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Animal behaviour
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Female genital mutilation and monandry
in an orb-web spider
Kensuke Nakata
Research
Cite this article: Nakata K. 2016 Female
genital mutilation and monandry in an
orb-web spider. Biol. Lett. 12: 20150912.
http://dx.doi.org/10.1098/rsbl.2015.0912
Received: 30 October 2015
Accepted: 3 February 2016
Subject Areas:
behaviour
Keywords:
genitalia, sexual conflict, epigynum,
mating frequency
Author for correspondence:
Kensuke Nakata
e-mail: [email protected]
Kyoto Women’s University, Kitahiyoshi-cho 35, Higashiyama-ku, Kyoto 605-8501, Japan
KN, 0000-0002-1207-3448
Monandry, in which a female has only one mating partner during the reproductive period, is established when a female spontaneously refrains from
re-mating, or when a partner male interferes with the attempts of a female to
mate again. In the latter case, however, females often have countermeasures
against males, which may explain why polyandry is ubiquitous. Here, I demonstrate that the genital appendage, or scape, of the female orb-web spider
(Cyclosa argenteoalba) is injured after her first mating, possibly by her first
male partner. This female genital mutilation (FGM) permanently precludes
copulation, and females appear to have no countermeasures. FGM is considered to confer a strong advantage to males in sexual conflicts over the
number of female matings, and it may widely occur in spiders.
1. Introduction
The mating system of animals, especially female mating frequency, has fitness
consequences for both sexes [1]. Monandry, in which a female has one mating
partner, is established when a female spontaneously refrains from mating with
multiple males, and/or when a male prevents a partner female from mating
with other males [2]. Males use various methods of interference, including
mate guarding, prolonged copulation, injecting chemical substances to decrease
the likelihood of a female re-mating, and mating plugs (which block the copulatory openings in the female) [3]. On the other hand, females often resist these
methods, and these methods do not always perfectly secure the paternity of the
males [4–7]. A recent study revealed that polyandry is ubiquitous in animals
[8], which suggests that the countermeasures of females are usually effective.
Genital coupling is necessary for successful mating in many animals, and it
would be impeded by genitalia damage. In fact, in some male spiders, genitalia
or parts of genitalia such as sclerites or embolus are broken after mating to form
mating plugs and cannot be used again [9]. On the other hand, studies about
the effect of female genital damage after mating on the subsequent copulation
ability of females are scarce.
In some female Araneidae spiders, a small projection, or scape, covers the
copulatory openings of the genitalia (epigynum); this appendage can be mutilated in many females [10,11]. Such mutilation may interfere with genital
coupling [12], and could be used to control female mating frequency. This
study examines this hypothesis using the orb-web spider, Cyclosa argenteoalba,
in a series of staged matings.
2. Methods
Electronic supplementary material is available
at http://dx.doi.org/10.1098/rsbl.2015.0912 or
via http://rsbl.royalsocietypublishing.org.
I used 84 virgin adult Cyclosa argenteoalba females from bamboo forests in Osaka and
Kyoto, Japan in 2014. Some of them were collected as penultimate subadult females,
and were kept until they made their final moult. Other adults were collected on their
moulting webs, which were characterized by their small size, reduced number of
radii, lack of spiral threads and presence of fibrous decorations [13], indicating
& 2016 The Author(s) Published by the Royal Society. All rights reserved.
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(a)
(b(i))
(c(i))
(b(ii))
0.2 mm
(d(ii))
0.2 mm
0.2 mm
Figure 1. (a) Lateral view of mated and mutilated female of Cyclosa argenteoalba. (b – d) Close-up photo of frontal ventral abdomen of (b) virgin female, (c) mated
and mutilated female and (d ) virgin and artificially mutilated female ( picture brightness was enhanced). (i) Ventral and (ii) lateral view. Epigynum with or without
scape is shown within a solid white or dotted circle, respectively. Spade-shaped projections seen in b(i) and b(ii) are scapes. Solid and dotted white or black arrows
indicate the basal part of the scape where the appendage is cut during mutilation. See also Tanikawa [11].
that the spiders had just moulted to adults. They were convincingly assumed to be virgins, because preliminary observations
had revealed that no female mates with a male when on a moulting web. The spiders were released in a fenced garden
observation area (9 2 m), located in Shimamoto, Osaka,
where the females built their webs. Individual spiders were distinguished by their photographs (C. argenteoalba exhibit
individual variation in silver and black marking patterns [14])
and web locations. Adult males were also collected and kept in
vials. The observation area was separated from the natural habitat of the spiders by residential buildings, and whereas
experimental spiders could emigrate from it, no immigration
occurred during the study. This set-up isolated subject females
from outside (non-experimental) males.
