Competition between males and their sperm over access to females and their eggs has resulted in manifold ways by which males try to secure paternity, ranging from physically guarding the female after mating to reducing her receptivity or her attractiveness to subsequent males by transferring manipulative substances or by mechanically sealing the female reproductive tract with a copulatory plug. Copulations may also result in internal damage of the female genitalia; however, this is not considered as a direct adaptation against sperm competition but as a collateral effect. Here, we present a drastic and direct mechanism for securing paternity: the removal of coupling structures on female genitalia by males. In the orb-weaving spider Larinia jeskovi males remove the scapus, a crucial coupling device on the female external genital region. Reconstruction of the coupling mechanism using micro-CT-scanned mating pairs revealed that several sclerites of the male genitalia interact to break off the scapus. Once it is removed, remating cannot occur due to mechanical coupling difficulties. In the field, male-inflicted genital damage is very prevalent since all female L. jeskovi were found to be mutilated at the end of the mating season. External genital mutilation is an overlooked but widely spread phenomenon since 80 additional spider species were found for which male genital manipulation can be suspected. Interlocking genitalia provide an evolutionary platform for the rapid evolution of this highly effective mechanism to secure paternity, and we suspect that other animal groups with interlocking genital structures might reveal similarly drastic male adaptations.

We unveiled the penile penetration mechanics of two earwig species, Echinosoma horridum, whose intromittent organ, termed virga, is extraordinarily long, and E. denticulatum, whose virga is conversely short. We characterised configuration, geometry, material and bending stiffness for both virga and spermatheca. The short virga of E. denticulatum has a material gradient with the stiffer base, whereas the long virga of E. horridum and the spermathecae of both species are homogeneously sclerotised. The long virga of E. horridum has a lower bending stiffness than the spermatheca. The virga of E. denticulatum is overall less flexible than the spermatheca. We compared our results to a previous study on the penetration mechanics of elongated beetle genitalia. Based on the comparison, we hypothesised that the lower stiffness of the male intromittent organ comparing to the corresponding female structure is a universal prerequisite for the penetration mechanics of the elongated intromittent organ in insects.

The broad-snouted caiman (Caiman latirostris) is a crocodilian species that inhabits South American wetlands. As in all other crocodilians, the egg incubation temperature during a critical thermo-sensitive window (TSW) determines the sex of the hatchlings, a phenomenon known as temperature-dependent sex determination (TSD). In C. latirostris, we have shown that administration of 17-β-estradiol (E2) during the TSW overrides the effect of the male-producing temperature, producing phenotypic females (E2SD-females). Moreover, the administration of E2 during TSW has been proposed as an alternative way to improve the recovery of endangered reptile species, by skewing the population sex ratio to one that favors females. However, the ovaries of E2SD-female caimans differ from those of TSD-females. In crocodilians, the external genitalia (i.e. clitero-penis structure or phallus) are sexually dimorphic and hormone-sensitive. Despite some morphological descriptions aimed to facilitate sexing, we found no available data on the C. latirostris phallus histoarchitecture or hormone dependence. Thus, the aims of this study were: (1) to establish the temporal growth pattern of the phallus in male and female caimans; (2) to evaluate histo-morphological features and the expression of estrogen receptor alpha (ERα) and androgen receptor (AR) in the phallus of male and female pre-pubertal juvenile caimans; and (3) to determine whether the phallus of TSD-females differs from the phallus of E2SD-females. Our results demonstrated sexually dimorphic differences in the size and growth dynamics of the caiman external genitalia, similarities in the shape and spatial distribution of general histo-morphological compartments, and sexually dimorphic differences in innervation, smooth muscle fiber distribution, collagen organization, and ERα and AR expressions. The external genitalia of E2SD-females differed from that of TSD-females in many histological features and in the expression of ERα and AR, resembling patterns described in males. Our results alert on the effects of estrogen agonist exposure during TSW and suggest that caution must be taken regarding the use of E2SD as a procedure for wildlife population management.

