Thalassas, 27 (2): 113-119
An International Journal of Marine Sciences
TIME FOR SEX CHANGE! 3D-RECONSTRUCTION OF THE
COPULATORY SYSTEM OF THE ’APHALLIC‘
Hedylopsis ballantinei (GASTROPODA, ACOCHLIDIA)
KOHNERT P, NEUSSER TP, JÖRGER KM & SCHRÖDL M
Key words: Mollusca, Panpulmonata, morphology, hypodermal injection, penial stylet, protandry,
sequential hermaphroditism.
ABSTRACT
Within hedylopsacean acochlidians an
evolutionary trait from a simple unarmed copulatory
system towards complex hypodermal injection
systems was recognized. This culminates in a
large, trap-like spiny rapto-penis of several limnic
Acochlidiidae having a sperm injection stylet plus
an additional injection system with an accessory
gland. The only exception was the mesopsammic
hedylopsacean species Hedylopsis ballantinei
Sommerfeldt & Schrödl, 2005, since it was assumed
to be aphallic. Specimens with mature autosperm
and oogonia in the hermaphroditic gonad showed no
trace of any male copulatory organs. Sperm transfer
via spermatophores was thus suggested, as known
to occur in the generally aphallic microhedylaceans.
The present study re-examines several series of
semithin sections used for the original description.
Additionally, one specimen of H. ballantinei was
Bavarian State Collection of Zoology. Münchhausenstr.
21, D-81247 Munich, Germany.
Email: [email protected], [email protected], [email protected], [email protected]
newly collected near the type locality in the Red Sea.
It is externally identical with but smaller than the
original specimens. The specimen was embedded
into Spurr’s resin and serially cut into semithin
histological sections. Reproductive systems were
compared in detail and that of a specimen in the
male phase was 3-dimensionally reconstructed
using AMIRA software. The copulatory organs
comprise the posterior-leading vas deferens passing
into a voluminous tubular prostate, a presumable
paraprostate and a bipartite penis with a large apical,
hollow penial stylet and with a cuticular, solid thorn
on top of the basal swelling. As already known
for H. spiculifera (Kowalevsky, 1901), its European
sister species, H. ballantinei thus is a sequential
hermaphrodite with sex change. The male phase
precedes the female one, in which male copulatory
organs completely disappear. Sperm transfer is likely
by hypodermal injection. Hedylopsis ballantinei in
the male phase has an external sperm groove, while
specimens in the female phase possess a ciliary field;
the latter may have a function related to building or
placing the egg mass. Hedylopsis ballantinei now
fits well with evolutionary traits observed within
other hedylopsacean acochlidians known in detail.
113
Kohnert P, Neusser TP, Jörger KM & Schrödl M
Figure 1:
Schematic overview of the male cephalic copulatory organs with associated glands of Hedylopsis ballantinei. Abbreviations: bs, basal swelling; ed,
ejaculatory duct; mgo, male gonopore; p, penis; ppd, paraprostatic duct; ppr, paraprostate; pr, prostate; ps, penial sheath; pst, hollow penial stylet;
sg, external sperm groove; th, solid thorn; ugm, unidentified glandular mass; vdp, posterior-leading vas deferens. Not to scale.
INTRODUCTION
Most recently, opisthobranch gastropods were
shown to be an artificial assemblage, with the
traditional order Acochlidia clustering within a (pan)
pulmonate relationship (Jörger et al., 2010; Schrödl
et al., this volume pags. 101-112). Both molecular
and morphology-based phylogenetic analyses (Jörger
et al., 2010; Schrödl & Neusser, 2010) indicate a
basal acochlidian split into generally regressive,
meiofaunal Microhedylacea (Neusser et al., 2009)
and morphologically and ecologically more variable
Hedylopsacea, including marine, brackish water and
limnic species of variable body sizes (e.g. Neusser
& Schrödl, 2007, 2009; Brenzinger et al., 2011).
Within hedylopsacean acochlidians an evolutionary
trait from a simple, unarmed copulatory system
towards complex hypodermal injection systems
was recognized (Schrödl & Neusser, 2010). This
culminates in the large, trap-like spiny rapto-penis
of several limnic Acochlidiidae, having a sperm
114
injection stylet plus an additional injection system
with an accessory gland (Haase & Wawra, 1996).
The only exception in this evolutionary scenario of
evolving a more and more complex and probably
violent copulatory apparatus was the mesopsammic
hedylopsacean species Hedylopsis ballantinei
Sommerfeldt & Schrödl, 2005, since it was assumed
to be aphallic. The few specimens available had
mature autosperm and oogonia in the hermaphroditic
gonad, but showed no trace of any copulatory organs
(Sommerfeldt & Schrödl, 2005). Sperm transfer via
spermatophores was thus suggested, as known to
occur in the generally aphallic microhedylaceans.
