Copulatory Behavior Research Articles

Vibrational signals differ between contests and copulatory courtship in the red milkweed beetle Tetraopes tetrophthalmus (Coleoptera: Cerambycidae)

Abstract Substrate-borne vibrational communication is commonly used by herbivorous insects to communicate intraspecific information. This form of communication is underexplored and many discoveries of the form and functional significance of these vibrations have yet to be made. Here, we add red milkweed beetles, Tetraopes tetrophthalmus (Coleoptera: Cerambycidae) to the record of arthropods that communicate with these plant-borne vibrations. Red milkweed beetles, like other cerambycids, commonly use stridulation to produce audible squeaks when in stressful situations and can also produce plant-borne vibrations when in contact with conspecifics. These beetles engage in fights for territory which ultimately results in mating opportunities (i.e., contests). We placed beetles into three sex-ratio treatments: male–male (contests), male–female (copulatory courtship), or male–male–female (contests and copulatory courtship). We first describe contests and copulatory courtship behaviours. We then used laser vibrometry and found that red milkweed beetles communicate by producing plant-borne vibrational signals composed of low-frequency rumbles and high-frequency clacks during male–male contests and copulatory courtship. We compared the spectral and temporal features of the vibrations between the two pre-copulatory contexts. We found that vibrational signals were shorter and had a higher fundamental and dominant frequencies when beetles engaged in contests compared to copulatory courtship behaviours. Our results show that these beetles use plant-borne vibrations as context-dependent signals that communicate different information under these two pre-copulatory scenarios. Our data suggests that these context-dependent signals may help beetles gain access to mates and prolong copulation.

Molecular phylogenetic relationships among lineages in the genus Carabus (Coleoptera: Carabidae) and radiation of the subgenus Apotomopterus

Abstract The genus Carabus (Coleoptera: Carabidae: Carabinae) is a group of predacious ground beetles found throughout Holarctic temperate zones. The diversification of this group is attributed not only to allopatric speciation but also to diversification in life-history traits and morphological traits that are involved in ecological adaptation and reproductive isolation. Precise phylogenetic reconstruction of Carabus is important to understanding the factors associated with species’ divergence in relation to such character divergence. Here, we analyse the phylogeny of Carabus among higher taxonomic groups and among species of the subgenus Apotomopterus, a hyper-diverse group endemic to East Asia. We used genome-wide sequence data from ultraconserved elements for phylogenetic reconstruction via maximum-likelihood and species-tree reconstruction methods. With regard to the relationships among higher taxonomic groups, the obtained phylogenetic trees resolved two structures with previously unresolved divergence. The subgenus Apotomopterus contained three lineages, i.e. the Vitalisi, Prodigus, and Sauteri groups. It showed diversification of the spinula, a sclerotized male genital structure, and variation in the female elytral notch (preapical emargination). These morphological traits may be involved in copulatory behaviour and potentially affect reproductive isolation, and their character state divergence may have facilitated species divergence of Apotomopterus.

Stimulatory Functions of Male Genitalia in Tipula (Triplicitipula) colei Alexander and Tipula (Lunatipula) translucida Doane (Diptera: Tipulidae) and Implications for Theories of Genital Evolution.

Male genitalia have been hypothesized to function as courtship devices during copulation, but it is difficult to use behavioral observations to test this hypothesis because male genitalia are usually hidden inside the female during copulation. In tipuloid flies, however, nearly all of the male's complex genital structures remain outside the female. Copulation behavior and genital morphology in Tipula (Triplicitipula) colei and T. (Lunatipula) translucida suggest that some male genital structures function to stimulate the female: male structures that contact the female bear tufts or dense arrays of modified setae on precisely the surfaces that contact the female; contact involves repeated, stereotyped rhythmic movements that include brushing, vibrating, scraping, and tapping; the movements are appropriately designed to utilize the morphology of the modified setae to stimulate the female; and the movements have little or no other perceptible mechanical effects on the female. The female structures contacted by these male genital movements fail to show the defensive designs predicted by the theories of genital evolution that are based on morphological species isolation or male-female morphological conflicts of interest; also unexplained by the conflict of interest hypothesis are female movements that seem designed to increase rather than avoid stimulation by the male.

