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Evolutionary biology
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First evidence of neurons in the male
copulatory organ of a spider
(Arachnida, Araneae)
Research
Elisabeth Lipke1, Jörg U. Hammel2,3 and Peter Michalik1,4
1
Cite this article: Lipke E, Hammel JU,
Michalik P. 2015 First evidence of neurons
in the male copulatory organ of a spider
(Arachnida, Araneae). Biol. Lett. 11: 20150465.
http://dx.doi.org/10.1098/rsbl.2015.0465
Department of General and Systematic Zoology, Zoological Institute and Museum,
Ernst-Moritz-Arndt-University of Greifswald, J.-S.-Bach-Straße 11/12, 17489 Greifswald, Germany
2
Institute of Materials Research, Outstation at DESY, Helmholtz-Zentrum-Geesthacht, Building 25c Notkestraße
85, 22607 Hamburg, Germany
3
Institut für Spezielle Zoologie und Evolutionsbiologie mit Phyletischem Museum, Friedrich-Schiller-Universität
Jena, Erbertstraße 1, 07743 Jena, Germany
4
Research Associate, Division of Invertebrate Zoology, American Museum of Natural History, New York, NY, USA
JUH, 0000-0002-6744-6811
Received: 28 May 2015
Accepted: 17 June 2015
Subject Areas:
evolution
Keywords:
reproductive biology, finite-element modelling,
arthropods, proprioreception,
ejaculate plasticity
Author for correspondence:
Elisabeth Lipke
e-mail: [email protected]
Electronic supplementary material is available
at http://dx.doi.org/10.1098/rsbl.2015.0465 or
via http://rsbl.royalsocietypublishing.org.
Spider males have evolved a remarkable way of transferring sperm by using a
modified part of their pedipalps, the so-called palpal organ. The palpal organ is
ontogenetically derived from tarsal claws; however, no nerves, sensory organs
or muscles have been detected in the palpal bulb so far, suggesting that the
spider male copulatory organ is numb and sensorily blind. Here, we document
the presence of neurons and a nerve inside the male palpal organ of a spider
for the first time. Several neurons that are located in the embolus are attached
to the surrounding cuticle where stresses and strains lead to a deformation
(stretching) of the palpal cuticle on a local scale, suggesting a putative proprioreceptive function. Consequently, the male copulatory organ of this species is
not just a numb structure but likely able to directly perceive sensory input
during sperm transfer. In addition, we identified two glands in the palpal
organ, one of which is located in the embolus (embolus gland). The embolus
gland appears to be directly innervated, which could allow for rapid modulation of secretory activity. Thus, we hypothesize that the transferred seminal
fluid can be modulated to influence female processes.
1. Introduction
Male fertilizing ability is strongly influenced by postcopulatory sexual selection,
i.e. sperm competition and cryptic female choice [1]. However, whenever females
mate with multiple males the ability of males to detect females as potential mating
partners with respect to e.g. age or mating status is of particular relevance [2].
Consequently, if resources are limited, or the mating effort is high, mate choice
and mating investment play a crucial role and males not only discriminate
between potential mates, but also are able to adjust their investment, e.g. quality
of ejaculates [3].
Males, however, initially need to identify and distinguish the quality of a
female, at the latest while mating. During copulation, spider males use a modified
part of their pedipalps, the so-called palpal organ, to transfer seminal fluid (sperm
and secretions). One major finding from several histological studies of the last
decades—including a considerable number of spider taxa—is the lack of nervous
tissue in the male palpal organ [4,5]. For this reason, it has been assumed that
spider males are not able to receive sensory input through their copulatory
organs while mating, despite the fact that males need to manoeuvre these
‘numb’ structures precisely into the female genitalia [4]. Although many spiders,
for example the Entelegynae, have evolved highly complex male pedipalps that
& 2015 The Author(s) Published by the Royal Society. All rights reserved.
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(a)
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Biol. Lett. 11: 20150465
(b)
Cu
Ne
So
So
?
Nv
?
