Traumatic Insemination Research Articles

Under the radar: Transcriptomic responses of bed bugs to an entomopathogen, environmental bacteria, and a human pathogen

Bed bugs (Hemiptera: Cimicidae) are widely distributed, obligately blood-feeding insects, but they have never been linked to pathogen transmission in humans. Most other hematophagous insects that frequently bite humans transmit pathogens, and it is unclear why bed bugs do not. One hypothesis is that bed bugs have evolved a highly robust immune system because their mating system, traumatic insemination, exposes females to consistent wounding and bacterial infections. Although this has been proposed, very little is known about the bed bug immune system and how bed bugs respond to microbial challenges introduced by wounding. Similarly, there is little known about how the bed bug immune system responds to human pathogens. Understanding the bed bug immune system could give insight to why bed bugs appear not to transmit disease and under what circumstances they could, while also facilitating biological control efforts involving microbes. To investigate the transcriptomic response of bed bugs to immune challenges, we exposed female bed bugs to three bacterial challenges. 1.) Pseudomonas fluorescens, an entomopathogen known to have harmful effects to bed bugs, 2.) bacteria cultured from a bed bug enclosure (99.9 % Bacillus spp.), likely encountered during traumatic insemination, and 3.) Borrelia duttoni, a human vector-borne pathogen that causes relapsing fever. We compared the transcriptomes of infected bed bugs with uninfected matched controls in a pairwise fashion, focusing on immune-related genes. We found many known antimicrobial effector genes upregulated in response to P. fluorescens and traumatic insemination-associated bacteria, but interestingly, not in response to B. duttoni. In the differentially expressed genes that were shared between experiments, we found significant overlap in the P. fluorescens treatment and the traumatic insemination bacteria treatment, and between the P. fluorescens and B. duttoni treatments, but not between the traumatic insemination bacteria treatment and the B. duttoni treatment. Finally, we identify previously overlooked candidates for future studies of immune function in bed bugs, including a peroxidase-like gene, many putative cuticle-associated genes, a laccase-like gene, and a mucin-like gene. By taking a comprehensive transcriptomic approach, our study is an important step in understanding how bed bugs respond to diverse immune challenges.

Sexual conflict and sexual networks in bed bugs: the fitness cost of traumatic insemination, female avoidance and male mate choice.

Sexual conflict is prevalent among animals and is primarily caused by the fact that the optimal mating rates are often higher in males than in females. While there is a growing appreciation that females can also gain from multiple matings, we still know relatively little about which sex controls the observed mating rates and how close it is to the optimal female mating rates. To address this issue, we tracked female bed bugs (Cimex lectularius) inseminated daily versus weekly and found that weekly inseminated females lived longer and produced over 50% more offspring. In a follow-up experiment employing a social network framework, we placed 24 bed bugs into a semi-naturalistic arena and recorded all sexual interactions. While recently inseminated females did not avoid males more often, they were more frequently rejected by males. Finally, we tracked avoidance behaviour in a single cohort of female bed bugs as they received six successive daily inseminations. Avoidance rates increased and insemination durations decreased with increasing number of prior inseminations. Overall, our results indicate high costs of polyandry. Although females possess some plastic avoidance strategies, the observed rates of insemination fall closer to the male rather than female optimum.

The puncture mechanics: an example from the bed bug Cimex lectularius showing traumatic insemination using the paramere.

Cimicidae are well-known for traumatic insemination, and males pierce females with their parameres and transfer sperm through them. The shape of parameres is relatively stable in the family, but in some genera, the paramere is elongated, appearing less resistant against lateral deflection. To understand the mechanical limitations of the paramere, we studied its penetration mechanics of the common bed bug, Cimex lectularius. We examined the post-abdominal morphology, paramere geometry and material properties and conducted breaking stress experiments on the paramere under wet and dry conditions. Mechanical property gradients are present with the paramere tip as the stiffest region and the base as the most flexible one. These mechanical properties relate to the presence of Ca, Zn and Si. The basal wing-shaped structure is flexible, enabling it to interlock with the anal region during mating. The paramere is slightly twisted; the tip region is circular in cross-section, and the geometry of the rest is rather complex. In the mechanical tests, wet parameres mainly buckled, while dried parameres broke off. The level of structural failures depended on directions from which the compression forces were applied. Structural, material and mechanical strengthening mechanisms preventing the paramere from mechanical failure are discussed.