Altered brain activity associated with premature ejaculation improved by electroacupuncture in rats.

Premature ejaculation (PE) is linked with abnormal brain activity that is modifiable by electroacupuncture (EA). In this study we aimed to explore the central pathological mechanism underlying EA in treating PE. Six-week-old male Sprague-Dawley rats were divided into a PE group (n = 8) and a control group (n = 8) according to ejaculatory frequency during copulatory behavior. All rats underwent EA at the Zusanli acupoint (ST-36) for 4weeks. Magnetic resonance imaging data were collected before and after EA. The behavioral parameters, plasma norepinephrine levels, fractional amplitude of low frequency fluctuation (fALFF), and regional homogeneity (ReHo) were evaluated. The PE group ejaculated more times with shorter latency compared with controls. After EA, the ejaculation frequency of the PE group decreased, and the ejaculation latency period increased, with no changes observed in the control group. Norepinephrine levels were higher in the PE group than in the controls and were positively correlated with ejaculation frequency and negatively correlated with ejaculation latency. The PE group showed lower fALFF in the right striatum and higher ReHo in the brainstem compared with controls. After EA, controls showed decreased fALFF in the right striatum, left olfactory bulb, and dorsal fornix and increased ReHo in the right interpeduncular nucleus, as well as decreased ReHo in the left striatum, prelimbic system, right basal forebrain region, septal region, and olfactory bulb, while the model group exhibited increased fALFF in the right hypothalamic region, decreased fALFF in the left globus pallidum and right basal forebrain region and increased ReHo in the right interpeduncular nucleus, as well as decreased ReHo in the left striatum, olfactory bulb, basal forebrain region, dentate gyrus, right dysgranular insular cortex, and striatum. Compared with the controls after EA, the model group showed increased ReHo of the right hypothalamic region and decreased ReHo of the right dysgranular insular cortex. These findings might enhance the understanding of PE and contribute to new, targeted therapies for PE. The therapeutic effects might be achieved by EA inhibiting the activity in brain regions involved in ejaculatory behavior. However, the curative effect of acupuncture might be underestimated due to some curative effects of sham acupuncture used in the control group. In conclusion, the ejaculatory frequency of rats may be reduced and ejaculation latency could be extended by EA at ST-36, which might be achieved by the effects of this treatment on brain activity.

Correction to: Copulation behaviour in the Southern Lapwing (Vanellus chilensis)

Correction to: Copulation behaviour in the Southern Lapwing (Vanellus chilensis)

Fecal testosterone levels positively correlated with sexually motivated behaviors in male Tibetan macaques (Macaca thibetana)

AbstractBackgroundTestosterone plays a crucial role in regulating sexual behavior among non‐human primates, working primarily by increasing copulatory behavior and sexual motivation. In this study, we analyzed fecal testosterone levels in five adult male Tibetan macaques (Macaca thibetana) living freely in the Huangshan National Reserve in Anhui Province. The aim was to investigate the relationships between fecal testosterone levels and sexually motivated behaviors.MethodsWe collected a total of 426 fecal samples and observed approximately 453 h of focal sampling behavioral data. Sexually motivated behaviors were categorized as sexual chase, grimace, and sexual‐inspection.ResultsThe results showed a positive correlation between sexually motivated behaviors and copulatory behavior. Furthermore, all the three sexually motivated behaviors, including sexual chase, grimace, and sexual‐inspection, were positively correlated with fecal testosterone levels.ConclusionOur results demonstrated positive correlations between sexually motivated behaviors and fecal testosterone in free‐ranging Tibetan macaques.