(c)
Mi
Ne
(d)
Ne
Ne
Ne
Ne
Ne
cuticle
glandular duct
spermophor
neurons and nerve
bulbus gland
embolus gland
Figure 1. Male of the Tasmanian cave spider, Hickmania troglodytes, in its natural habitat ( photograph courtesy of M. J. Ramı́rez, & M. J. Ramı́rez), three-dimensional
model representation of the male copulatory organ and fine structural details of the neurons and the nerve. (a) The tube-like spermophor extends into the palpal organ. In
addition to two distinct glands, two distinct clusters of neurons, one of which is located near the blind end of the spermophor (asterisk) can be distinguished. A second
cluster of neurons is located in the furthermost part of the palpal organ in front of the embolus gland. Schematic drawings—cellular extensions that arise from some of
these neurons are likely attached to the cuticle. Consecutive sections reveal a very thin cuticle in the region of these extensions when compared with adjacent sections. The
properties of the thin cuticle with respect to stress and strain further support the presumed proprioreceptive function of the associated neurons, respectively. (b) Numerous
neurites join to form a small nerve that projects through the palpal organ. (c,d) Some of these neurites bear a distinct amount of clear vesicles (arrow), in addition to
mitochondria. Cu, part of the cuticular lining of the spermophor; Mi, mitochondrion; Ne, neurite; Nv, nerve; So, soma of neuron. (Online version in colour.)
are equipped with prelocking structures that may aid in mating,
many other spiders are equipped only with simple pedipalps
that lack such structures and could certainly profit from sensory
input. Here, we demonstrate the presence of neurons in the
simple male palpal organ of such a spider, the Tasmanian
cave spider Hickmania troglodytes (Higgins & Petterd, 1883),
for the first time (we believe). We discuss the potential role of
these neurons in processing sensory information during copulation and in mediating the interaction between male and
female genitalia. In addition, we provide evidence for a directly
innervated gland in the furthermost part of the palpal organ,
the embolus, which releases its secretions into a distinct glandular duct. This configuration might enable the male to directly
adjust the transferred ejaculate during copulation.
2. Material and methods
We investigated adult males of the Tasmanian cave spider by
means of histological and ultrastructural analyses, as well as X-ray
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60
50
40
30
20
10
0
–2
Figure 2. Surface model analysed with FEM displaying von Mises stress (N m ) and load (grey arrows). Cut planes of the copulatory organ near the tip of the
nerve are colour-coded according to the third principal stress (N m – 2). Arrows indicate the principal stress and strain directions. (Online version in colour.)
micro-computed tomography. Furthermore, we provide a detailed
three-dimensional reconstruction using AMIRA v. 5.6.0. (FEI, Visualization Science Group) and performed a finite-element modelling
(FEM) analysis of the male palpal organ. For details of our methods
and collection sites, see the electronic supplementary material (S1).
Voucher specimens are deposited in the Zoological Museum of
the University of Greifswald (ZIMG).
3. Results
The major findings of this study are (i) the presence of a nerve,
projecting as far as the embolus and (ii) the presence of two
clusters of neurons within the male palpal bulb (figure 1).
A first cluster of neurons is located near the blind end of the
internal cuticular tube containing the seminal fluid, the spermophor (figure 1a, asterisk, b), while a second one is situated
in the intromitting part of the organ, the embolus (figure 1a).
In addition, transmission electron microscopy analysis
revealed the presence of large neurites, forming a small nerve
that projects through the palpal organ (figure 1b,c), as well as
putative neurotransmitter (clear vesicles) inside some of these
neurites (figure 1d).
We furthermore revealed the presence of two glands
inside the male palpal organ. A prominent gland, which
occupies the main part of the bulb, is associated and likely
connected to the spermophor, through a porous portion at
the base of the latter (data not shown). A second, smaller
gland is located in the embolus and is provided with its
own glandular duct. The spermophor runs through this
duct, which opens in combination with the spermophor at
the tip of the embolus (figure 1a).
Although only the tip of the embolus is inserted in the
female genitalia during mating, the stresses and strains in
the embolus expanding from the contact area of the tip and
the female genitalia are distributed along the entire structure.
As a result of the geometry, materials properties and wall
thickness of the cuticle the embolus gets stretched or compressed in certain areas depending on the acting forces
(figure 2). We found a pronounced stretching in the cuticle
wall in the area next to the tip of one of the neuron branches
(figure 2). Thus, neurons located in the embolus might have a
proprioceptive function, enabling males of H. troglodytes to
perceive sensory input with respect to stress and strain.
