pump, through which they directly transfer seminal fluid
Received 8 December 2018 to the female spermatheca. However, the sperm pump has not been studied in Panorpodes to date. Here,
Accepted 14 March 2019 the structure of the sperm pump and the internal coupling of genitalia were investigated in the short-
faced scorpionfly Panorpodes kuandianensis Zhong, Zhang, and Hua, 2011 using light and scanning
electron microscopy. The sperm pump mainly consists of a piston, a pumping chamber, the anterior
Keywords:
region of the aedeagal complex, the posterior region of the ejaculatory sac, and associated muscles. The
Antliophora
piston as a propulsion apparatus is controlled by levator and depressor muscles. Its posterior region
Copulation
Copulatory mechanism
connects dorsally to the aedeagus via a joint. The pumping chamber is located between the piston and
Genitalia the aedeagus. The dorsal and ventral parameres were attached by retractor muscles. During copulation,
Mating the male phallotreme connects to the female copulatory pore to transfer sperm. Male gonostyli and
Sperm ejaculation parameres grasp the female to restrict the genitalia movement and impede her medigynium from
retreating. The sperm ejaculatory mechanism of Panorpodes and the evolution of sperm transfer mode in
insects are briefly discussed based on the structure of the sperm pump and the internal coupling of
genitalia.
© 2019 Elsevier Ltd. All rights reserved.

1. Introduction Sperm pumps are found in all other families of Mecoptera

Sperm transfer is one of the most important processes in animal
mating behaviors and is usually achieved by the physical contact of
male and female genitalia (Arnqvist, 1997; Crudgington and Siva-
Jothy, 2000; Simmons, 2001; Gwynne, 2009; Hotzy and Arnqvist,
2009; Chapman, 2013). Use of a spermatophore for sperm trans-
mission is widespread in arthropods, including scorpions (Jacob et al.,
2004), spiders (Legendre, 1981), and insects (Lewis et al., 2014).
However, in the Endopterygota (Insecta), most males of Mecoptera,
Siphonaptera, and Diptera transfer seminal fluid directly into the
female reproductive tract (Kristensen, 1975); this highly effective
ejaculatory mode is confirmed to be achieved by a propulsion
apparatus or a sperm pump (Gerber, 1970; Mann, 1984; Hünefeld and
Beutel, 2005; Gao and Hua, 2015). Owing to the presence of the
sperm pump, these three orders are grouped into the superorder
Antliophora (Kristensen, 1975). The sperm pump of Antliophora is
suggested to be derived independently in , Diptera, and Mecoptera
(Mickoleit, 2008; Boudinot, 2018).
* Corresponding author.
E-mail address: huabzh@nwafu.edu.cn (B.-Z. Hua).
except Boreidae (Mickoleit, 2008; Willmann, 1981), the male of
which transfers sperm via a spermatophore (Mickoleit, 1974). The
sperm pumping complex in Nannochoristidae is likely the inter-
mediate between Boreidae and other families, because it lacks a
piston but has an oblong tegmen as the device for pressing out
sperm (Mickoleit, 2008; Boudinot, 2018). In Bittacidae the piston of
the sperm pump is fused with a piston-carrying sclerite, and the
pumping chamber is enclosed by the genital fold (Gao and Hua,
2015). The movement of the sperm pump is controlled by levator
muscles of the piston and two antagonistic muscles attached to the
ostial sclerite (Willmann, 1982; Gao and Hua, 2015; Boudinot,
2018). The well-studied sperm pump of Panorpidae is derived
from the genital fold and is largely composed of a piston, a pumping
chamber and the posterior region of the ejaculatory sac (Grell,
1942; Hünefeld and Beutel, 2005; Shen and Hua, 2013). The
sperm transfer is primarily controlled by levator and depressor
muscles of the piston. The structure of the sperm pump, however,
remains undocumented in other groups of Mecoptera, including
Panorpodidae.