Age-related mating rates among ecologically distinct lineages of bedbugs, Cimex lectularius

Understanding how many mates an animal has in its lifetime is a critical factor in sexual selection. At the same time, differences in an organism's ecology, such as the quantity and quality of food, could be reflected in different mating rates. Mating rate had a significant effect on female net fitness (i.e., lifetime offspring production), however, laboratory measurements cannot well mirror the situation in wild. The common bedbug (Cimex lectularius) is a well-established model for studying traumatic insemination and sexual conflict. The species comprises two host lineages that feed on bats (BL) or humans (HL). HL can constantly feed on human hosts throughout the year, while BLs feed only during summer months when their bat hosts occupy the roosts. Because mating in female bedbugs is closely linked to foraging, this system provides a valuable model to study mating variation in the field. We established a new method for estimating age-dependent mating rates of females in the wild by relating the fluorescent pigment accumulation in the eyes of females to the number of mating scars that manifest as melanized spots caused by the injection of sperm through the wall of the female abdomen by the male into the spermalege. In addition, using laboratory bedbugs we found that three and a half observed matings on average lead to one observed melanized mating scar. Although young BL and HL females (with low pteridine concentrations) did not differ in the number of matings, the mating rate increased with age only in HL but not in BL females. We sampled on average older BL than HL females. The lack of access to food (bat blood) during winter could explain the lack of increase in the number of scars with age in BL. In species where mating leaves visible marks, using fluorescent pigments to determine female age (applicable to most arthropods) could be an important tool to study sexual selection and mating rate in the wild. The method can help formulate sustainable and biologically lucid approaches for their control.

‘Stab, chase me, mate with me, seduce me’: how widespread is traumatic insemination in Strepsiptera?

Abstract Traumatic insemination refers to mating in which males pierce the female’s integument with his penis for insemination. Strepsiptera are often listed as an example for this mode of copulation. However, while traumatic insemination in Mengenillidae with free-living females is undisputed, its occurrence in Stylopidia with permanent endoparasitic females – 97% of the known species of Strepsiptera – has remained unclear. Rather, observations from a single study on Xenos vesparum (Xenidae) that questioned traumatic insemination in this species became generalized for Stylopidia. Here we show that integration of data from various imaging methods provides convincing evidence for traumatic insemination being phylogenetically widespread in Strepsiptera. Specifically, we provide the first evidence of injury wounds from traumatic insemination in species of Mengenillidae, Corioxenidae, Elenchidae, Halictophagidae and Xenidae. Using three-dimensional models of copulating pairs of Stylops ovinae (Stylopidae) and X. vesparum, we visualize the physical piercing of the female’s integument by the male’s penis. Finally, we show in species of Mengenillidae, Xenidae and Stylopidae that traumatic mating is associated with the injection of sperm in the female’s haemocoel. Our results significantly alter the understanding of the reproductive biology of Strepsiptera and imply that traumatic insemination has been the ancestral mode of copulation and retained in most, if not all, extant families.

Molecular Phylogeny of Cimicoidea (Heteroptera: Cimicomorpha) Revisited: Increased Taxon Sampling Reveals Evolution of Traumatic Insemination and Paragenitalia

The molecular phylogeny of the Cimicoidea was reconstructed from an expanded sampling based on mitochondrial (16S, COI) and nuclear (18S, 28SD3) genes. The data were analyzed using maximum likelihood (ML), maximum parsimony (MP), and Bayesian inference (BI) phylogenetic frameworks. The phylogenetic relationships inferred by the model-based analyses (ML and BI) were largely congruent with those inferred by the MP analysis in terms of the monophyly of most of the higher taxonomic groups and the species-level relationships. The following clades were recovered in all analyses: Cimiciformes; Nabidae: Prostemmatinae; Nabidae: Nabinae; Plokiophilidae; Microphysidae; Lasiochilidae; Cimicidae: Cacodminae; Cimicidae; Lyctocoridae; Anthocoridae s. str.; Cardiastethini excluding Amphiareus; Almeidini; Scolopini; Anthocorini; Oriini; Curaliidae + Lasiochilidae; Almeidini + Xylocorini; Oriini + Cardiastethini; and Anthocorini + Amphiareus. Reconstructions of ancestral copulation states based on Bayesian and parsimony inference indicated that at least one shift from standard insemination (SI) to traumatic insemination (TI) occurred within Cimicoidea, and an investigation of the evolutionary correlation between TI and paragenitalia (PG) revealed that the acquisition of PG in cimicoid females was correlated with the TI habit. Additionally, our morphological examination of various types of PG suggested that even the same PG type may not constitute a homologous feature at various taxonomic levels, indicating the convergent evolution of female morphology to adapt to TI.