The present study examines old and new material of
different-sized H. ballantinei from serial histological
sections for the presence of reproductive organs.
Male copulatory organs were identified, labeled
and 3-dimensionally reconstructed using AMIRA
software, and compared to other hedylopsacean
copulatory systems.
Time for sex change! 3D-reconstruction of the copulatory system of the ’aphallic‘
Hedylopsis ballantinei (Gastropoda, Acochlidia)
MATERIAL AND METHODS
One specimen of Hedylopsis ballantinei was
newly collected approx. 600 m north of the type
locality (Inmo Reef) in Mashraba (28°29`42``
N, 34°31`04`` E), Dahab, Egypt in August 2009.
A sample of coarse coral sand was obtained by
snorkeling from 6 m depth by night. The specimen
was extracted from the sand sample according
to the method described by Schrödl (2006). The
specimen was relaxed with isotonic MgCl 2-solution
and was preserved in 4 % glutardialdehyde buffered
in 0.2 M sodium cacodylate (0.1 M NaCl and
0.35 M sucrose, pH 7.2). Following a post-fixation
in buffered 1 % OsO4 for 1.5 h in the dark, the
specimen was decalcified in 1 % ascorbic acid
overnight and dehydrated in an acetone series (30,
50, 70, 90, 100 %). For semithin sectioning the
specimen was embedded in Spurr’s low viscosity
resin (Spurr, 1969) and a series of ribboned serial
semithin sections of 1.5 µm thickness was prepared
using a diamond knife (Histo Jumbo, Diatome,
Biel, Switzerland) and contact cement on the lower
cutting edge to form ribbons (Ruthensteiner, 2008).
Finally, the sections were stained with methyleneazure II (Richardson et al., 1960) and were deposited
at the Mollusca Section of the Bavarian State
Collection of Zoology (ZSM), Germany (ZSM
Mol 20100856). Additionally, we (re-) examined
five series of serial semithin sections (2 µm) of
Hedylopsis ballantinei which were available at the
ZSM by light microscopy: ZSM Mol 20100855,
ZSM Mol 20004766/1, ZSM Mol 20004767, ZSM
Mol 20004768 and ZSM Mol 20004769. The series
N° 20100855 revealed H. ballantinei to possess
mature male copulatory organs. Digital photographs
of every slice of the latter series were taken with a
CCD microscope camera (Spot Insight, Diagnostic
Instruments, Sterling Heights, USA) mounted on
a DMB-RBE microscope (Leica Microsystems,
Wetzlar, Germany). The image resolution was
reduced to 50 % and images were contrast enhanced,
unsharp masked and converted to 8bit greyscale
format with standard image editing software. A
detailed computer-based 3D-reconstruction of the
body surface and the male reproductive system
was performed using the software AMIRA 5.2.2
(Visage Imaging GmbH, Germany) as outlined by
Ruthensteiner (2008).
RESULTS
The re-examination of the semithin section
series used for the original description of Hedylopsis
ballantinei (see Sommerfeldt & Schrödl, 2005)
and for the examination of the excretory system
(Fahrner & Haszprunar, 2002, as Hedylopsis sp.),
did not provide new data on the male reproductive
system. The newly collected specimen was in the
female phase with mature female reproductive
organs, but lacking any male copulatory organs. In
contrast, the examination of a series of semi- and
ultrathin sections (ZSM Mol 20100855) showed
a male specimen of H. ballantinei with mature
complex copulatory organs. The 3D reconstruction
by Amira and the following description of the male
genital system of H. ballantinei is based on series
N° 20100855.
Hedylopsis ballantinei is a sequential, protandric
hermaphrodite with an external sperm groove (Figs.
1; 2A,B) in the male phase and a ciliary field in the
female phase. The external sperm groove connects
the posterior reproductive system from the female
gonopore (Fig. 2D) to the male gonopore (Fig. 1) and
the cephalic male copulatory organs (Figs. 1; 2A-C).
The latter include a large bipartite penis with an
apical hollow stylet, a very voluminous prostate, a
potential paraprostate and an accessory gland (Figs.
1; 2C) with unknown function and homology.
The posterior-leading vas deferens (Figs. 1;
2A,B) leads from the male genital opening (Fig. 1)
which is situated at the base of the right rhinophore,
to the tubular, glandular prostate (Figs. 1; 2A,B,F).