https://doi.org/10.1016/j.asd.2019.03.002
1467-8039/© 2019 Elsevier Ltd. All rights reserved.
16 X. Tong, B.-Z. Hua / Arthropod Structure & Development 50 (2019) 15e23
The small family Panorpodidae is the sister group of Panorpidae
in Mecoptera (Willmann, 1987; Friedrich et al., 2013), containing 13
extant species in two genera: Brachypanorpa and Panorpodes
(Pollmann et al., 2008; Hu and Hua, 2016). The mating pattern of
Panorpodes is interesting in that the mating position changes from a
V-shape to tail-to-tail with pulling behavior and the female
abdominal segments VIIeIX are provisionally twisted by 180
(Tong
et al., 2017). The copulatory mechanism of Panorpodes was
described in detail by Tong et al. (2017) based on the morphological
observation of genitalia and freeze-fixed pairs in copula. However,
the internal coupling of genitalia is difficult to examine, because the
genital internal components are usually shielded from view by
external structures. The structure of the sperm pump and the
coupling of genitalia have not been satisfactorily elucidated in
Panorpodes to date.
In this study, we investigated the morphology and histology of
the genitalia and freeze-fixed pairs in copula of the short-faced
scorpionfly Panorpodes kuandianensis (Zhong, Zhang, and Hua,
2011 et al., 2011) using light and scanning electron microscopy to
describe the structure of the sperm pump and to reveal the internal
connection of male and female genital components during
copulation.
2. Materials & methods
2.1. Adult collecting
Live adults of P. kuandianensis were collected at the Quanshan
Forest Park (41
060 N, 125
020 E, elev. 650e800 m), Kuandian
County, Liaoning Province in northeastern China from July to early
August in 2015 and 2016.
2.2. Adult rearing
The adults collected were transported to the laboratory in the
Quanshan Forest Park. The rearing method followed Zhong and Hua
(2013) and Jiang et al. (2014). Ten males and 10 females were
maintained in one gauze cage (40 cm  40 cm  60 cm) that
contained twigs with leaves to simulate the habitat. Each cage was
supplied with flowers and five drops of honey every day to prevent
low nutritional status. Absorbent cotton containing water was
provided in a Petri dish.
2.3. Freeze-fixation of pairs in copula
Adults were observed every 30 min from daybreak to midnight.
Copulating pairs were frozen immediately by carbon dioxide
aerosol spray compressed in a hydraulic can. Then the shock-frozen
pairs were fixed in Carnoy's solution (100% ethanol: glacial acetic
acid ¼ 3:1, v/v) for 24e48 h and afterwards preserved in 75%
ethanol.
2.4. Scanning electron microscopy (SEM)
The male genitalia fixed in Carnoy's solution were dissected
under a Nikon SMZ1500 microscope (Nikon, Tokyo, Japan). The
dissected genital structures were ultrasonically cleaned in 75%
ethanol for 1 min to remove dust, dehydrated in a graded ethanol
and tert-butyl alcohol series (ethanol: 80% for 15 min, 90% for
20 min, and 100% for 30 min twice; mixtures of ethanol and tert-
butyl alcohol: 3:1, 1:1, and 1:3 for 15 min, pure tert-butyl alcohol
for 15 min twice), and freeze-dried in a critical point-drier for 3 h.
Then the samples were coated with gold in a sputter coater and
observed in a Hitachi S-3400N scanning electron microscope
(Hitachi, Tokyo, Japan) at 5 kV.
2.5. Light microscopy
Live adults were fixed in Dietrich's fluid (formalin: 95% ethanol:
glacial acetic acid: distilled water ¼ 6:15:1:80, v/v) and dissected
under a Nikon SMZ1500 microscope (Nikon, Tokyo, Japan). Photo-
graphs were taken with a QImaging Retiga 2000R Fast 1394 Digital
Camera (QImaging, Surrey, Canada) attached to the microscope and
were stacked with Syncroscopy Auto-Montage software (Syncro-
scopy, Cambridge, UK).