A new remarkable cimicoid genus and species (Hemiptera, Heteroptera, Cimicomorpha) from mid-Cretaceous Burmese amber, with implications for its aberrant male genitalia

A new genus and species of cimicoid true bug,Ecpaglocoris ditomeusYamada & Yamamoto,gen. et sp. nov., is described and illustrated from mid-Cretaceous (Cenomanian–Albian) amber in the Kachin State of northern Myanmar (Burma). This new fossil genus and species is reminiscent of members of Anthocoridae by the strongly flattened and elongated body, four-segmented labium, distinct costal fracture and presence of fossula spongiosa on fore tibiae, but should not be ascribed to this family. The new taxon cannot be placed in any extant cimicoid families, based upon hemelytral, male genital and other morphological structures. Based on the hemelytral membrane venation and presence of dorsal laterotergites on abdominal segments I to VIII, it can be assumed that this new genus belongs to the extinct family Vetanthocoridae.Ecpaglocoris ditomeusgen. et sp. nov.has aberrant male genitalia characterised by sickle-shaped left and right parameres and grooves running throughout the paramere. This characteristic indicates that traumatic insemination occurred in this genus. The peculiar combination of male genital characteristics seen inEcpaglocorisgen. nov.prevents its placement in any of the extant cimicoid families.

The social consequences of sexual conflict in bed bugs: social networks and sexual attraction

Sexual conflict is ubiquitous across the animal kingdom and often involves costly sexual harassment of females by males. An overlooked outcome of sexual conflict is its potential impact on social behaviour. Due to their seemingly harmful mode of copulation (traumatic insemination) and tendency to form aggregations, bed bugs are an ideal model for studying the social implications of sexual conflict. Repeated traumatic inseminations are known to reduce some aspects of female fitness, so we expected the benefits to males and the high costs for females of frequent mating to result in divergent social preferences between the sexes. To examine the impact of sexual harassment on social structure, we devised a novel experimental arena with either two shelters or 12 shelters and continuously tracked sexual and social interactions between individually marked bed bugs over 6 days. By constructing aggregation networks, we examined whether female bed bugs occupied more peripheral network positions compared to males as well as whether females preferentially associated with other females as a strategy to reap the benefits of group living while mitigating the costs of unsolicited sexual attention. We found no evidence that females shape their social environment to evade associating with males. However, when tested individually in a follow-up experiment, mated females showed a strong preference for social cues from females over social cues from males. Our results therefore suggest that males and females may be in conflict over the composition of social associations and highlight the importance of both examining behaviour at the individual level and tracking larger groups of freely interacting populations in more complex environments. • Sexual conflict may differentially influence social behaviour of females and males. • We constructed bed bug social networks to see whether females socially evade males. • Aggregation networks did not show female avoidance through decreased sociality. • Social networks were also not assorted by sex. • However, females preferred social cues associated with other females over males.

Have female twisted-wing parasites (Insecta: Strepsiptera) evolved tolerance traits as response to traumatic penetration?

Traumatic insemination describes an unusual form of mating during which a male penetrates the body wall of its female partner to inject sperm. Females unable to prevent traumatic insemination have been predicted to develop either traits of tolerance or of resistance, both reducing the fitness costs associated with the male-inflicted injury. The evolution of tolerance traits has previously been suggested for the bed bug. Here we present data suggesting that tolerance traits also evolved in females of the twisted-wing parasite species Stylops ovinae and Xenos vesparum. Using micro-indentation experiments and confocal laser scanning microscopy, we found that females of both investigated species possess a uniform resilin-rich integument that is notably thicker at penetration sites than at control sites. As the thickened cuticle does not seem to hamper penetration by males, we hypothesise that thickening of the cuticle resulted in reduced penetration damage and loss of haemolymph and in improved wound sealing. To evaluate the evolutionary relevance of the Stylops-specific paragenital organ and penis shape variation in the context of inter- and intraspecific competition, we conducted attraction and interspecific mating experiments, as well as a geometric-morphometric analysis of S. ovinae and X. vesparum penises. We found that S. ovinae females indeed attract sympatrically distributed congeneric males. However, only conspecific males were able to mate. In contrast, we did not observe any heterospecific male attraction by Xenos females. We therefore hypothesise that the paragenital organ in the genus Stylops represents a prezygotic mating barrier that prevents heterospecific matings.