No abstract available

Armatures of the male intromittent copulatory structures have been surmised to increase male fitness by imposing physiological costs on female re-mating. Female kicking could, consequently, be a counterstrategy to avoid wounding or to prevent males from mating. The membranous endophallus of male Acanthoscelidesobtectus (Say, 1831) is armed with denticles. Checking if these denticles penetrate the wall of the female genital tract during copulation revealed that only the tip of the median lobe of the aedeagus is intromitted into the female genital opening during copulation. The everted endophallus extends over the full length of the ovipositor, and the spermatophore is placed in the bursa. Identification by means of light microscopy and Micro-CT of the exact relative position of male and female copulatory organs while mated confirmed that the denticles do not cause wounds in the vagina wall. Parts of the inner wall of the bursa copulatrix are covered with inward pointing denticles. Already mated females kick mounting males by vehement movements of their hind legs, thereby preventing mating. In contrast, virgin females usually accept the first male they encounter and terminate copulation by slower movements of their hind legs. The same applied to females who accepted re-mating the second day after the first copulation. Acanthoscelidesobtectus females kick males off to prevent rather than to terminate copulation. Copulatory structures as well as behaviour may have different functional roles in different beetle species, even within the Bruchinae.

In Calopteryx damselflies, males remove rivals' sperm stored by the female, thereby reducing sperm competition. This behaviour may create a sexual conflict, because females could lose the sperm stored in the spermatheca, used for long-term storage. Comparative evidence suggested antagonistic coevolution between sexes, which might prompt the evolution of narrow spermathecal ducts, or longer spermathecae, hindering sperm removal. Calopteryx haemorrhoidalis and C. splendens coexist and sometimes hybridize. Therefore, here I predicted that if females coevolve with conspecific males, heterospecific males should have an advantage when interspecific matings occur because females will show less resistance to them than to conspecific males. By hand-pairing females to males of both species, I found that in intraspecific and interspecific matings, sperm was almost completely removed from the bursa (97-100%), but only partially from the spermathecae, with more spermathecal removal in interspecific (63-71%) than intraspecific matings (14-33%). This suggests that heterospecific males are more efficient in sperm removal as predicted by a sexually-antagonistic coevolutionary scenario. Furthermore, in most cases, only the left spermatheca was emptied, suggesting that the evolution of more than one spermatheca might also be a female counter-adaptation to regain control over fertilization.

The specification and morphogenesis of an organ requires the coordinate deployment and integration of regulatory information, including sex specific information when the organ is sex specific. Only a few gene networks controlling size and pattern development have been deciphered, which limits the emergence of principles, general or not, underlying the organ-specifying gene networks. Here we elucidate the genetic and molecular network determining the control of size in the Drosophila abdominal A9 primordium, contributing to the female genitalia. This network requires axial regulatory information provided by the Hox protein Abdominal-BR (Abd-BR), the Hox cofactors Extradenticle (Exd) and Homothorax (Hth), and the sex specific transcription factor Doublesex Female (DsxF). These factors synergize to control size in the female A9 by the coordinate regulation of the Decapentaplegic (Dpp) growth pathway. Molecular dissection of the dpp regulatory region and in vivo protein interaction experiments suggest that Abd-BR, Exd, Hth and DsxF coordinately regulate a short dpp enhancer to repress dpp expression and restrict female A9 size. The same regulators can also suppress dpp expression in the A8, but this requires the absence of the Abd-BM isoform, which specifies A8. These results delineate the network controlling female A9 growth in Drosophila.

The LHCGR gene encodes a G-protein coupled receptor that plays a pivotal role in sexual differentiation in males, ovarian development in females and in fertility via its interaction with luteinizing hormone and chorionic gonadotropin. Inactive variants of the LHCGR gene cause Leydig cell hypoplasia (LCH), which is a rare disease and one of the causes of disorder of sexual differentiation (DSD) in males. The aim of this work was to clarify the clinical and molecular characteristics of a 2.75 year old patient with type 1 LCH. Whole exome sequencing was performed for the patient family and variants in the LHCGR gene were validated by Sanger sequencing. Pathogenicity of the missense variant was evaluated by multiple in silico tools. Our Chinese patient, who exhibited DSD, had female external genitalia (normal labia majora and minora, external opening of urethra under the clitoris and blind-ended vagina) and bilateral testis tissues in the inguinal region. Genetic sequencing revealed compound heterozygous variants in the LHCGR gene in the patient, including a novel missense variant in exon 4 (c.349G>A, p.Gly117Arg) and a novel nonsense variant in exon 10 (c.878C>A, p.Ser293*). The missense variant is in the first leucine-rich repeat domain of the LHCGR protein, which is predicted to affect ligand recognition and binding affinity and thus protein function. The patient is molecularly and clinically diagnosed with type 1 LCH, which is caused by novel, compound heterozygous variants of the LHCGR gene. We believe this report will serve to expand the genotypic spectrum of LHCGR variants.