Developmental exposure to environmentally relevant doses of phthalates alters the neural control of male and female reproduction in mice

The present study aims to analyze the effects of developmental exposure to phthalates at environmentally relevant doses on the neural control of male and female reproduction. For this purpose, C57Bl/6J mice were exposed to di-(2-ethylexyl) phthalate (DEHP) alone (5 or 50 μg/kg/d), or DEHP (5 μg/kg/d) in a phthalate mixture. Exposure through diet started 6 weeks before the first mating and lasted until weaning of litters from the second gestation (multiparous dams). Analyses of offspring born from multiparous dams exposed to DEHP alone or in a phthalate mixture showed that females experienced a delayed pubertal onset, and as adults they had prolonged estrous cyclicity and reduced Kiss1 expression in the preoptic area and mediobasal hypothalamus. Male littermates showed a reduced anogenital distance and delayed pubertal onset compared with controls. However, in adulthood the weight of androgen-sensitive organs and hypothalamic Kiss1 expression were unaffected, suggesting normal functioning of the male gonadotropic axis. Developmental exposure to DEHP alone or in a phthalate mixture reduced the ability of intact males and ovariectomized and hormonally primed females to attract a sexual partner and to express copulatory behaviors. In addition, females were unable to discriminate between male and female stimuli in the olfactory preference test. Social interaction was also impaired in females, while locomotor activity and anxiety-like behavior in both sexes were unaffected by the treatment. The sexual deficiencies were associated with reduced expression of the androgen receptor in the preoptic area and progesterone receptor in the mediobasal hypothalamus, the key regions involved in male and female sexual behavior, respectively. Thus, the neural structures controlling reproduction are vulnerable to developmental exposure to phthalates at environmentally relevant doses in male and female mice. Adult females had an impaired gonadotropic axis and showed more affected behaviors than adult males.

Copulation behaviour in the Southern Lapwing (Vanellus chilensis)

Copulation behaviour in the Southern Lapwing (Vanellus chilensis)

Reproductive behavior of Erythrodiplax abjecta (Rambur, 1842) from Andean Mountains

Research about the behavior of Neotropical species is crucial to understand how the rapid environmental changes in the Neotropics affect the reproduction of various organisms. The reproductive behavior of insects in tropical ecosystems, such as those belonging to the order Odonata, is as yet scarcely known. In this article, the reproductive behavior of Erythrodiplax abjecta is described from several localities in the Colombian Andean Mountains. Playing out between 10:00 and 14:30 h, male individuals of this species may exhibit either territorial or satellite behavior. Sexually mature males and females engage in tandem and copulation behaviors, which are brief and may occur either perched or in flight. Females of E. abjecta oviposit directly in the water at various times, alternating with periods of rest. During oviposition, males stand guard over females, perched or hovering, in a quest to protect them against other males. Conspecific males have been observed to engage in sieges of perching or ovipositing females, as well as mating pairs. Following a siege, either the female or the original pair flees, or the besieging male will succeed in outcompeting the original male, take the female in tandem, copulate with her, and then guard her while she oviposits. If the female flees, she may perch elsewhere, continue ovipositing, or may be taken by another male. The intense male guarding behavior during oviposition could represent an adaptation to temporarily high male densities at oviposition sites and a high level of competition during the short period in which active copulation takes place.

Kisspeptin administration may promote precopulatory behavior in male rats independently or supplementally to testosterone and contribute to proceptive behavior in female partners, reducing mating failure

Kisspeptin is a peptide that plays an important role through its effects on the hypothalamus-pituitary–gonadal (HPG) axis. It has also been implicated in sexual behavior. The present study investigated whether the relationship between kisspeptin and sexual behavior is independent of the HPG axis, i.e., testosterone. Sexual behavior was examined after the administration of kisspeptin to gonadally intact male rats and gonadectomized male rats that received testosterone supplementation. Other male rats were also observed for sexual behavior once a week from 2 to 5 weeks after gonadectomy and receiving kisspeptin for the sixth postoperative week. Sexual behavior in female rats serving as the partner for each male was also observed. Female rats were not administered kisspeptin in the present study. The results obtained showed that the administration of kisspeptin increased precopulatory behavior in gonadally intact male rats and gonadectomized male rats that received testosterone supplementation and proceptive behavior in their female partners. Precopulatory behavior in males and receptive behavior in females increased, while copulatory behavior in males and receptive behavior in females remained unchanged. Furthermore, the administration of kisspeptin increased precopulatory behavior in gonadectomized males, but did not affect receptive behavior in females. These results suggest that kisspeptin affected males independently and/or supplementally to testosterone, and also that changes in the presence of testosterone in males had an impact on proceptive behavior in their female partners. In conclusion, kisspeptin may involve an as-yet-unidentified neural pathway in sexual desire independently of the HPG axis.