Frequent origins of traumatic insemination involve convergent shifts in sperm and genital morphology.

Traumatic insemination is a mating behavior during which the (sperm) donor uses a traumatic intromittent organ to inject an ejaculate through the epidermis of the (sperm) recipient, thereby frequently circumventing the female genitalia. Traumatic insemination occurs widely across animals, but the frequency of its evolution, the intermediate stages via which it originates, and the morphological changes that such shifts involve remain poorly understood. Based on observations in 145 species of the free‐living flatworm genus Macrostomum, we identify at least nine independent evolutionary origins of traumatic insemination from reciprocal copulation, but no clear indication of reversals. These origins involve convergent shifts in multivariate morphospace of male and female reproductive traits, suggesting that traumatic insemination has a canalizing effect on morphology. We also observed sperm in both the sperm receiving organ and within the body tissue of two species. These species had intermediate trait values indicating that traumatic insemination evolves through initial internal wounding during copulation. Finally, signatures of male‐female coevolution of genitalia across the genus indicate that sexual selection and sexual conflict drive the evolution of traumatic insemination, because it allows donors to bypass postcopulatory control mechanisms of recipients.

Refutation of traumatic insemination in the Drosophila bipectinata species complex

Traumatic insemination (TI) is a rare reproductive behaviour characterized by the transfer of sperm to the female via puncture wounds inflicted across her body wall. Here, we challenge the claim made by Kamimura (Kamimura 2007 Biol. Lett. 3, 401–404. (doi:10.1098/rsbl.2007.0192)) that males of species of the Drosophila bipectinata complex use a pair of claw-like processes (claws) to traumatically inseminate females: the claws are purported to puncture the female body wall and genital tract, and to inject sperm through the wounds into the lumen of her genital tract, bypassing the vaginal opening. This supposed case of TI is widely cited and featured in prominent subject reviews. We examined high-resolution scanning electron micrographs of the claws and failed to discover any obvious ‘groove’ for sperm transport. We demonstrated that sperm occurred in the female reproductive tract as a single-integrated unit, inconsistent with the claim that sperm are injected via paired processes. Laser ablation of the sharp terminal ends of the claws failed to inhibit insemination. We showed that the aedeagus in the complex delivers sperm through the vaginal opening, as in other Drosophila. The results refute the claim of TI in the Drosophila bipectinata species complex.

No vagina, one vagina, or multiple vaginae? An integrative study of Pseudaxine trachuri (Monogenea, Gastrocotylidae) leads to a better understanding of the systematics of Pseudaxine and related genera.

The presence/absence and number of vaginae is a major characteristic for the systematics of the Monogenea. Three gastrocotylid genera share similar morphology and anatomy but are distinguished by this character: Pseudaxine Parona & Perugia, 1890 has no vagina, Allogastrocotyle Nasir & Fuentes Zambrano, 1983 has two vaginae, and Pseudaxinoides Lebedev, 1968 has multiple vaginae. In the course of a study of Pseudaxine trachuri Parona & Perugia 1890, we found specimens with structures resembling “multiple vaginae”; we compared them with specimens without vaginae in terms of both morphology and molecular characterisitics (COI barcode), and found that they belonged to the same species. We also investigated the male copulatory organ (MCO) of this species, the accuracy of the original description of which is known to be a matter of debate. We found that the genital atrium is armed with 12 hooks arranged as a single circle and a central hollow stylet which is probably involved in traumatic insemination. We redescribed Pseudaxine trachuri based on newly collected specimens from off the coast of Algeria and Museum specimens from off France. Specimens from the type-host, Trachurus trachurus, were found to be similar, for both molecular sequences and morphology, to those found on Boops boops. We can therefore confirm, for the first time with molecular evidence, that B. boops is a host of this parasite. We consider that Pseudaxinoides was erected on the basis of an erroneous interpretation of structures which are not vaginae and, consequently, propose the transfer of most of its species to Pseudaxine, as P. australis (Lebedev, 1968) n. comb., P. bychowskyi (Lebedev, 1977) n. comb., P. caballeroi (Lebedev, 1977) n. comb., P. cariacoensis (Nasir & Fuentes-Zambrano, 1983) n. comb., and P. vietnamensis (Lebedev, Parukhin & Roitman, 1970) n. comb. We also propose Allogastrocotyle dillonhargisorum nom. nov. for Pseudaxine bivaginalis Dillon & Hargis, 1965 to avoid a secondary homonymy.