For histological observations, male genitalia and freeze-fixed
pairs in copula were rinsed six times in phosphate buffered saline
(0.1 M, pH 7.2) and dehydrated through a graded ethanol series
following Wang and Hua (2018). Then the samples were infiltrated
in mixtures of ethanol and LR White resin (3:1 for 2 h, 1:1 for 4 h,
and 1:3 for 12 h), subsequently with pure LR White resin for 24 h at
18
C twice. Finally, the samples were embedded in pure LR White
resin and polymerized at 55
C for 48 h (Liu and Hua, 2018).
After complete polymerization, the samples were cut into
1.0 mm thick sections with a glass knife on the Leica EM UC7 ul-
tramicrotome (Leica, Nussloch, Germany) and stained with 0.5%
toluidine blue. The sections were observed under a Nikon Eclipse
80i light microscope (Nikon, Tokyo, Japan) and photographs were
taken with a Nikon DS-Fil digital camera (Nikon, Tokyo, Japan)
attached to the microscope.
2.6. Terminology
The terms used for male reproductive system follow Potter
(1938). The terminology used for male and female genitalia
mainly follows Zhong et al. (2011), and that used for structures of
the sperm pump comes from Shen and Hua (2013). The terms used
for muscles of the male genitalia follow Boudinot (2018).
3. Results
Male and female genitalia of P. kuandianensis couple with each
other to transfer sperm during copulation (Fig. 1). The female
genitalia are composed of a sclerotized medigynium (genital plate,
term from Mickoleit, 1976) and a lingulate subgenital plate (the
ninth sternite). The medigynium is located in the genital chamber
between the subgenital plate and the ninth tergum.
The male genitalia are composed of a dorsal epandrium (the
ninth tergite), a ventral hypandrium (the ninth sternite), a pair of
Fig. 1. Pair in copula of Panorpodes kuandianensis (Photo by Lu Jiang).
 X. Tong, B.-Z. Hua / Arthropod Structure & Development 50 (2019) 15e23 17

two-segmented gonopods (gonocoxite and gonostylus), and a
central aedeagal complex (parameres and aedeagus).
The sperm pump of P. kuandianensis is located between the male
aedeagal complex and the gonopods. It is derived from the ejacu-
latory sac and the genital fold. The sperm pump mainly consists of a
piston (P), the anterior part of the aedeagal complex (Pm and Ae),
the posterior region of the ejaculatory sac (ES), a pumping chamber
(PC), and associated muscles (Fig. 2).
3.1. The piston
The piston (P) is a heavily sclerotized propulsion apparatus with
paired lateral and front processes (LPr and FP) (Figs. 2 and 3). Its
distal part connects dorsally with the apical part of the aedeagus
(Ae) (Fig. 3A, D). The piston moves upwards and forwards recip-
rocally to seal and open the pumping chamber from the front by the
control of two antagonistic muscles through the joint of the piston
and the aedeagus.
3.2. The aedeagal complex
The aedeagal complex is composed of the aedeagus (Ae) and the
parameres (Pm). The distal parts of the aedeagus and the para-
meres are associated intimately (Fig. 2). The sclerotized structures
of the aedeagus consist of a pair of dorsal valves and a pair of
ventral valves (VV) (Fig. 3C, D). The paramere consists of a basal
stalk (BSP), a lateral process (LP), and dorsal and ventral parameres
(DPm and VPm) (Fig. 2). The dorsal and ventral parameres are
attached by paired muscles (Fig. 2B).