Chronic exposure to gestodene impairs reproductive system in adult female zebrafish (Daniarerio)

Gestodene (GES) is widely used in human therapy and animal husbandry and is frequently detected in aquatic environments. Although GES adversely affects aquatic organisms at trace levels, its effects on the reproductive biology of fish remain inconclusive. In this study, female zebrafish (Danio rerio) were exposed to environmentally relevant levels of GES for the evaluation of the effects of GES on the reproductive system by using endpoints including gene expression, plasma steroid concentrations, histological and morphological analyses, copulatory behavior, and reproductive output. Adult female zebrafish exposed to environmentally relevant concentrations of GES (4.0, 40.2, and 372.7 ng/L) for 60 d demonstrated stagnant ovarian oocyte development, evidenced by an increase in the percentage of perinuclear and atretic oocytes and a decrease in the percentage of late vitellogenic oocytes. GES-exposed females were less attractive to males and had lower copulatory intimacy than females in control. Consequently, spawning (44.3–49.2 %) and egg fertilization rates (27.9–32.0 %) were decreased. The decreased survival of fertilized eggs and hatching rates were accompanied by increased malformations. These negative effects were associated with abnormal transcriptional levels of gonadal steroid hormones, which were regulated by genes (Hsd17β3, Hsd11β2, Hsd20β, Cyp19a1a, and Cyp11b). Overall, our findings suggest that GES impairs the reproductive system of zebrafish, which may threaten population stability.

Copulation record of Tropidurus imbituba (Squamata: Tropiduridae)

Tropidurus imbituba is a lizard with endemic distribution. Data on the species are scarce as it is a new species and has the only known habitat to date. Understanding reproductive aspects is important for their conservation. Thus, the present study presents the first records of the courtship and copulation behavior of T. imbituba. Sampling was carried out in spring 2022 and summer 2023, between September 24, 2022 and March 19, 2023. Monitoring was adapted from the Squamata Reptile Monitoring Protocol in Federal Conservation Units of the Caatinga Biome, following the active search method without time limitation (BANLT), in order to register T. imbituba individuals without any type of physical restraint. The individuals viewed had their coordinates taken with GPS, photographed with a DSLR camera – 65x zoom with a 21 mm ultra-wide angle lens. Viewing was done with the naked eye and with the aid of 8x40 binoculars. In areas with a concentration of lizards, the fixed point methodology was also used for five to 10 minutes, to observe the animals and other important data, where, at opportune moments, filming and photographs of courtship or copulation behavior were recorded.

A long-read draft assembly of the Chinese mantis (Mantodea: Mantidae: Tenodera sinensis) genome reveals patterns of ion channel gain and loss across Arthropoda.

Praying mantids (Mantodea: Mantidae) are iconic insects that have captivated biologists for decades, especially the species with cannibalistic copulatory behavior. This behavior has been cited as evidence that insects lack nociceptive capacities and cannot feel pain; however, this behaviorally driven hypothesis has never been rigorously tested at the genetic or functional level. To enable future studies of nociceptive capabilities in mantids, we sequenced and assembled a draft genome of the Chinese praying mantis (Tenodera sinensis) and identified multiple classes of nociceptive ion channels by comparison to orthologous gene families in Arthropoda. Our assembly-produced using PacBio HiFi reads-is fragmented (total size = 3.03 Gb; N50 = 1.8 Mb; 4,966 contigs), but is highly complete with respect to gene content (BUSCO complete = 98.7% [odb10_insecta]). The size of our assembly is substantially larger than that of most other insects, but is consistent with the size of other mantid genomes. We found that most families of nociceptive ion channels are present in the T. sinensis genome; that they are most closely related to those found in the damp-wood termite (Zootermopsis nevadensis); and that some families have expanded in T. sinensis while others have contracted relative to nearby lineages. Our findings suggest that mantids are likely to possess nociceptive capabilities and provide a foundation for future experimentation regarding ion channel functions and their consequences for insect behavior.