Sexual conflict during Mesozoic: The first Cretaceous damsel bug in Burmese amber (Hemiptera: Heteroptera: Nabidae)

The oldest Nabidae, Cretanazgul camillei gen. et sp. nov., is described from the mid-Cretaceous Burmese amber and attributed to the extant subfamily Prostemmatinae. This new record is in accordance with the emergence of the Nabidae between 150 and 130 Ma. This new fossil should help to precise future dating of this family. As the amber area of Myanmar was probably an island of the Thetys Ocean during the mid-Cretaceous, originating from the Gondwana, it is possible that the Nabidae have a Gondwanian origin. Cretanazgul possibly practiced traumatic insemination as all extant Prostemmatinae, but it was not possible to find the male internal organ linked to this behavior in this fossil despite 3D reconstruction with CT-scan data and fluorescence imaging.

Female bed bugs (Cimex lectularius L) anticipate the immunological consequences of traumatic insemination via feeding cues

Not all encounters with pathogens are stochastic and insects can adjust their immune management in relation to cues associated with the likelihood of infection within a life cycle as well as across generations. In this study we show that female insects (bed bugs) up-regulate immune function in their copulatory organ in anticipation of mating by using feeding cues. Male bed bugs only mate with recently fed females and do so by traumatic insemination (TI). Consequently, there is a tight temporal correlation between female feeding and the likelihood of her being infected via TI. Females that received predictable access to food (and therefore predictable insemination and infection cycles) up-regulated induced immunity (generic antibacterial activity) in anticipation of feeding and mating. Females that received unpredictable (but the same mean periodicity) access to food did not. Females that anticipated mating-associated immune insult received measurable fitness benefits (survival and lifetime reproductive success) despite laying eggs at the same rate as females that were not able to predict these cycles. Given that mating is a time of increased likelihood of infection in many organisms, and is often associated with temporal cues such as courtship and/or feeding, we propose that anticipation of mating-associated infection in females may be more widespread than is currently evidenced.

How Efficient Is Fertilization by Traumatic Insemination in Orius insidiosus (Hemiptera: Anthocoridae)?

Traumatic insemination (TI) can be injurious to females, and females have evolved various paragenital structures to mitigate these impacts. We examined the mating behavior of Orius insidiosus (Say) and the consequences of single and double matings for female fitness. A total of 100 virgin females (4-6-d old) were directly observed while they mated with virgin males. Some of these females were mated a second time with a different, nonvirgin male 3-5 d later, after they oviposited in sunflower stems. Females were held in isolation, fed eggs of Ephestia kuehniella Zeller, and reproductive success was tracked for 30 d. Six females died during their first copulation (6%), and another within 48 h, without laying eggs. Four percent of the females died during their second copulations. Copulations lasting less than 90 s usually did not result in successful fertilization, and duration of copula was positively correlated with egg fertility in singly-mated females. Duration of copula was more than halved in second matings, twice as variable, and negatively correlated with 30 d fecundity. Thirty-seven percent of singly-mated females and 31% of twice-mated females were infertile, with fewer than half of all females producing 88% of all eggs. We conclude that O. insidiosus females are likely monandrous in the wild, and that TI in this species is inefficient, contributing to high variation in female fitness. Thus, mating involves a significant mortality risk for females, despite their possession of complex paragenital structures that ostensibly mitigate copulatory injury.

Males increase their fitness by choosing large females in the common bedbug Cimex lectularius

Abstract Mate choice is often a role assigned to females. Already Darwin realised that males are eager to copulate, and females are choosy. However, male mate choice is not as rare as assumed. Males should choose females if females vary in quality, i.e., fecundity. Indeed, males often choose larger mates and through this preference increase fitness benefits. In addition, if mating costs reduce the number of copulations a male can potentially perform, he should be choosy. Bedbug females vary in their fecundity and female size is positively related to fecundity. Male bedbugs are limited in seminal fluid availability and, hence, the number of consecutive matings they can perform. Traumatic insemination gives males full control over mating, therefore low female mating resistance could further allow males to be choosy. Here, using mate choice arenas, we investigated if male bedbugs prefer to mate with large females. We observed mating behaviour and measured female fecundity to investigate potential male fitness benefits. Males chose to mate with large females 1.8 times more often than small females and large females laid significantly more eggs than small females. Our study provides first evidence for male mate choice based on female body size in bedbugs and males can increase their fitness by mating large females. It has to be further established if male mate choice is driven by mating costs in terms of ejaculate investment and if such male mate choice based on female size could be a driver of sexual size dimorphism in bedbugs.