3.3. The ejaculatory sac
The male reproductive system of P. kuandianensis consists of a
pair of testes, the paired epididymides, the paired vasa deferentia
(VD), a seminal vesicle with mesadenia, and an ejaculatory sac (ES)
without ectadenia (Fig. 2A). The ejaculatory sac connects anteriorly
with the paired vasa deferentia, which are separated by depressor
muscles of the piston (DMP) and curve ventrally to form a Z-shape
(Fig. 2A). The ejaculatory sac consists of a central lumen (Lu) and a
symmetric sac (SS). At the anterior region, the ejaculatory sac is
small and all components are pressed together to cause the central
lumen to become narrower (Fig. 4A, B). At the median region, the
sac is plump and the lumen is relatively wide (Fig. 4C, D). At the
posterior region, the sac is compressed (Fig. 5AeD) with the lumen
decreasing gradually to form a conical cavity, which opens poste-
riorly into the pumping chamber.
3.4. The pumping chamber
The pumping chamber (PC) is a triangular cavity and is located
between the aedeagus (Ae) and the piston (P) (Figs. 3B, C and 5E, F).
The pumping chamber is closed by the membranes connected with
the ejaculatory sac, the piston, and the aedeagus. It opens poste-
riorly between the dorsal and ventral valves of the aedeagus
(Figs. 3D and 5F).
3.5. The associated muscles
Two antagonistic muscles (levator and depressor muscles) are
attached to the piston. The levator muscles of the piston (LMP)
originate on the dorsal cuticle of the gonocoxites perpendicularly
and are inserted on the lateral processes of the piston (LPr)
(Figs. 2B, 3B and 5D, E). The depressor muscles of the piston (DMP)
originate on the dorsal cuticle of the basal region of the gonocoxites
and are inserted on the front processes of the piston (FP) (Figs. 2, 3B
and 4). The paired retractor muscles are attached between the
dorsal and ventral parameres (RMDV). They originate from the
dorsal region of the dorsal parameres (DPm) and are inserted on
the dorsal region of the ventral parameres (VPm) (Fig. 2B).
Two antagonistic muscles (protractor and retractor muscles) are
attached to the basal stalk of the parameres (BSP). The protractor
muscles of parameres (PMP) originate from the ventral cuticle of the
basal part of the gonocoxites and are inserted on the basal stalk of
the parameres (Fig. 2A, B). The retractor muscles of parameres (RMP)
originate from the ventral cuticle of the gonocoxites near the
gonostyli and are inserted onto the basal stalk of the parameres
(Fig. 2A). Two pairs of abductor and adductor muscles are attached to
the paired gonostyli. The abductor muscles of gonostyli originate
from the dorsal surface of the gonocoxites and are inserted on the
dorsal surface of the gonostyli. The adductor muscles (AdM) fill most
volume of the gonocoxites ventrally and laterally and are inserted on
the ventral surface of the gonostyli (Figs. 4 and 5). Based on the
investigation of the structure of the sperm pump, a schematic dia-
gram of the sperm pump of P. kuandianensis is drawn (Fig. 6).
3.6. Internal coupling of genitalia
During copulation, the male inserts his aedeagus (Ae) into the
female genital chamber (Fig. 7), which can be stretched to deform

Fig. 2. Light micrographs of the associated structures of the sperm pump in Panorpodes kuandianensis. (A) The ejaculatory sac, the aedeagal complex, the piston and associated
muscles in lateral view. (B) The same with the ejaculatory sac removed to show the retractor muscles between dorsal and ventral parameres. Ae, aedeagus; BSP, basal stalk of
paramere; DMP, depressor muscle of the piston; DPm, dorsal paramere; ES, ejaculatory sac; FP, front process of the piston; LMP, levator muscle of the piston; LP, lateral process of
paramere; LPr, lateral process of the piston; P, piston; PMP, protractor muscle of the paramere; RMDV, retractor muscle between dorsal and ventral parameres; RMP, retractor
muscle of the paramere; VD, vas deferens; VPm, ventral paramere. Scale bars: (A) and (B) ¼ 0.5 mm.