Long-term neuropeptide modulation of female sexual drive via the TRP channel in Drosophila melanogaster

Connectomics research has made it more feasible to explore how neural circuits can generate multiple outputs. Female sexual drive provides a good model for understanding reversible, long-term functional changes in motivational circuits. After emerging, female flies avoid male courtship, but they become sexually receptive over 2 d. Mating causes females to reject further mating for several days. Here, we report that pC1 neurons, which process male courtship and regulate copulation behavior, exhibit increased CREB (cAMP response element binding protein) activity during sexual maturation and decreased CREB activity after mating. This increased CREB activity requires the neuropeptide Dh44 (Diuretic hormone 44) and its receptors. A subset of the pC1 neurons secretes Dh44, which stimulates CREB activity and increases expression of the TRP channel Pyrexia (Pyx) in more pC1 neurons. This, in turn, increases pC1 excitability and sexual drive. Mating suppresses pyx expression and pC1 excitability. Dh44 is orthologous to the conserved corticotrophin-releasing hormone family, suggesting similar roles in other species.

Sox21b underlies the rapid diversification of a novel male genital structure between Drosophila species

The emergence and diversification of morphological novelties is a major feature of animal evolution.1,2,3,4,5,6,7,8,9 However, relatively little is known about the genetic basis of the evolution of novel structures and the mechanisms underlying their diversification. The epandrial posterior lobes of male genitalia are a novelty of particular Drosophila species.10,11,12,13 The lobes grasp the female ovipositor and insert between her abdominal tergitesand, therefore, are important for copulation and species recognition.10,11,12,14,15,16,17 The posterior lobes likely evolved from co-option of a Hox-regulated gene network from the posterior spiracles10 and have sincediversified in morphology in the D.simulans clade, in particular, over the last 240,000 years, drivenby sexual selection.18,19,20,21 The genetic basis of this diversification is polygenic but, to the best ofour knowledge, none of the causative genes have been identified.22,23,24,25,26,27,28,29,30 Identifying the genes underlyingthe diversification of these secondary sexual structures is essential to understanding theevolutionary impact on copulation and species recognition. Here, we show that Sox21b negatively regulates posterior lobe size. This is consistent with expanded Sox21b expression in D.mauritiana, which develops smaller posterior lobes than D.simulans. We tested this by generating reciprocal hemizygotes and confirmed that changes in Sox21b underlie posterior lobe evolution between these species. Furthermore, we found that posterior lobe size differences caused by the species-specific allele of Sox21b significantly affect copulation duration. Taken together, our study reveals the genetic basis for the sexual-selection-driven diversification of a novel morphological structure and its functional impact on copulatory behavior.

Replenishment of Drosophila Male Pheromone After Mating.

Insect exocrine gland products can be involved in sexual communication, defense, territory labelling, aggregation and alarm. In the vinegar fly Drosophila melanogaster the ejaculatory bulb synthesizes and releases 11-cis-Vaccenyl acetate (cVa). This pheromone, transferred to the female during copulation, affects aggregation, courtship and male-male aggressive behaviors. To determine the ability of male flies to replenish their cVa levels, males of a control laboratory strain and from the desat1 pheromone-defective mutant strain were allowed to mate successively with several females. We measured mating frequency, duration and latency, the amount of cVa transferred to mated females and the residual cVa in tested males. Mating duration remained constant with multiple matings, but we found that the amount of cVa transferred to females declined with multiple matings, indicating that, over short, biologically-relevant periods, replenishment of the pheromone does not keep up with mating frequency, resulting in the transfer of varying quantities of cVa. Adult responses to cVa are affected by early developmental exposure to this pheromone; our revelation of quantitative variation in the amount of cVa transferred to females in the event of multiple matings by a male suggests variable responses to cVa shown by adults produced by such matings. This implies that the natural role of this compound may be richer than suggested by laboratory experiments that study only one mating event and its immediate behavioral or neurobiological consequences.

Unraveling the hierarchical structure of posture and muscle activity changes during mating of Caenorhabditis elegans.