Some sexual traits, including genitalia, have undergone coevolutionary diversification toward exaggerated states in both sexes among closely related species, but the underlying genetic mechanisms that allow correlated character evolution between the sexes are poorly understood. Here, we studied interspecific differences in gene expression timing profiles involved in the correlated evolution of corresponding male and female genital parts in three species of ground beetle in Carabus (Ohomopterus). The male and female genital parts maintain morphological matching, whereas large interspecific variation in genital part size has occurred in the genital coevolution between the sexes toward exaggeration. We analyzed differences in gene expression involved in the interspecific differences in genital morphology using whole transcriptome data from genital tissues during genital morphogenesis. We found that the gene expression variance attributed to sex was negligible for the majority of differentially expressed genes, thus exhibiting sex-concordant expression, although large variances were attributed to stage and species differences. For each sex, we obtained co-expression gene networks and hub genes from differentially expressed genes between species that might be involved in interspecific differences in genital morphology. These gene networks were common to both sexes, and both sex-discordant and sex-concordant gene expression were likely involved in species-specific genital morphology. In particular, the gene expression related to exaggerated genital size showed no significant intersexual differences, implying that the genital sizes in both sexes are controlled by the same gene network with sex-concordant expression patterns, thereby facilitating the coevolution of exaggerated genitalia between the sexes while maintaining intersexual matching.

Two new species of genus Oecleopsis Emeljanov, 1971, O. laminatus Zhi & Chen, sp. n. and O. productus Zhi & Chen, sp. n., and a new record, O. yoshikawai (Ishihara, 1961), are described and illustrated from China. Female genitalia of five species within this genus are compared morphologically: O. laminatus Zhi & Chen, sp. n., O. mori (Matsumura, 1914), O. productus Zhi & Chen, sp. n., O. sinicus (Jacobi, 1944) and O. yoshikawai (Ishihara, 1961). A key to five Chinese species based on female genitalia, and a key to all known species of Oecleopsis based on male genitalia, are provided.

The evolution of asymmetry in male genitalia is a pervasive and recurrent phenomenon across almost the entire animal kingdom. Although in some taxa the asymmetry may be a response to the evolution of one-sided, male-above copulation from a more ancestral female-above condition, in other taxa, such as Mammalia and Coleoptera, this explanation appears insufficient. We carried out an informal assessment of genital asymmetry across the Coleoptera and found that male genital asymmetry is present in 43% of all beetle families, and at all within-family taxonomic levels. In the most diverse group, Cucujiformia, however, genital asymmetry is comparatively rare. We also reconstructed the phylogeny of the leiodid tribe Cholevini, and mapped aspects of genital asymmetry on the tree, revealing that endophallus sclerites, endophallus, median lobe and parameres are, in a nested fashion, increasingly unlikely to have evolved asymmetry. We interpret these results in the light of cryptic female choice versus sexually antagonistic coevolution and advocate further ways in which the phenomenon may be better understood.This article is part of the themed issue 'Provocative questions in left-right asymmetry'.

Two new species of cixiid planthoppers genus Tsauria Koçak & Kemal, Tsauriabrevispina Zhi & Chen, sp. nov. and T.longispina Zhi & Chen, sp. nov., are described and illustrated from China and T.transspinus (Zhang & Chen, 2011) was removed to give the genus four species in total. The female genitalia of three species are described and illustrated for the first time. A key to all known species of Tsauria based on male genitalia, and a key to three species (except for T.major) based on female genitalia, are provided.

Triatoma is the most diversified and one of the most important genera from an epidemiological perspective. Given the difficulty in identifying some species of the Triatoma genus, morphological, histological, and morphometric studies were performed to provide new characters that make it possible to differentiate T. garciabesi, T. guasayana, T. patagonica, and T. sordida sensu stricto, triatomines that overlap geographically and have vector potential. Through the external female genitalia, as well as morphology, morphometry, and histology of eggshells, it was possible to discriminate the four species. In addition, this study reinforces the taxonomic validity of T. garciabesi and provides new data for discussion on systematic issues of T. guasayana and T. patagonica.