18 X. Tong, B.-Z. Hua / Arthropod Structure & Development 50 (2019) 15e23

Fig. 3. SEM micrographs of the associated structures of the sperm pump in Panorpodes kuandianensis. (A) The aedeagus, the piston, and the muscles in dorsal view, * showing the
joint of the piston and the aedeagus. (B) The same in ventral view to show the pumping chamber and the lateral and front processes of the piston connected to the levator and
depressor muscles. (C) The aedeagus and the piston in ventral view to show the pumping chamber and the phallotreme. Inset shows the piston. (D) The same in lateral view, *
showing the joint of the piston and the aedeagus. Ae, aedeagus; DMP, depressor muscle of the piston; DP, dorsal process of the aedeagus; DPm, dorsal paramere of the aedeagus; FP,
front process of the piston; LMP, levator muscle of the piston; LP, lateral process of the paramere; LPr, lateral process of the piston; P, piston; PC, pumping chamber; Pt, phallotreme;
VV, ventral valve. Scale bars: (A) and (B) ¼ 0.3 mm; (C) and (D) ¼ 0.2 mm.

(Fig. 7CeF). The male aedeagus is surrounded completely by the
female genital chamber (Fig. 7). The lateral processes (LP) of the
parameres are restricted by the pleural membranes (LM) of the
genital chamber (Fig. 7A, B). The male gonostyli with finger-like
basal processes and lobe-like basal teeth (BT), clasp the interseg-
mental membrane (IM) of segments VIII and IX to cause it to
become narrower (Fig. 7). The paired basal teeth (BT) of male
gonostyli block both sides of the anterior region of the female
medigynium (Mg) (Fig. 7E, F). The posterior region of the dorsal
parameres (DPm) grasps the distal part of the medigynium
(Fig. 7DeF). The male aedeagus (Ae) couples physically with the
female medigynium (Mg) (Fig. 7E, F) with the distal part of the
aedeagus engaged with the rear region of the medigynium
(Fig. 7DeF). At this moment, the male phallotreme (Pt) perfectly
connects to the female copulatory pore (Fig. 7D, E).
4. Discussion
The sperm pump of P. kuandianensis consists of a piston, a
pumping chamber, the anterior region of the aedeagal complex, the
posterior portion of the ejaculatory sac, and associated muscles.
During copulation, the male phallotreme connects to the female
copulatory pore. Male gonostyli and parameres grasp the female
genital segment and medigynium to restrict the genitalia move-
ment. The sperm pump of Panorpodes is distinctly different from
that of Bittacidae (Willmann, 1982; Gao and Hua, 2015; Boudinot,
2018), but is very similar to that of Panorpidae (Grell, 1942;
Willmann, 1981; Hünefeld and Beutel, 2005; Mickoleit, 2008;
Shen and Hua, 2013; Boudinot, 2018).
The piston is controlled by levator and depressor muscles in
Panorpodes, but is controlled only by levator muscles in Bittacidae
(Willmann, 1982; Gao and Hua, 2015; Boudinot, 2018). Probably
replacing the function of the depressor muscles of the piston, two
antagonistic muscles (retractor and protractor) of Bittacidae attach
to the ostial sclerite, which serves as the discharge valve to open
and close the pumping chamber (Gao and Hua, 2015). The sperm
pump of Panorpodes, however, only bears a pair of retractor muscles
attached between the dorsal and ventral parameres. The con-
tracting and relaxing of the retractor muscles may indirectly pull
the aedeagus to move ventrally and dorsally to influence the
pumping chamber. The activity of the sperm pump in Panorpodes is
likely to be promoted by the combination of the levator and
depressor muscles of the piston and the retractor muscles between
dorsal and ventral parameres.
