Research Article |
Corresponding author: Michael Schmitt ( michael.schmitt@uni-greifswald.de ) Academic editor: Jorge Santiago-Blay
© 2015 Michael Schmitt, Gabriele Uhl.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Schmitt M, Uhl G (2015) Functional morphology of the copulatory organs of a reed beetle and a shining leaf beetle (Coleoptera: Chrysomelidae: Donaciinae, Criocerinae) using X-ray micro-computed tomography. In: Jolivet P, Santiago-Blay J, Schmitt M (Eds) Research on Chrysomelidae 5. ZooKeys 547: 193–203. https://doi.org/10.3897/zookeys.547.7143
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For more than 100 years it has been known that the sclerotised median lobe of beetles harbours a membranous structure (the "internal sac" or "endophallus") which is everted during copula inside the female genital tract. In order to explore the functional role of this structure and those associated with it, we cryofixed copulating pairs of Donacia semicuprea and Lilioceris lilii and studied the relative position of the elements of the copulatory apparatus of males and females by micro-computer-tomography.
We found that the everted endophallus fills the lumen of the bursa copulatrix completely. Our data suggest that in L. lilii the tip of the sclerotised distal part of the ejaculatory duct, the flagellum, is positioned exactly over the opening of the spermathecal duct inside the bursa copulatrix. The mouth of the bursa copulatrix in D. semicuprea is armed with a strong muscle ring, and the whole wall of the bursa is covered externally with a layer of muscle fibres. These morphological differences correspond with differences in mating behaviour: In reed beetles (Donaciinae), females seemingly can control mating to a higher degree than in lily beetles (Lilioceris spp.).
Aedeagus, endophallus, flagellum, bursa copulatrix, spermatheca, sperm transfer
As the primary role of copulatory organs is to secure transfer of sperm from males to females, they could, in principal, be shaped very simply. A tube, rigid or elastic, and a corresponding basket would do. The fact that copulatory organs are often complex and species-specific has traditionally been explained as a lock-and-key device that guarantees the preservation of the species and prevents waste of time, energy, and sperm by copulations between allospecific partners (
In beetles, the form of the male copulatory apparatus (aedeagus), especially its median lobe, has found the lively interest of taxonomists (e.g., Kraatz 1881;
Schematic drawing of the sclerotised part of a Donaciinae aedeagus. The given terms of orientation do not refer to the position within the male abdomen nor within the female during copulation since these structures are rotated during mating. The median lobe is shaded grey. Paramere, tegminal ring and manubrium compose the tegmen.
However, only few investigators have studied the functional roles of the different elements of the male copulatory apparatus (e.g.
During copulation, a membranous sac, the endophallus, is everted through the ostium of the median lobe (fig. 1) inside the female bursa copulatrix. The ejaculatory duct transverses the endophallus, ending in a sclerotized tube, the flagellum. In addition, the wall of the endophallus bears sclerites in several beetle species studied so far (
In earlier papers,
The mating behaviour of leaf beetles in general was described by
We investigated cryofixed pairs of copulating leaf beetles. Male shining leaf beetles (Criocerinae) lack parameres while reed beetles (Donaciinae) possess a so-called complete cucujoid aedeagus. We focused especially on the relative position of male and female genitalia during copulation. We had observed earlier that copulating pairs of reed beetles quickly separate when disturbed, while copulating lily beetles (Lilioceris lilii) can only be separated by applying considerable force (
We collected numerous individuals of Donacia semicuprea Panzer, 1796 and Lilioceris lilii (Scopoli, 1763) in the area of Greifswald (northeast Germany). Copulating pairs were fixed using 70% ethanol at -12 °C (D. semicuprea) or liquid nitrogen (L. lilii). The fixed pairs were stored in 80% ethanol at -40 °C for at least ten days. We prepared them for X-ray micro-computed tomography (micro-CT) analysis by critical point drying (BAL-TEC CPD 030), glued them head downwards on the tip of a little plastic rod of 2 mm in diameter, with the tip of the female abdomen as close to the rotation axis as possible. Three pairs of D. semicuprea and two pairs of L. lilii were scanned under an Xradia Micro XCT-200 (Carl Zeiss X-ray Microscopy Inc.), using the 4× or 10× object lens units, at 30kV and 4W, with a pixel size of 5.36 µm or 2.34 µm. Tomography projections were reconstructed using the reconstruction software provided by XRadia. Volume rendering of image stacks was performed by using Amira 5.4.5 and Amira 5.6.0 (FEI Visualization Science Group, Burlington, USA) using the "Volren" or "Voltex" function.
We use the morphological terms as given in Fig.
Lilioceris lilii: The median lobe of the aedeagus is inserted in the female abdomen, the endophallus is everted and inflated. We did not make an attempt to trace the ejaculatory duct because we focused on the relative position of male and female copulatory organs. Of the ejaculatory duct only the sclerotised distal part, the flagellum, gets into contact with the female body. The bursa copulatrix is nearly globular (length/height: 1.25/1), its opening is situated at half the length of the last sternite (Figs
The tip of the small flagellum is positioned exactly opposite the opening of the spermathecal duct, see Fig.
Fig.
Lilioceris lilii, copulating pair. Volume rendering of the virtual sections right to the median, digitally stained. The terminal part of the spermathecal duct can be seen immediately left to the bursa wall. The shape of the bursa is nearly globular. B: bursa copulatrix; C: the compound muscle inserting at the manubrium and extending to the lateral rims of the basal orifice of the median lobe; E: endophallus; M: median lobe.
Donacia semicuprea: The median lobe is inserted into the female body while the paramere remains outside (Fig.
Donacia semicuprea. Volume rendering of the virtual sections – sagittal, right to the median – through the abdomina of a mating pair. The opening of the bursa copulatrix (circle) is armed with a conspicuous ring muscle. C: the compound muscle inserting at the manubrium and extending to the lateral rims of the basal orifice of the median lobe; M: median lobe; P: paramere.
The tip of the median lobe is inserted into the bursa copulatrix. There is a strong muscular ring around the mouth of the bursa (Fig.
Donacia semicuprea, Volume rendering of ca. 390 virtual sections – sagittal, paramedian, tilted to the right – through the abdomina of a mating pair. The bursa is elongate, as seen from the proximal wall (arrow). The yellow shining areas inside the female abdomen are parts of the left ovary. P: paramere.
When interpreting the morphological data, we have to take into account that the copulating partners may be fixed in different stages of, e.g. the intromission of the median lobe or endophallus inflation. Moreover, we cannot be certain that the interacting male and female copulatory organs remained exactly as they were in the millisecond when the beetles were cryofixed.
Already
Our observation that the paramere of D. semicuprea remains outside the female body is in concordance with the earlier report of
In species with a "complete cucujoid aedeagus", the compound muscle (Fig.
The morphological difference between the two species corresponds with differences in mating behaviour. It suggests that in D. semicuprea – and probably in all donaciine species – females control admittance of males for mating and the duration of the copulation to a higher degree than in L. lilii (and probably in all Criocerinae).
We thank Marina Ide and Janine Dombrowski for their help in the cryofixation of the Donacia pairs, Elisabeth Lipke and Philip Steinhoff (all Greifswald) for assisting with the Amira application, Irina A. Kaygorodova (Irkutsk, Russia) and Christopher Lyal (London, UK) for valuable feedback on the manuscript, and the Deutsche Forschungsgemeinschaft (German Science Foundation, Uh87/ 7-1, INST 292/119-1 FUGG, and INST 292/120-1 FUGG) for financial support.
Extended version of a presentation to the 2nd European Symposium on the Chrysomelidae, York (England), August 4, 2014