(a) General procedure
The genitalia of each female was inspected under a dissection
microscope before she was allowed to mate. She was then returned
to her web. After she regained her sitting posture, a male was introduced. Her response, the occurrence of copulation and sexual
cannibalism, and the number and duration (the time between
genital contact and separation) of palpal insertions, were recorded
using normal-speed (Pentax Optio WG-2, 30 frames per second
(FPS)) and high-speed cameras (Casio EXILIM EX-F1, 300 FPS).
Normally, a copulation bout in this species consists of two separate
palpal insertions (see Results). Copulation was judged unsuccessful when, despite male courtship, no palpal insertion was observed
for 1 h, or the male left the web without palpal insertion.
I inspected females after mating to verify whether the scape
remained or not. Each male was returned to a vial.
(b) Experiment 1: copulation ability after genital
mutilation
Each of 54 virgin females was paired with a male for staged
mating. For five pairs, copulation was interrupted after the first
palpal insertion because of sexual cannibalism or strong wind,
and among the remaining 49 females, 13 disappeared from the
observation area soon after the first mating, perhaps because of
emigration. The remaining 36 mated females (five with a scape
and 31 without a scape) were coupled with another male to
determine if they could copulate.
(c) Experiment 2: artificial genital mutilation
Ten virgin females were anaesthetized with CO2, and their scapes
were cut using fine scissors to mimic natural genital mutilation
(figure 1d). Damage from the procedure appeared negligible, as
no haemolymph was observed at the site, and the spiders subsequently resumed normal web building and prey capture. The
mutilation apparently did not cause pain, because the scape has
no sensory hair [12] and anaesthesia appeared effective. Ten control virgin females were also anaesthetized with CO2 but were
not mutilated. Females from the experimental and control
groups were both coupled with a male, and their ability to copulate
was observed.
(d) Experiment 3: the timing of mutilation in a
copulation bout
Ten virgin females were subjected to staged mating. After the
first palpal insertion, I removed the female from the web and
looked for her scape. During this time, the male continued courting for the second insertion in eight pairs. I returned the female
to her web and observed whether the second insertion occurred.
3. Results
Every virgin female had a scape on the basal plate of the
epigynum (figure 1b). Scapes were wrinkled spade-shaped
projections, and were bent in the middle. Copulation occurred
on a mating thread spun by the male and the males courted
females by vibrating this thread. After several courting trials,
all females exhibited an accepting posture by exposing their
genitalia to the males. The males then attempted to insert one
of their two pedipalps. When the insertion was successful,
Biol. Lett. 12: 20150912
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(c(ii))
0.2 mm
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0.2 mm
0.2 mm
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(d(i))
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A series of staged matings of C. argenteoalba revealed that one
mating bout consisted of two palpal insertions, and was in
most cases accompanied by female genital mutilation (FGM),
by removal of the scape. Once mutilated, either naturally or
artificially, no female successfully mated. Mutilated females
did not appear to stop re-mating spontaneously to avoid
harassment from males, because males pursued and courted
both mutilated and unmutilated females similarly. The response
of all mated females to these subsequent courtships instead
suggests that whereas females pursue re-mating, males engage
in FGM to enforce lifetime monandry in C. argenteoalba. The
scape is presumably an essential appendage that the male genitalia must clasp before palpal insertion [15], and mutilation
potentially destroys this fine mechanism of genital coupling. In
most cases, FGM occurred after the second palpal insertion,
which is logical, because if mutilation occurred after the first
insertion, then even the partner male could not complete the
second insertion, as observed twice in experiment 1. These findings imply that the mutilation is an intended act and that the
scape is not accidentally damaged.
Recently, FGM was also found to secure paternity in Larinia
jeskovi, another species of orb-weaving spider [16]. FGM in
L. jeskovi is very similar to that in C. argenteoalba. In L. jeskovi,
the tegular apophysis of a pedipalp cut the side of the basal
3
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4. Discussion
part of the scape halfway [16]. The same mechanism may
also exist in C. argenteoalba, and it explains why two palpal
insertions are necessary for successful mutilation: at each insertion, males possibly cut each of the right and left halves of the
scape, although mutilation apparently does not always occur
after two insertions in L. jeskovi.
Mutilated females were frequently (90% of all females at a
maximum) found in the field (K.N. 2011–2014, personal observation), indicating that FGM is not an artefact of the experiment.
Why did mutilated females respond to courting even though
they could not re-mate? The females may not have recognized
that they were mutilated. To date, there is no evidence that
the scape has a sensory system [12]. Although spider females
are considered at increased risk of predation and have
decreased foraging opportunity during the mating trial [17],
these costs may be negligible to C. argenteoalba, and therefore
females do not stop responding to males. Alternatively, the
females may attempt to capture and eat the second male
(sexual cannibalism), but this is unlikely. Overall, the rate of
sexual cannibalism was very low; among 120 mating trials in
this study, cannibalism occurred only four times. These four
cases of cannibalism, in addition to five other cannibalism
events (K.N. 2014, unpublished observation), occurred after
palpal insertions, suggesting that pre-copulation cannibalism
does not occur, or occurs rarely, in this species. As mutilated
females do not complete copulation, they do not have the
opportunity to capture and cannibalize males, making it unlikely that cannibalism is their motivation for courting males.