The ejaculatory duct (Fig. 1) emerges from the latter
and enters the muscular penis (Figs. 1; 2A-C). A
second glandular mass, the sac-like paraprostate
115
Kohnert P, Neusser TP, Jörger KM & Schrödl M
Figure 2:
3D-reconstruction and histological semithin sections of the male reproductive system of Hedylopsis ballantinei. A, Hermaphroditic reproductive
system (ventral view); B, Male cephalic copulatory organs (right view); C, Penis and basal swelling with glands and armature (anterior view); D,
Body with ovotestis and female glands (right anterolateral view); E, Penis, penial stylet and basal thorn; F, Ovotestis, prostate and female glands.
Abbreviations: bs, basal swelling; dg, digestive gland; f, foot; fgl, female glands; fgo, female gonopore; lt, labial tentacle; ov, ovotestis; p, penis; pd,
prostatic duct; plg, pleural ganglion; ppd, paraprostatic duct; ppr, paraprostate; pr, prostate; ps, penial sheath; pst, hollow penial stylet; sg, external
sperm groove; th, solid thorn; ugm, unidentified glandular mass; vdp, posterior-leading vas deferens; vh, visceral hump.
116
Time for sex change! 3D-reconstruction of the copulatory system of the ’aphallic‘
Hedylopsis ballantinei (Gastropoda, Acochlidia)
(Figs. 1; 2A-C,E), is much smaller than the prostate
and connected to the penis via the paraprostatic
duct (Figs. 1; 2C). The latter enters the penis in
the upper part and joins the ejaculatory duct.
Together they discharge at the top of the penial
papilla into a curved, hollow penial stylet (Figs.
1; 2A,C,E) of approx. 160 µm length. A muscular
basal swelling with a solid thorn of approx. 40 µm
(Figs. 1; 2A,C,E) is attached to the base of the penis.
Near the muscular penis an additional, unidentified
glandular mass (Figs. 1; 2B,C,E) with yet unknown
function was detected. The bipartite penis and the
unidentified glandular mass are surrounded by the
thin-walled penial sheath (Figs. 1; 2E).
DISCUSSION
Among hedylopsacean acochlidians, H.
ballantinei was exotic in lacking any detectable
cephalic male reproductive organs. The presence
of mature autosperm and egg cells in the
hermaphroditic gonad of aphallic specimens
led Sommerfeldt & Schrödl (2005) to assume
that H. ballantinei is an aphallic hermaphrodite
species rather than a sequential hermaphrodite
as Hedylopsis spiculifera. However, our results
show a specimen of H. ballantinei having
complex male reproductive organs, while others
do not possess any. We thus conclude that H.
ballantinei is a sequential hermaphrodite with a
male, phallic phase preceding a female, aphallic
phase, just as it was described for H. spiculifera
by Wawra (1989). The function, if any, of testis
remainders in aphallic, early (?) female stages
is unknown. All hedylopsacean species known
to date thus have copulatory organs, in contrast
to microhedylaceans that are all aphallic during
their entire ontogeny (e.g. Neusser et al., 2009).
The external sperm groove of Hedylopsis in the
male phase is likely to transform into the ciliary
field that was observed in the female phase of
specimens of H. ballantinei by Sommerfeldt &
Schrödl (2005); a function related to handling the
egg mass can be inferred.
Sequential hermaphroditism with complete
reduction of copulatory organs occur in some,
but not all hedylopsacean clades, i.e. in the genus
Hedylopsis, Strubellia, and possibly in Tantulum
(Wawra, 1989; Neusser & Schrödl, 2007; Brenzinger
et al., 2011). In contrast, Pseudunela, Acochlidium and
Palliohedyle may be protandric but then simultaneous
hermaphrodites during most of their ontogeny
(Bücking, 1933; Haynes & Kenchington, 1991;
Wawra, 1980; Neusser & Schrödl, 2009; Neusser et
al., 2009). Mapping this feature on an acochlidian
consensus tree (Neusser et al., 2009) reveals an
ambiguous scenario. Possibly, hedylopsaceans are
sequential hermaphrodites either ancestrally or
evolved ontogenetic resorption of copulatory systems
after the offshoot of Tantulum from the stemline, with
re-evolution of simultaneous hermaphroditism in
Pseudunela and the common ancestor of Acochlidium
and Palliohedyle.
The anterior male copulatory system of H.
ballantinei is quite complex, resembling that of
its congener H. spiculifera in having an external
sperm groove leading to a cephalic posteriorleading vas deferens with a well-developed prostate
and a muscular penial papilla tipped with a hollow
stylet. The dimensions of the penial stylets cannot
be compared due to lacking data on the stylet
length of H. spiculifera. Obviously, sperm is
transferred to the mate via injection rather than
via spermatophores as assumed originally for H.
ballantinei (see Sommerfeldt & Schrödl, 2005). In
absence of any allosperm receptacles (Sommerfeldt
& Schrödl, 2005), hypodermal injection is likely.
