Sexual selection on bushcricket genitalia operates in a mosaic pattern.

In most species with internal fertilization, male genitalia evolve faster than other morphological structures. This holds true for genital titillators, which are used exclusively during mating in several bushcricket subfamilies. Several theories have been proposed for the sexual selection forces driving the evolution of internal genitalia, especially sperm competition, sexually antagonistic coevolution (SAC), and cryptic female choice (CFC). However, it is unclear whether the evolution of genitalia can be described with a single hypothesis or a combination of them. The study of species-specific genitalia action could contribute to the controversial debate about the underlying selective evolutionary forces. We studied female mating behaviors in response to experimentally modified titillators in a phylogenetically nested set of four bushcricket species: Roeseliana roeselii, Pholidoptera littoralis littoralis, Tettigonia viridissima (of the subfamily Tettigoniinae), and Letana inflata (Phaneropterinae). Bushcricket titillators have several potential functions; they stimulate females and suppress female resistance, ensure proper ampulla or spermatophore attachment, and facilitate male fixation. In R. roeselii, titillators stimulate females to accept copulations, supporting sexual selection by CFC. Conversely, titillator modification had no observable effect on the female's behavior in T. viridissima. The titillators of Ph. l. littoralis mechanically support the mating position and the spermatophore transfer, pointing to sexual selection by SAC. Mixed support was found in L. inflata, where manipulation resulted in increased female resistance (evidence for CFC) and mating failures by reduced spermatophore transfer success (evidence for SAC). Sexual selection is highly species-specific with a mosaic support for either cryptic female choice or sexually antagonistic coevolution or a combination of both in the four species.

Female perception of copulatory courtship by male titillators in a bushcricket.

Males of the bushcricket Metrioptera roeselii bear paired titillators that are spiny genital structures supposedly functioning as copulatory courtship devices. During copulation, the male inserts its titillators into the female's genital chamber, where they rhythmically tap on the sensilla-covered dorsal surface of the genital fold. Here, we investigated the stimulatory function of male titillators during mating in M. roeselii Tracer backfills of presumptive mechanosensory sensilla at the female genital fold revealed a thick bundle of sensory axons entering the last unfused abdominal ganglion (AG-7). Electrophysiological recordings of abdominal nerves demonstrated that females sense mechanical stimulation at their genital fold. The mechanosensory responses, however, were largely reduced by the insecticide pymetrozine that selectively blocks scolopidia of internal chordotonal organs but not campaniform and hair sensilla on the outer cuticle surface. In mating experiments, the females showed resistance behaviours towards males with asymmetrically shortened titillators, but the resistance was largely reduced when mechanoreceptors at the female's genital fold were either pharmacologically silenced by pymetrozine or mechanically blocked by capping with UV-hardened glue. Our findings support the hypothesis that the male titillators in these bushcrickets may serve as copulatory courtship devices to mechanically stimulate the female genitalia to reduce resistance behaviour.

Copulatory courtship by internal genitalia in bushcrickets.

Male genital organs are among the fastest evolving morphological structures. However, large parts of the male's genitalia are often hidden inside the female during mating. In several bushcricket species, males bear a pair of sclerotized genital appendices called titillators. By employing synchrotron-based in vivo X-ray cineradiography on mating couples, we were able to visualize titillator movement and spermatophore attachment inside the female. Titillators are inserted and retracted rhythmically. During insertion the titillator processes tap the soft and sensillae-covered dorsal side of the female's flap-like genital fold, which covers the opening of the female's genitalia, without tissue penetration. Titillators thus appear to be initially used for stimulation; later they may apply pressure that forces the female's genital fold to stay open, thereby aiding mechanically in spermatophore transfer.

Copulatory courtship by bushcricket genital titillators revealed by functional morphology, µCT scanning for 3D reconstruction and female sense structures.

Genitalia are rapidly evolving morphological structures most likely under sexual selection. Due to their internal nature they are often hidden inside the body, thus morpho-functional studies of animal genitalia are broadly lacking. Males of some bushcricket taxa bear paired genital appendices called titillators, the exact function of which is unknown since they are obscured inside the female body during pairing. To investigate titillator morphology and possible function during copulation, we studied the bushcricket Metrioptera roeselii (Hagenbach, 1822) using a novel combination of independent, yet complementary, techniques. Copulating pairs were snap-frozen and scanned by X-ray micro-computed tomography (µCT) to visualize the coupling of male and female genitalia in situ. Video recordings of copulating pairs also showed rhythmical insertion of male titillators into the female's genital chamber, where they percuss a softened structure on the female's subgenital plate. Movements did not induce damage to the female's structure, which lacks any sclerotized genital counterparts. Instead, scanning electron microscopy and histological sections show the female subgenital plate to be covered with two different types of sensory receptors at the contact zone between the male's titillator and the female genital chamber. We interpret the non-harmful function of the titillator processes, the lack of a genital counter-structure and the presence of sensory cells on the female's subgenital plate as indicators of a copulatory courtship function of titillators, subject to sexual selection by female choice.