 X. Tong, B.-Z. Hua / Arthropod Structure & Development 50 (2019) 15e23 19

Fig. 4. Series of cross-section of male genitalia of Panorpodes kuandianensis from anterior to middle regions. (A) and (B) Basal region of the male genitalia to show the anterior part
of the ejaculatory sac, the vas deferens, and the depressor muscle. (C) and (D) Middle region of the male genitalia to show the middle part of the ejaculatory sac, the vas deferens,
and the depressor muscle. AdM, abductor muscles of gonostyli; DMP, depressor muscle of the piston; ES, ejaculatory sac; FP, front process of the piston; Lu, lumen; SS, symmetric
sac; VD, vas deferens. Scale bars ¼ 0.2 mm.

The sperm pump of Panorpodes is morphologically similar to
that of Panorpidae in that the piston bears levator and depressor
muscles, and the dorsal and ventral parameres are attached by
retractor muscles (Grell, 1942; Willmann, 1981; Mickoleit, 2008;
Hünefeld and Beutel, 2005; Shen and Hua, 2013; Boudinot, 2018).
The retractor muscles between parameres are easily neglected in
the sperm pump of Panorpidae (Hünefeld and Beutel, 2005; Shen
and Hua, 2013). However, they may also function to control the
aedeagal complex of Panorpidae and indirectly control the pump-
ing chamber to fit in with the posterior of the ejaculatory sac
(Mickoleit, 2008; Boudinot, 2018). Similar sperm pumps may pro-
vide additional evidence to support the close relationship between
Panorpodidae and Panorpidae.
However, the ejaculatory sac of Panorpodes is distinctly larger
than that of Panorpidae. The ectadenia in accessory glands are
usually well-developed in Panorpidae (Shen and Hua, 2013; Lyu
and Hua, 2017), but are lacking in Panorpodes. Accessory glands
(including ectadenia and mesadenia) are important abdominal
gland systems (Leopold, 1976; Chapman, 2013) and their secretions
can influence the reproductive behavior of some insects (Gillott,
2003; Baldini et al., 2012; Yu et al., 2014; Yamane et al., 2015). In
Mecoptera the secretions of ectodermal glands are considered to
have two functions: 1) providing energy to the male whose
abdominal segments VIIeVIII are elongated and constricted (Lyu
and Hua, 2017), because the liquid sperm need to pass through
long vasa deferentia; 2) offering lubricant to the sperm pump to
prevent it from damage, for the piston as a reciprocating plunger
waves at a high frequency during copulation (Shen and Hua, 2013;
Lyu and Hua, 2017). Lacking ectadenia in male Panorpodes is
probably related to the abdomen and the ejaculatory sac. The
abdominal segments VIIeVIII are shortened without constriction in
male Panorpodes (Zhong et al., 2011). Thus, the liquid sperm passing
through vasa deferentia may require lower energy. The ejaculatory
sac is considered as a secretory organ in panorpids (Shen and Hua,
2013), and is distinctly larger in Panorpodes. Therefore, secretions of
the ejaculatory sac in Panorpodes are likely strong enough to pro-
vide sufficient energy for sperm transfer and to offer lubricant to
protect the sperm pump. The ejaculatory sac of Panorpodes may
replace the function of ectadenia in accessory glands.
Maintaining the stability of the connection to the female is
critical to sperm transfer. Although the internal process is often
difficult to observe directly, it is conceivable that males can evolve
various strategies to enhance their fertilization success (Gwynne,
2008; Hotzy and Arnqvist, 2009; Chapman, 2013; Uhl et al., 2014;
Wulff and Lehmann, 2016; Woller and Song, 2017; Wulff et al.,
2018). For example, the male of seed beetles usually utilize the
endophallus barbs to anchor the female genital tract to ensure that
genital coupling is established tightly (Crudgington and Siva-Jothy,
2000; Ro €nn et al., 2007; Hotzy and Arnqvist, 2009). However,
panorpid males lack the endophallus and transfer liquid sperm
directly through the male aedeagus coupled with the female
medigynium (Fig. 7) (Ma et al., 2010; Tong et al., 2017, 2018). This
20 X. Tong, B.-Z. Hua / Arthropod Structure & Development 50 (2019) 15e23

Fig. 5. Series of cross-section of male genitalia of Panorpodes kuandianensis from middle to posterior regions. (A) Posterior part of the male genitalia to show the ejaculatory sac and
the depressor muscle connected to the front process of the piston. (B) and (C) Posterior part of the male genitalia to show the piston and the ejaculatory sac. (D) and (E) Posterior
region of the male genitalia to show the piston, the pumping chamber, the levator muscle. (F) Posterior region of the male genitalia to show the aedeagus and the pumping chamber.