Another question raised in this study is why females accepted
all courting males, even though they mate with only one male
throughout their lives. The sex ratio, mate-encounter rate and
variation in male quality are areas for future investigation to
explain this lack of choosiness in females.
If FGM is a mechanism for males to restrict the re-mating of
females, then it is more efficient than other previously known
mechanisms, including mate guarding, manipulation by chemical substances or placing mating plugs. Indeed, no mutilated
female re-mated, and copulation ability, once lost, was not
restored. Whereas the mutilation rate was high (approx. 90%),
the cost for males to restrict the re-mating of females in
this manner is considered low. In contrast to other species,
C. argenteoalba males do not have to spend a long time deterring
rival males, or sacrifice their mating ability by plugging female
genitalia with broken pieces of their pedipalps [9]. Females
appear to have no efficient way to counteract FGM. Sexual cannibalism might protect females if it occurred before mutilation,
but it would not be a significant countermeasure, as the cannibalism rate was very low. Why do females accept mutilation?
One answer may be that FGM is a recently developed trait,
and the females may not have had time to evolve countermeasures. This explanation seems unlikely, because FGM is
observed in other spiders of genus Cyclosa [11], which suggests
that mutilation may be an ancestral trait. Alternatively, females
may seldom have the opportunity to mate with a second male.
Information about sex ratio and mate-encounter rate in the field
will be required to validate this hypothesis.
FGM in C. argenteoalba (this study) and L. jeskovi [16] is a
previously unknown mechanism to realize monandry in
animals. In bean weevils [18] and dung flies [19], female genitalia are damaged during copulation, but this injury does not
inhibit re-mating [20]. In some ponerine ants, mutilation
deprives the females of their mating ability, but the mutilated
organ is a pair of vestigial wings, not genitalia [21]. Female
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the pair separated and the male courted the female again for
the second insertion.
In experiment 1, among the 49 females that completed
copulation, five retained their scapes, and all other females
were mutilated, i.e. their scapes were cut at their basal part
(figure 1a,c). The mutilation rate was approximately 90%
(44/49). Two mutilated females received only one palpal insertion; the partner males continued courting after the first
insertion and attempted the second insertion repeatedly, but
all trials failed. The remaining 47 females received two insertions. Median duration of insertion and its interquartile range
were 0.89 and 0.73–1.27 s, respectively, for the first insertion,
and 0.85 and 0.62–1.07 s, respectively, for the second insertion.
At the re-mating trial, all males courted females, irrespective of being mutilated or not, and all females exhibited
accepting posture to males. However, all mutilated females
failed copulation, despite the repeated attempts of the
males to insert their pedipalps (electronic supplementary
material, video S1). Copulation was successful in four of
five unmutilated females, which had scapes (electronic supplementary material, video S2). The difference in mating
success was significant (Fisher’s exact test, p , 0.0001). Average courting durations were 8.01 min for mutilated (n ¼ 30)
and 5.00 min for unmutilated females (n ¼ 5).
In experiment 2, all artificially mutilated females failed
copulation. For the control virgin females, copulation was successful in nine cases, and failed in only one case. The success
ratio was significantly different (Fisher’s exact test, p , 0.001).
In experiment 3, all females retained their scapes after the
first palpal insertion. Recoupling resulted in successful
second insertion in seven pairs, and all females lost their
scapes after this coupling. In the other three pairs, the second
insertion did not occur, because males stopped courting in
two cases and a female did not respond to courting in one
case. Females in these three cases retained their scapes.
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Ethics. The experiments were performed in accordance with the
guidelines for ethological studies from the Japan Ethological Society.
Data accessibility. All essential data are available in the text.
Competing interests. I declare I have no competing interests.
Funding. This study was partly supported by a JSPS grant-in-aid for
Scientific Research (C) (no. 26440251).
Acknowledgements. I thank Midori Nakata, Dr Keizo Takasuka and
Dr Atushi Ushimaru for their help during the study. I also thank
Dr Matthias Foellmer and three anonymous reviewers for their
helpful comments.
References
2.
3.
4.
5.
6.
7.
Slatyer RA, Mautz BS, Backwell PRY, Jennions MD.
2012 Estimating genetic benefits of polyandry
from experimental studies: a meta-analysis. Biol.
Rev. 87, 1–33. (doi:10.1111/j.1469-185X.2011.
00182.x)
Arnqvist G, Andrés JA. 2006 The effects of
experimentally induced polyandry on female
reproduction in a monandrous mating system.