4. Discussion
Our data provide the first evidence of neuronal tissue in
the spider male copulatory organ. Thus, this spider’s male
genitalia are likely not numb and sensory input could play
an important role in copulation and consequently in male
mating investment and/or securing paternity. The majority
of comparative studies suggest that female spiders are in control of processes regarding sperm utilization [6]. A putative
sensitive palpal organ might be an advantage as it could be
used to alter the male’s copulatory behaviour in order to
stimulate the female for the male’s own benefit (e.g. [7]).
Alternatively, a sensory copulatory organ might enable the
male to assess female parameters and adjust his investment
accordingly. However, this remains speculative at present,
as there are no data relating male copulatory behaviour to
paternity success in H. troglodytes.
Our study revealed the presence of two glands inside the
palpal organ, the spermophor gland and a gland that we
called the embolus gland. Based on the presence of neurotransmitter our data suggest that at least the embolus gland is likely
directly innervated. The spermophor gland of H. troglodytes is
associated with the base of the spermophor and therefore similar to the organization described for other spider taxa. Here, the
glandular tissue is expected to play an important role during
sperm uptake and transfer into the female during copulation [4].
Glands accessory to copulation, such as the embolus
gland we have described here, are usually thought to play
a role in assuring paternity [8–10]. For example, secretions
from such glands are often associated with the production
of mating plugs [11]. However, there is no evidence for
mating plugs in H. troglodytes [12] and thus the gland present
in the embolus would appear to serve other function(s). In
theory, males should invest more sperm in high-quality
females, but take advantage of any further mating. Thus,
males of H. troglodytes might be able to attenuate the seminal
fluid with additional secretions as shown for the accessory
glands of other animal taxa. Such secretions can have a
variety of functions influencing female processes to secure
paternity. For example, male accessory gland secretions in
insects are known to affect the receptivity to remate, to modulate oviposition behaviour and to influence egg development
[13]. So far, in spiders, only indirect evidence from the wolf
spider Schizocosa malitiosa suggests a positive correlation
Biol. Lett. 11: 20150465
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wrote the manuscript. J.U.H. performed the FEM analysis, P.M. collected
and dissected the specimens. J.U.H. and P.M. contributed to the writing
of the manuscript. All authors gave final approval for publication.
Competing interests. The authors have no competing interests.
Ethics. All specimens were collected in Tasmania under permit no. FA
Acknowledgements. We are grateful to Dave Lee for his help to collect a
12287.
Data accessibility. The images stacks obtained by X-ray micro-computed
Authors’ contributions. E.L. and P.M. designed the study. E.L. performed the
histological and X-Ray micro-CT studies, analysed the data, compiled
the three-dimensional reconstructions and schematic drawings and
Foundation to P.M. (DFG Mi 1255/5-1). The micro-computed tomography was funded by the state of Mecklenburg-Vorpommern and the
German Research Foundation (DFG INST 292/119-1 FUGG and DFG
INST 292/120-1 FUGG), which is gratefully acknowledged.
male in the Marakoopa Cave and Arthur Clarke for providing access
to and help in collecting on his private land in Francistown. We thank
Martı́n J. Ramı́rez, Christian Wirkner and Stefan Richter for their help
with the collecting and the logistics in Tasmania. In addition, we are
indebted to Michael Gebhardt, Roland Melzer, as well as Steffen
Harzsch, Andy Sombke and Gabriele Uhl for inspiring discussions.
Martı́n J. Ramı́rez, as well as two anonymous reviewers provided
helpful suggestions on the manuscript. We thank Martı́n J. Ramı́rez
for allowing us to use his photograph of a male Tasmanian cave
spider.
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Biol. Lett. 11: 20150465
tomography is stored in MorphDBase (www.morphdbase.de/?E_
Lipke_20150323-M-30.1; www.morphdbase.de/?E_Lipke_20150323M-31.1). A detailed description of collection sites and methods and
an additional figure supporting this article have been uploaded as
part of the electronic supplementary material.
Funding. Funding for this research was provided by the German Research
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of male transferred substances and female reluctance to
remate after a successful mating [14].
In conclusion, the possibility for sensory reception in males of
H. troglodytes, together with the directly innervated embolus
gland, suggests the presence of alternative mechanisms for securing paternity unknown for spiders. Future studies will reveal if
similar mechanisms are also present in other spider species.