AdM, abductor muscle of the gonostylus; DMP, depressor muscle of the piston; DPm, dorsal paramere; ES, ejaculatory sac; FP, front process of the piston; LMP, levator muscle of the
piston; LP, lateral process of the paramere; P, piston; PC, pumping chamber. Scale bars ¼ 0.2 mm.

kind of copulation is usually very unstable. Non-genital grasping
structures (e.g., the notal organ and anal horn) and genital claspers
(including epandrium, hypandrium, and basal process on gono-
stylus) derived in Panorpidae may play an important role in helping
males to maintain copulation and obtain advantage in sperm
competition (Mickoleit, 1971; Kock et al., 2009; Zhong and Hua,
2013; Zhong et al., 2015a, b; Tong et al., 2018). However, abdom-
inal claspers are lacking in Panorpodes males. Alternatively, the
well-developed basal processes and basal teeth on gonostyli grasp
the female genital segment tightly. The male of P. kuandianensis
even can change the mating position and keep on pulling the mate
during copulation (Tong et al., 2017). The well-developed gonostyli
in Panorpodidae may functionally replace the abdominal grasping
structures of Panorpidae, both being adaptations for controlling
female movement and obtain advantage in sperm competition.
Combined with the investigation of the sperm pump and the
internal coupling of genitalia, the sperm ejaculatory mechanism of
P. kuandianensis is inferred as follows. When depressor muscles of
the piston contract, the piston is pulled anteriorly, compressing the
apical and median regions of the ejaculatory sac. Meanwhile,
retractor muscles between dorsal and ventral parameres contract
to indirectly pull the aedeagus ventrally, leading to the anterior
region of the pumping chamber connecting to the posterior region
of the ejaculatory sac. Thus, the sperm stored in the ejaculatory sac
is transferred into the pumping chamber. Then levator muscles of
the piston contract to pull the piston to move upwards. The
retractor muscles between dorsal and ventral parameres relax to
allow the aedeagus to move dorsally. In this case the rear region of
the piston can seal the pumping chamber from the front. Because of
this, the liquid sperm in the pumping chamber is rapidly ejaculated
into the female spermathecal duct through the connection of the
phallotreme to the copulatory pore. The basal processes and basal
teeth on male grasp the anterior region of the female genital
segment to restrict the genitalia movement. The dorsal parameres
 X. Tong, B.-Z. Hua / Arthropod Structure & Development 50 (2019) 15e23 21

Fig. 6. Schematic diagram of the sperm pump of Panorpodes kuandianensis. AE, aedeagus; BSP, basal stalk of paramere; DMP, depressor muscle of the piston; DP, dorsal process;
DPm, dorsal paramere; ES, ejaculatory sac; LMP, levator muscle of the piston; OES, opening of the ejaculatory sac; P, piston; PC, pumping chamber; PMP, protractor muscle of the
paramere; RMDV, retractor muscle between dorsal and ventral parameres; RMP, retractor muscle of the paramere; SPG, sex pheromone gland; VD, vas deferens; VPm, ventral
paramere; VV, ventral valve. Scale bars ¼ 0.5 mm.