Ethology 112, 748–756. (doi:10.1111/j.1439-0310.
2006.01211.x)
Arnqvist G, Rowe L. 2013 Sexual conflict. Princeton,
NJ: Princeton University Press.
Avila FW, Sirot LK, LaFlamme BA, Rubinstein CD,
Wolfner MF. 2011 Insect seminal fluid proteins:
identification and function. Annu. Rev. Entomol.
56, 21 –40. (doi:10.1146/annurev-ento-120709144823)
Johnsen A, Lifjeld JT, Rohde PA, Primmer CR,
Ellegren H. 1998 Sexual conflict over fertilizations:
female bluethroats escape male paternity guards.
Behav. Ecol. Sociobiol. 43, 401–408. (doi:10.1007/
s002650050507)
Koprowski JL. 1992 Removal of copulatory plugs by
female tree squirrels. J. Mammal 73, 572–576.
(doi:10.2307/1382026)
Foellmer MW. 2008 Broken genitals function as
mating plugs and affect sex ratios in the orbweb spider Argiope aurantia. Evol. Ecol. Res. 10,
449–462.
8.
9.
10.
11.
12.
13.
14.
Taylor ML, Price TAR, Wedell N. 2014 Polyandry in
nature: a global analysis. Trends Ecol. Evol. 29,
376 –383. (doi:10.1016/j.tree.2014.04.005)
Uhl G, Nessler S, Schneider J. 2010 Securing
paternity in spiders? A review on occurrence
and effects of mating plugs and male genital
mutilation. Genetica 138, 75 –104. (doi:10.1007/
s10709-009-9388-5)
Levi H. 1995 The neotropical orb-weaver genus
Metazygia (Araneae: Araneidae). Bull. Mus. Comp.
Zool. 154, 63 –151.
Tanikawa A. 1992 A revisional study of the Japanese
spiders of the genus Cyclosa MENGE (Araneae:
Araneidae). Acta Arachnol. 41, 11 –85. (doi:10.
2476/asjaa.41.11)
Eberhard W, Huber B. 2010 Spider genitalia: precise
maneuvers with a numb structure in a complex
lock. In The evolution of primary sexual characters
in animals, pp. 249–284. Oxford, NY: Oxford
University Press.
Takasuka K, Yasui T, Ishigami T, Nakata K,
Matsumoto R, Ikeda K, Maeto K. 2015 Host
manipulation by an ichneumonid spider
ectoparasitoid that takes advantage of
preprogrammed web-building behaviour for its
cocoon protection. J. Exp. Biol. 218, 2326–2332.
(doi:10.1242/jeb.122739)
Nakata K, Shigemiya Y. 2015 Body-colour variation
in an orb-web spider and its effect on predation
15.
16.
17.
18.
19.
20.
21.
success. Biol. J. Linn. Soc. 116, 954 –963. (doi:10.
1111/bij.12640)
Uhl G, Nessler SH, Schneider J. 2007 Copulatory
mechanism in a sexually cannibalistic spider with
genital mutilation (Araneae: Araneidae: Argiope
bruennichi). Zoology 110, 398 –408. (doi:10.1016/j.
zool.2007.07.003)
Mouginot P, Prügel J, Thom U, Steinhoff Philip OM,
Kupryjanowicz J, Uhl G. 2015 Securing paternity by
mutilating female genitalia in spiders. Curr. Biol.
25, 2980 –2984. (doi:10.1016/j.cub.2015.09.074)
Herberstein M, Schneider J, Elgar M. 2002 Costs
of courtship and mating in a sexually cannibalistic
orb-web spider: female mating strategies and their
consequences for males. Behav. Ecol. Sociobiol. 51,
440–446. (doi:10.1007/s00265-002-0460-8)
Crudgington HS, Siva-Jothy MT. 2000 Genital
damage, kicking and early death. Nature 407,
855–856. (doi:10.1038/35038154)
Ward P, Hemmi J, Roosli T. 1992 Sexual conflict
in the dung fly Sepsis cynipsea. Funct. Ecol. 6,
649–653. (doi:10.2307/2389959)
Hosken DJ, Martin OY, Born J, Huber F. 2003 Sexual
conflict in Sepsis cynipsea: female reluctance,
fertility and mate choice. J. Evol. Biol. 16, 485 –490.
(doi:10.1046/j.1420-9101.2003.00537.x)
Fukumoto Y, Abe T, Taki A. 1989 A novel form
of colony organization in the ‘queenless’ ant
Diacamma rugosum. Physiol. Ecol. Jpn 26, 55 –61.
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1.
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genital damage has been found to occur in many Araneoidea
species [16], and more FGM-driven monandry is expected to
be found in these species. Comparison of species with and
without FGM is anticipated to advance our understanding
of the evolution of social systems and sexual conflict.