Animal morphology and behavior often appear to evolve cooperatively. However, it is difficult to assess how strictly these two traits depend on each other. The genitalia morphologies and courtship behaviors in insects, which vary widely, may be a good model for addressing this issue. In Diptera, phylogenetic analyses of mating positions suggested that the male-above position evolved from an end-to-end one. However, with this change in mating position, the dorsoventral direction of the male genitalia became upside down with respect to that of the female genitalia. It was proposed that to compensate for this incompatibility, the male genitalia rotated an additional 180° during evolution, implying evolutionary cooperativity between the mating position and genitalia direction. According to this scenario, the proper direction of male genitalia is critical for successful mating. Here, we tested this hypothesis using a Drosophila Myosin31DF (Myo31DF) mutant, in which the rotation of the male genitalia terminates prematurely, resulting in various deviations in genitalia direction. We found that the proper dorsoventral direction of the male genitalia was a prerequisite for successful copulation, but it did not affect the other courtship behaviors. Therefore, our results suggested that the male genitalia rotation and mating position evolved cooperatively in Drosophila.

The external female genitalia of 29 species belonging to three genera of Megalopodidae and 80 species belonging to 61 genera of another four families in Chrysomeloidea were studied. The external female genitalia within the superfamily Chrysomeloidea can be divided into a cerambycid type and a chrysomelid type. The comparative study of external female genitalia shows Megalopodidae is more closely related to the family Cerambycidae than to the family Chrysomelidae s.l. Among five subfamilies of Cerambycidae we studied, the subfamily Lamiinae is most closely allied to Megalopodidae. An evolutionary path is proposed for the spiculum gastrale in Chrysomeloidea: the characteristic state of the spiculum gastrale without a joint is primary, and that with a joint is secondary. The family Orsodacnidae has probably evolved in isolation from the early chrysomelids, due to their shared external female genitalia (cerambycid type). In the family Chrysomelidae, the structure of external female genitalia and ovipositing behavior show that the subfamily Synetinae is closer to the Camptosomata than the subfamily Eumolpinae. In general, the shape of the terminal ovipositor is palp-like in the Chrysomeloidea. Terminal ovipositors are generally palp-shaped in Chrysomeloidea except for those that are lamellate in the genus Callispa and the subfamily Cassidinae who produce egg-sheaths.

Five DSD heifers underwent genetic analysis in the present study. We cytogenetically analyzed in vitro cultured leukocytes and searched for SRY, AMELX/AMELY and ZFX/ZFY genes in leukocytes and hair follicles, finding that four of the studied heifers were freemartins (XX/XY leukocyte chimerism). The fifth case had an underdeveloped vulva localized ventrally and cranially to the mammary gland, a normal female sex chromosome complement (60,XX) in the leukocytes, and a lack of Y-chromosome-derived genes in the leukocytes and hair follicles. Postmortem anatomical examination of this heifer revealed the presence of normal ovaries with follicles, uterus, and oviducts, but molecular detection of the SRY, ZFX, ZFY,AMELX, and AMELY genes in these organs indicated the presence of a cell line carrying the Y chromosome. Further analysis of twelve microsatellite markers revealed the presence of additional variants at six loci in DNA samples derived from the reproductive organs; XX/XY chimerism was thus suspected in these samples. On the basis of the detection of AMELY (Y-linked) versus AMELX (X-linked) and SOX9 (autosomal) versus AMELY genes by droplet digital PCR (ddPCR), the Y/X and Y/autosome ratios were evaluated; they indicated the presence of XX and XY cell lines in the reproductive tissues. Our study showed that XX/XY chimerism can be present in the internal reproductive organs of the virilized heifers with a normal female set of sex chromosomes (60,XX) and a lack of Y-chromosome-derived genes in the leukocytes. The etiology of this phenomenon remains unknown.

