Research Article |
Corresponding author: Yoko Matsumura ( yoko.matumura.hamupeni@gmail.com ) Academic editor: Michael Schmitt
© 2017 Yoko Matsumura, Haruki Suenaga, Yoshitaka Kamimura, Stanislav N. Gorb.
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:
Matsumura Y, Suenaga H, Kamimura Y, Gorb SN (2017) Traumatic mating by hand saw-like spines on the internal sac in Pyrrhalta maculicollis (Coleoptera, Chrysomelidae, Galerucinae). In: Chaboo CS, Schmitt M (Eds) Research on Chrysomelidae 7. ZooKeys 720: 77-89. https://doi.org/10.3897/zookeys.720.13015
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Morphology of the aedeagus and vagina of Pyrrhalta maculicollis and its closely related species were investigated. The internal sac of P. maculicollis bears hand saw-like spines, which are arranged in a row. Healing wounds were found on the vagina of this species, whose females were collected in the field during a reproductive season. However, the number of the wounds is low in comparison to the number of the spines. In addition, males of P. tibialis bear one spinous sclerite on the internal sac, but the female of this species show no wounds on the vagina. The vaginal wall is thicker in P. maculicollis and P. tibialis in comparison to other studied species, whose males bear no spinous sclerite. This thickening in P. maculicollis is hypothesized that they prevent damaging their own internal sac during everting and withdrawing the internal sac with the spines.
Copulation, genitalia, insect, internal sac, leaf beetles, mating systems
Traumatic mating is one of the well–observed phenomena in invertebrate mating systems (
In insects, male trauma inflicting structures usually locate in aedeagi, whose morphology is usually one of the best diagnoses especially in beetle systematics (
The male and female genitalia of the following four species were examined: Pyrrhalta maculicollis, P. humeralis, P. tibialis, and Tricholochmaea semifulva species complex with a special focus on P. maculicollis, which has spines on the internal sac. In regards to the scientific name, P. maculicollis had been treated as Xanthogaleruca maculicollis previously (e.g.
Examined specimens were mainly collected in the Okayama prefecture, Japan with some exceptions. For P. maculicollis we used individuals also from the Kanagawa prefecture, Japan, for P. tibialis two specimens from the Hokkaido prefecture, for P. humeralis one specimen from the Ehime prefecture, and for T. semifulva species complex two samples from the Oita prefecture.
To show general morphology and measure body sizes and the dimensions of genital spines, we dissected and observed samples under the stereomicroscopes (Nikon SMZ 745: Nikon Corporation, Tokyo, Japan; Olympus SZX12: Olympus Corporation, Tokyo, Japan; Leica M205 A with the camera Leica DFC420 and the software LAS 3.8: Leica Microscopy GmbH, Wetzlar, Germany) and the light microscope Zeiss Axioplan equipped with the camera Zeiss Axio Cam MRc (Carl Zeiss Microscopy GmbH, Jena, Germany). Then the sizes were measured with aids of the software Fiji (
To understand three-dimensional configuration of the aedeagus of Pyrrhalta maculicollis the aedeagus was dissected out from 99.5 % ethanol preserved specimens, dehydrated up to 100 %, and dried with a critical point drier (E3100 CPDA/Quorum Technologies LTD, Kent, UK). Then the sample was glued onto a thin–wall borosilicate glass capillary (120 × 1 mm, Hirschmann–Laborgeräte GmbH & Co. KG, Eberstadt, Germany) with super glue and scanned using the high–resolution micro–computed tomography (µCT) SkyScan 1172 (RJL Micro & Analytic GmbH, Karlsdorf–Neuthard, Germany) with a current of 250 µA and a voltage of 40 kV. Segmentation of each structure was carried out using the software Amira 5.4 (Visualization Sciences Group, Mérignac, France).