Fig. 7. Section series of coupling genitalia of Panorpodes kuandianensis, with the female on the left and colored in purple. (A) Ventral portion of the coupling genitalia to show the
narrow female intersegmental membrane and the piston. (B) The same to show the lateral process of the paramere stuck by the pleural membrane of the genital chamber. (C) The
same to show the pumping chamber. (D) The male aedeagus is engaged with the female medigynium. The phallotreme connects to the copulatory pore. The aedeagus is clung by
the female genital chamber. (E) Dorsal portion of the coupling genitalia to show the distal part of the dorsal paramere grasped the rear region of the medigynium and the basal teeth
block both sides of the medigynium. (F) The same to show the basal teeth and the female pleural membranes of the genital chamber. Ae, aedeagus; BT, basal tooth; DPm, dorsal
paramere; Gcx, gonocoxite; Gst, gonostylus; IM, intersegmental membrane; LM, lateral membrane; LP, lateral process of paramere; Mg, medigynium; P, piston; PC, pumping
chamber; Pt, phallotreme; VV, ventral valve. Scale bars ¼ 0.1 mm.
22 X. Tong, B.-Z. Hua / Arthropod Structure & Development 50 (2019) 15e23
and the basal teeth control the female medigynium to impede it
from retreating. The piston moves reciprocally to ejaculate sperm
till the male loses control of the female.
Variation in sperm ejaculation across insects is almost certainly
the result of evolutionary adaptation. Insects are considered to be
derived from marine arthropods, which release sperm directly into
water (Regier et al., 2010). Therefore, as an adaptation to terrestrial
life, insects evolve spermatophores to protect sperm and maintain
the humid environment (Klowden, 2009). For primitive wingless
insects (Archaeognatha and Zygentoma) and some bushcrickets of
Tettigoniidae (Orthoptera), their males indirectly transfer sper-
matophores, which are placed on the substrate (Gwynne, 2009;
Klowden, 2009) or on the female abdomen (Alexander and Otte,
1967; Lehmann, 2012). This mode of sperm transfer may cause
the males to have limited control over spermatophores and bear a
great risk to waste sperm. For most pterygote insects, the males
directly transfer spermatophores through physical contact of male
€nger and Naumann, 1998; Arnqvist and
and female genitalia (Fa
Rowe, 2002; Wulff et al., 2015). This may help the males control
spermatophores and benefit them, such as by hurting the mate,
thus reducing reproductive capacity, or by removal of the rival's
spermatophores to reduce sperm competition (Gage, 1992; Hayashi
and Tsuchiya, 2005; Hotzy and Arnqvist, 2009; Foraita et al., 2017).
For many Neoptera (such as Antliophora and Heteroptera), their
males make more efficient mechanical adaptation in genitalia to
directly transfer liquid sperm during copulation (Kinzelbach, 1971;
Hennig, 1973; Cheetham, 1988; Hünefeld and Beutel, 2005; Shen
and Hua, 2013; Gao and Hua, 2015; Boudinot, 2018). This sperm
transfer mode is likely to benefit the male to directly disturb the
rival's sperm and help the male control the volume of the sperm
transfer to reduce resource wasting. Sperm competition, resource
limitation, and the better control of sperm may all promote the
evolution of direct sperm transfer.
As technology improves, a number of approaches have been
developed to examine the internal structure of genitalia such as
micro-CT scanning for 3D reconstruction (Willkommen et al., 2015;
Wulff et al., 2015; Antunes-Carvalho et al., 2017; Helm et al., 2018)
and X-ray cineradiography (Wulff et al., 2017). Nevertheless, we
conducted only histological serial sections to explain the structure
of sperm pump and the internal genital connection. The copulatory
mechanism may be more directly perceived through a 3D recon-
struction of copulas and genitalia.
Acknowledgments
We are grateful to Lu Jiang for providing a live photo of Panor-
podes kuandianensis in copulation. We also thank Lu Liu for the help
in semithin section. Our special thanks go to three anonymous
reviewers for valuable comments and suggestions in the revision of
the manuscript. This research was funded by the National Natural
Science Foundation of China (grant no. 31672341).
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