Insect genitalia exhibit rapid divergent evolution. Truly extraordinary structures have evolved in some groups, presumably as a result of postmating sexual selection. To increase our understanding of this phenomenon, we studied the function of one such structure. The male genitalia of Callosobruchus subinnotatus (Coleoptera: Bruchinae) contain a pair of jaw-like structures with unknown function. Here, we used phenotypic engineering to ablate the teeth on these jaws. We then experimentally assessed the effects of ablation of the genital jaws on mating duration, ejaculate weight, male fertilization success and female fecundity, using a double-mating experimental design. We predicted that copulatory wounding in females should be positively related to male fertilization success; however, we found no significant correlation between genital tract scarring in females and male fertilization success. Male fertilization success was, however, positively related to the amount of ejaculate transferred by males and negatively related to female ejaculate dumping. Ablation of male genital jaws did not affect male relative fertilization success but resulted in a reduction in female egg production. Our results suggest that postmating sexual selection in males indeed favors these genital jaws, not primarily through an elevated relative success in sperm competition but by increasing female egg production.

In the last 240,000 years, males of the Drosophila simulans species clade have evolved striking differences in the morphology of their epandrial posterior lobes and claspers (surstyli). These appendages are used for grasping the female during mating and so their divergence is most likely driven by sexual selection. Mapping studies indicate a highly polygenic and generally additive genetic basis for these morphological differences. However, we have limited understanding of the gene regulatory networks that control the development of genital structures and how they evolved to result in this rapid phenotypic diversification. Here, we used new D. simulans/D. mauritiana introgression lines on chromosome arm 3L to generate higher resolution maps of posterior lobe and clasper differences between these species. We then carried out RNA-seq on the developing genitalia of both species to identify the expressed genes and those that are differentially expressed between the two species. This allowed us to test the function of expressed positional candidates during genital development in D. melanogaster. We identified several new genes involved in the development and possibly the evolution of these genital structures, including the transcription factors Hairy and Grunge. Furthermore, we discovered that during clasper development Hairy negatively regulates tartan (trn), a gene known to contribute to divergence in clasper morphology. Taken together, our results provide new insights into the regulation of genital development and how this has evolved between species.

Sexually dimorphic outgrowth and differentiation of the embryonic genital tubercles (GTs) give rise to the penis in males and the clitoris in females. Defects in androgen production or in response to androgen signaling can lead to various congenital penile anomalies in both mice and humans. Due to lack of a high-throughput screening system, identification of crucial regulators of GT sexual differentiation has been slow. To overcome this research barrier, we isolated embryonic GT mesenchymal (GTme) cells to model genitalia growth and differentiation in vitro. Using either a mechanical or fluorescence-activated cell sorting-assisted purification method, GTme cells were isolated and assayed for their proliferation using a microscopy and image analysis system, on a single cell level over time. Male and female GTme cells inherently exhibit different cellular dynamics, consistent with their in-vivo behaviors. This system allows for the rapid quantitative analyses of numerous drug treatments, and enables the discovery of potential genetic modulators of GT morphogenesis on a large scale. Using this system, we completed a 438-compound library screen and identified 82 kinase inhibitor hits. In mice, in-utero exposure to one such candidate kinase inhibitor, Cediranib, resulted in embryos with severe genitalia defects, especially in males. Gene silencing by RNAi was optimized in this system, laying the foundation for future larger-scale genetic screenings. These findings demonstrate the power of this novel high-throughput system to rapidly and successfully identify modulators of genitalia growth and differentiation, expanding the toolbox for the study of functional genomics and environmental factors.

Sperm removal behaviour (SRB) is known in many animals, and male genital structures are often involved in the SRB, e.g. rubbing female genitalia vigorously. However, it remains unclear how those male genital structures function properly without severe genital damage during SRB. In the present study, we focused on the bushcricket Metaplastes ornatus and examined the biomechanics of male and female genital structures, involved in their SRB as a model case. During an initial phase of mating, males of this species thrust their subgenital plate with hook-like spurs and many microscopic spines into the female genital chamber. By moving the subgenital plate back-and-forth, males stimulate females, and this stimulation induces the ejection of sperm previously stored in females. We aimed to uncover the mechanics of the interaction between the subgenital plate and genital chamber during SRB. The genital morphology and its material composition were investigated using modern imaging and microscopy techniques. The obtained results showed a pronounced material heterogeneity in the subgenital plate and the genital chamber. The material heterogeneity was completely absent in that of a second bushcricket species, Poecilimon veluchianus, which does not exhibit SRB. Finite element simulations showed that the specific material heterogeneity can redistribute the stress in the subgenital plate of M. ornatus and, thereby, reduces stress concentration during SRB. This may explain why only a few examined males had a broken spur. We suggest that the observed structural features and material heterogeneity in M. ornatus are adaptations to their SRB.