Some additional internal sac specimens of Pyrrhalta maculicollis were also dried with the same methods and sputter coated with gold–palladium (ca. 10 nm thickness) using the Leica EM SCD 500 High Vacuum Sputter Coater (Leica Microscopy GmbH) for detailed surface investigations using the Hitachi S4800 and TM3000 scanning electron microscopes (Hitachi High–Tech. Corp., Tokyo, Japan) at an accelerating voltage of 3 kV and ca. 15 kV, respectively. For interspecific comparisons, we also used internal sac samples dried at room temperature.
For interspecific comparisons of the vaginal wall thickness, we dissected female vaginas from freshly killed samples in phosphate–buffered saline (PBS; Carl Roth GmbH & Co. KG, Karlsruhe, Germany) and fixed with 2.5 % glutaraldehyde for one to three weeks. Two females per species were fixed except for P. tibialis, for which only one sample was treated. The samples were washed with PBS at least three times, dehydrated with a series of ethanol up to 100 % ethanol. Then they were gradually replaced with Epon 812 (Glycidether 100; Carl Roth GmbH & Co. KG), and finally the samples were embedded in the Epon resin. All procedures were processed at room temperature, but polymerisation was done at 60 °C for two days. Semi–thin sections (ca. 300–700 nm) were prepared using the Leica EM UC7 ultramicrotome (Leica Microscopy GmbH). Sections were stained with 0.1 % toluidine blue for three to four hours, and overstained dye was removed by retaining the slices in glycerine for two days. Images of the sections were then taken with the light microscope Zeiss Axioplan equipped with the camera Zeiss Axio Cam MRc. Following the method of
A Fisher’s exact probability test was adopted for comparing the occurrence rate of mating trauma among species. All statistical analyses were carried out using R 3.2.0 (R Core Team 2015).
The males have a relatively stout aedeagus (Fig.
Aedeagus of Pyrrhalta maculicollis in the lateral view 1 relative size of the aedeagus compared to body size 2 the aedeagus and a part of the ejaculatory duct, muscles completely macerated. Two arrows point to the membranous areas of the median lobe 3 a micro CT scanned and segmented aedeagus and a part of the ejaculatory duct. The green structure represents the part of the ejaculatory duct with well–developed circular muscles, the yellow one represents the median lobe, and the red ones represent spines on the internal sac 4 the internal sac at rest with a part of the ejaculatory duct (ed) 5 schemes of the aedeagus at rest (left) and with partly evaginated internal sac during copulation (right). Abbreviations: aed, aedeagus; ed, ejaculatory duct; is, internal sac; ml, median lobe.
The internal sac membrane and spines of Pyrrhalta maculicollis. 6, 7 the sample was cut laterally, opened, dehydrated, and dried at the critical point 8 the sample dried at room temperature. The surfaces of the spines is slightly shrunken in comparison to that depicted in 7. Abbreviations: mp, membranous projections; s, spines; each is highlighted with blue and pink, respectively.
The measurements of the male spines in Pyrrhalta maculicollis, Kanagawa population in Japan.
N | Mean ± S.D. (min.–max.) | |
Body size (mm) | 6 | 6.00 ± 0.12 (5.84–6.11) |
Spine number | 7 | 23.6 ± 2.4 (20– 8) |
Spine size (μm) | 7 | 126.2 ± 10.7 (110.8–145.0) |
Internal sac length (μm) | 7 | 2305 ± 83.9 (2204–2432) |
The vaginas of 13 female Pyrrhalta maculicollis were examined, collected during the reproductive season in the field (Kanagawa, Japan) and probably had already copulated. In most females (N = 11), 11.8 ± 6.3 (mean ± SD) wounds (2–25) were detected on both the ventral and dorsal sides of the vaginal wall, whereas no wound was found in the other two females. The sizes of wounds varied considerably and some were large enough to be visible under the stereomicroscope (Figs
Wounds observed on the vagina of Pyrrhalta maculicollis 9 schemes of three female vaginas, which had largest numbers of wounds, are shown. Each symbol represents one individual 10 one representative with four wounds 11, 12 enlarged images of the wounds. Abbreviations: sc: spermathecal capsule.
The shape of the median lobe of the studied species is similar except for Tricholochmaea semifulva species complex, whose median lobe is relatively slender.