Opportunities for female choice in the bed bug Cimex lectularius

AbstractBed bugs are cited as exemplars of sexual conflict because mating can only occur via traumatic insemination. However, past antagonistic coevolution between the sexes does not necessarily preclude current female choice. Here, we investigate opportunities for precopulatory female choice in bed bugs. We examined whether females seek out mating opportunities when they gain the most benefit: when females are virgin and/or have recently fed. But, we found that female mating and feeding status had little effect on female attraction to males and male odor. To determine whether females approach male harborages (home crevices) to seek matings in nature, we investigated where matings occurred among unfamiliar pairs of bed bugs. We found that, despite female attraction to male odor, matings were most likely to take place in the female's harborage rather than the male's harborage. We also examined the effect of feeding on male and female ability to mate. Whereas previous research reported that engorgement impaired female ability to refuse matings, we found that male feeding status had a larger effect on the success of mating encounters than female feeding status. Fed males had poor mating success, suggesting that males may be faced with a trade‐off between mating and feeding.

Traumatic insemination is not the case in three Orius species (Heteroptera: Anthocoridae).

Traumatic insemination (TI) is an extraordinary style of mating behavior wherein the female integument is pierced by the male extragenital structure to transfer the spermatozoa into the female’s body through wounding. Flower bugs of the genus Orius belong to the family Anthocoridae (Heteroptera), which is referred to as the “TI family”. Males possess sharp shaped extragenitalia, and females receive the extragenitalia using the copulatory tubes, which are specialized extragenital structures in Orius species. Since TI is not well studied in insects possessing the copulatory tube, we examined the genital structures and copulatory processes of three species, Orius strigicollis, O. sauteri, and O. minutus. Scanning electron microscopic observations revealed the positions of male extragenital structures during copulation. A needle-like flagellum was deeply inserted into the female intersegment between the abdominal VII and VIII segments, while the curved part of a sickle-like cone forced the intersegment to expand. No scars were detected around the copulation region after copulation. The copulatory tube adhered to the interior of segment VII, and the interior integument around the copulatory tube remained intact after copulation. On the basis of these results, TI does not occur in these Orius species. A pair of seminal conceptacles, which exists in typical TI insects, was found at the base of the oviducts in O. strigicollis. The distal end of the copulatory tube connected to a closed bag with a double-membrane, termed the sperm pouch. The sperm pouch was filled with filamentous structures after copulation and structures with equivalent forms were observed in adult male testis. These structures, considered to be spermatozoa, persisted in the pouch for at least two weeks after copulation, suggesting that the pouch is a long-term spermatozoa storage organ.

Bed Bugs

Bed bugs are blood sucking arthropod parasites and are found in tropical and temperate regions. They are small oval-shaped flat reddish brown wingless insects. They possess a segmented abdomen and exhibit ant-like movement. They mate by traumatic insemination. Bed bugs have adapted to the human environment. They get distributed from human travel. They bite human hosts and suck the blood. They feed during night times without being noticed. Bite of bed bugs result in cutaneous and systemic reactions. They are not known to transmit or spread diseases. Antihistaminics control urticarial itchy lesions. Improved cleanliness will help in the control of beg infestations.

Traumatic insemination and female counter-adaptation in Strepsiptera (Insecta).

In a few insect groups, males pierce the female’s integument with their penis during copulation to transfer sperm. This so-called traumatic insemination was previously confirmed for Strepsiptera but only in species with free-living females. The more derived endoparasitic groups (Stylopidia) were suggested to exhibit brood canal mating. Further, it was assumed that females mate once and that pheromone production ceases immediately thereafter. Here we examined Stylops ovinae to provide details of the mating behaviour within Stylopidia. By using μCT imaging of Stylops in copula, we observed traumatic insemination and not, as previously suggested, brood canal mating. The penis is inserted in an invagination of the female cephalothorax and perforates its cuticle. Further we show that female Stylops are polyandrous and that males detect the mating status of the females. Compared to other strepsipterans the copulation is distinctly prolonged. This may reduce the competition between sperm of the first mating male with sperm from others. We describe a novel paragenital organ of Stylops females, the cephalothoracic invagination, which we suggest to reduce the cost of injuries. In contrast to previous interpretations we postulate that the original mode of traumatic insemination was maintained after the transition from free-living to endoparasitic strepsipteran females.