Imprecise sperm transfer into the body cavity was
observed from H. spiculifera by Wawra (1989)
who detected a penial stylet in the visceral sac
of a mature female specimen. In both species the
penis is bipartite having a basal swelling with a
solid, cuticular thorn. The copulatory organs of
H. ballantinei differ from those of H. spiculifera
by the presence of a rather well-developed gland,
a putative paraprostate, which connects through
a duct to the ejaculatory duct within the penis.
117
Wawra E (1980). Acochlidium bayerfehlmanni spec. nov., (Gastropoda: Opisthobranchia:
Acochlidiacea) from Palau Islands. Veliger, 22: 215-220.
Kohnert P, Neusser TP, Jörger KM & Schrödl M
Wawra E (1989). Zur Kenntnis der interstitiellen Opisthobranchierart Hedylopsis spiculifera
(Kowalevsky) (Mollusca, Gastropoda). Zoologica Scripta, 18: 397-403.
Table 1:
Comparison of the male genital system within Hedylopsis. (? = no data available).
Table 1: Comparison of the male genital system within Hedylopsis. (? = no data available).
Data source
Hedylopsis
spiculifera
(Kowalevsky,
1901)
Wawra (1989)
Type of
hermaphroditism
Hedylopsis ballantinei Sommerfeldt & Schrödl,
2005
Sommerfeldt &
Schrödl (2005)
present study
sequential,
protandric
simultaneous
sequential, protandric
Complex,
cephalic male
copulatory
organs
penis with
hollow stylet
and basal thorn,
prostate, penial
gland of
unknown
function and
homology
absent
large bipartite penis with
apical hollow penial
stylet (approx. 160 µm)
and basal thorn (approx.
40 µm), voluminous
prostate, potential
paraprostate, plus
accessory gland of
unknown function and
homology
Sperm transfer
via
hypodermic
injection
spermatophore
hypodermic injection
for handling
spermatophore
probably involved in egg
mass deposition
Function of ciliary ?
field
Specimens of H. spiculifera have a small “penial
gland” in a corresponding location that, however,
opens separately at the base of the penial stylet.
A comparison of the male reproductive features
within Hedylopsis is given in Table 1.
Potentially homologous, more elaborate
paraprostatic systems present in higher
hedylopsaceans (Neusser & Schrödl, 2009; Neusser
et al., 2009; Brenzinger et al., 2011) are separated
from the ejaculatory duct and exit via own stylets
on the tip of the basal swelling that is developed into
a larger, so-called basal finger (according to Haase
& Wawra, 1996). The copulatory system found in
H. ballantinei thus represents a formerly unknown,
intermediate condition in hedylopsaceans and is in
line with the idea of progressively evolving more and
more elaborate copulatory organs with various glands
and injection systems (Neusser et al., 2009; Schrödl
& Neusser, 2010).
118
CONCLUSIONS
1. Hedylopsis ballantinei is a sequential protandric
hermaphrodite with sex change.
2. H. ballantinei has a large and complex cephalic
copulatory organ with an apical hollow stylet, a
solid thorn and two accessory gland systems, all
of which completely disappear in the early female
phase. Some male parts of the gonad, however, may
still persist after the loss of the copulatory organs.
3. The presence of an apical penial stylet and a basal
thorn resembles that of Hedylopsis spiculifera;
but the arrangement of glands is unique.
4. As a phallic species transferring sperm via
hypodermic impregnation and lacking any
allosperm receptacles, H. ballantinei now much
better resembles its Mediterranean/ eastern
Atlantic sister species H. spiculifera, and fits
well with evolutionary traits observed within
hedylopsacean acochlidians.
Time for sex change! 3D-reconstruction of the copulatory system of the ’aphallic‘
Hedylopsis ballantinei (Gastropoda, Acochlidia)
ACKNOWLEDGEMENTS
We thank the organizing team of the 3rd
International Workshop on Opisthobranchs in Vigo.
We are grateful to Christian Alter at the RSEC (Red
Sea Environmental Center) for support during field
work and collecting permits. This study was financed
by DFG projects (SCHR667/3,4) to MS, and by a PhD
grant by the Volkswagen Foundation to KJ. Amira
software was supported by the GeoBio Center (LMU
Munich). Bastian Brenzinger (ZSM) and an unknown
referee gave valuable comments on the manuscript.
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119