The internal sac of Pyrrhalta tibialis and P. humeralis is broader than that of P. maculicollis and Tricholochmaea semifulva species complex (Figs
Schemes of hypothetical couplings between male and female genitalia, light microscopy (LM) and scanning electron microscopy (SEM) images of the male internal sac 13 Pyrrhalta maculicollis 14 P. tibialis 15 P. humeralis 16 Tricholochmaea semifulva species complex. The schemes were created using information on morphology and dimensions of both male and female genitalia, but the genital coupling has not been experimentally proven. The meshed areas in the male internal sacs show areas covered by tiny projections, as shown in the SEM images. All samples were dried at room temperature. Abbreviations: is, internal sac; mp, membranous projection; s, sclerite; sc, spermathecal capsule; sd, spermathecal duct; v, vagina.
The vaginal wall of Pyrrhalta maculicollis and P. tibialis shows relatively well developed epidermis and cuticle layers, if compared to that of the other species (Figs
Female reproductive systems: 17–19 Pyrrhalta maculicollis 20–22 P. tibialis 23 P. humeralis 24–25 Tricholochmaea semifulva species complex. 18, 21, 23, 25 show the cross sections of the lower part of the vagina, where wounds were found in P. maculicollis; each cuticular, epidermal, and muscular layer are partly highlighted in the upper right corner. 19, 22 show enlarged parts of the cuticular, epidermal, and muscular layers; these samples were embedded in glycerine for two days before taking the images. Arrowheads in 22 point to the strip stained with toluidine blue. Abbreviations: c, cuticle; e, epidermis; m, muscles; sc, spermathecal capsule.
The measurements of the areas of the vagina muscles and epidermal plus cuticular layers.
Species | N | Areas of muscles (µm2) | Areas of epidermis and cuticle (µm2) |
Pyrrhalta maculicollis | 2 | 102545 / 45674 | 180528 / 84607 |
Pyrrhalta tibialis | 2 | 12574 / 40076 | 73012 / 88799 |
Pyrrhalta humeralis | 1 | 27599 | 16555 |
Tricholochmaea semifulva species complex | 2 | 38635 / 13166 | 23952 / 3601 |
Any possible healing wounds were not found in the vagina of P. tibialis (N = 5) and P. humeralis (N = 4), indicating that mating trauma, if any, occurs significantly less frequently in these species than in P. maculicollis (Fisher’s exact probability test: P = 0.0025–0.0063). However, five small melanised patches were found in one out of four females of the T. semifulva species complex examined. Those patches were similar to the small ones in Figs
Among the examined Pyrrhalta spp., wounds were found significantly more in the vagina of P. maculicollis. This finding supports the view that the hand saw-like spines of the internal sac, which is characteristic of this species, are responsible for traumatic mating. Although we have neither compared virgin females with mated ones nor examined the genital coupling of the species, it is reasonable to estimate that the regularly arranged male spines are everted and face to the vaginal wall during copulations (Fig.
As in the cases of seed beetles (
The internal sac of P. maculicollis is relatively narrow in comparison to other Pyrrhalta species. The narrow internal sac would be problematic for bearing the spiny sclerites, since the spines may harm the internal sac, especially its dorsal surface, during its eversion and withdrawal (Fig.
In comparison to the seed beetles, whose spinous sclerites on the internal sac are arranged three dimensionally (
We thank J. Guhl (Kiel, Germany) for her assistance with experiments during her internship at Kiel University, E. Appel and J. Michels (Kiel University, Germany) for technical support in preparing histological sections, and H. Yoshitomi (Ehime University, Japan) for discussion on terminology. We also thank to J. Bezděk (Mendel University, Czech Republic) and H. Takizawa (Saitama Pref., Japan) for providing essential information on Pyrrhalta and Tricholochmaea species and two anonymous reviewers for their careful reading of our draft. This study was party supported through funding from the Japan Society of the Promotion of Science (postdoctoral fellowship, grant no. 15J03484) to YM and from the German Research Foundation (µCT) to SNG.