One goal of neurobiology is to explain how decision-making in neuromuscular circuits produces behaviors. However, two obstacles complicate such efforts: individual behavioral variability and the challenge of simultaneously assessing multiple neuronal activities during behavior. Here, we circumvent these obstacles by analyzing whole animal behavior from a library of Caenorhabditis elegans male mating recordings. The copulating males express the GCaMP calcium sensor in the muscles, allowing simultaneous recording of posture and muscle activities. Our library contains wild type and males with selective neuronal desensitization in serotonergic neurons, which include male-specific posterior cord motor/interneurons and sensory ray neurons that modulate mating behavior. Incorporating deep learning-enabled computer vision, we developed a software to automatically quantify posture and muscle activities. By modeling, the posture and muscle activity data are classified into stereotyped modules, with the behaviors represented by serial executions and transitions among the modules. Detailed analysis of the modules reveals previously unidentified subtypes of the male's copulatory spicule prodding behavior. We find that wild-type and serotonergic neurons-suppressed males had different usage preferences for those module subtypes, highlighting the requirement of serotonergic neurons in the coordinated function of some muscles. In the structure of the behavior, bi-module repeats coincide with most of the previously described copulation steps, suggesting a recursive "repeat until success/give up" program is used for each step during mating. On the other hand, the transition orders of the bi-module repeats reveal the sub-behavioral hierarchy males employ to locate and inseminate hermaphrodites.

The ultrastructure of nymphal dermal pores and the genitalia of adult male of Phenacoccus solenopsis (Hemiptera: Pseudococcidae)

The cotton mealybug, Phenacoccus solenopsis, has established itself as an invasive insect pest worldwide. It causes structural and physiological damage to various crops and can cause substantial financial losses in their production. The successful reproduction of this pest under a wide range of conditions is a key to its success. Despite this, the morphology of its genitalia, genital sensilla, and wax-producing dermal pores has received little attention, with little descriptions of their ultrastructure. By investigating those features with SEM, the present study revealed considerable new insights into the identification of the nymphal and adult stages of P. solenopsis. In addition, the description of the ultrastructural genital morphology of the immature stages of P. solenopsis has revealed characteristics that facilitate their discrimination. Trilocular pores were observed on both sides of the body, while the quinquelocular pores were distributed only on the ventral surface in both the first and second nymphal instars. The adult male is characterized by two pairs of waxy caudal filaments surrounded by clusters of 55 to 60 stellate pores, and each pregenital segment bears a pair of stellate pores composed of 4 or 5 peripheral loculi. Sensilla trichodea and numerous microtrichia are present on the pregenital segments. The penile sheath bears three subtypes of sensilla basiconica and also campaniformia, whereas the style bears three subtypes of sensilla campaniformia. The findings of this study could assist in the identification of the adult and nymphal stages of P. solenopsis, and also provide insights into the structures found on the genitalia of the adult male that possibly have an important role in mating events and copulatory behavior. Furthermore, these findings were able to contribute to better understanding the functional morphology of P. solenopsis.

First record of fossil psocodeans in copula from mid-Cretaceous Burmese amber.

We report the first discovery of barklice preserved in copula from amber dating back to the mid-Cretaceous, thus documenting the oldest preserved reproductive behaviour of Psocodea. The new finding provides new insight into the evolution of copulatory behaviour in Trogiomorpha. Moreover, we describe and illustrate the new fossil material of Burmempheria densuschaetae (= Latempheria kachinensis) in the extinct family Empheriidae and Longiantennum fashengi in the extinct family Archaeatropidae, two diverse and widely distributed groups during the Cretaceous. No significant sexual dimorphism is recorded, apart from variation in size where males are larger. The taxonomy of Cretaceous empheriids from Burmese amber is revised and discussed.

Callosobruchus maculatus males and females respond differently to grandparental effects.

In this study, we used the cowpea weevil Callosobruchus maculatus (Coleoptera: Chrysomelidae) and two essential oils (mint and rosemary) to investigate the effect of the parents (F0) exposure to a sublethal dose of essential oil on grand offspring (F2) encountering the same essential oil. Then we evaluated biological parameters, including immature development time, sex ratio, adult emergence, egg number, egg hatch, longevity, and mating behaviors in three generations (F0, F1, and F2). Results showed when F0 experienced essential oil in the embryonic stage, parental and grandparental effects were more severe than adulthood experiences. Also, grandparental effects increased or decreased reactions of F2 generation when faced with a similar essential oil, depending on grand offspring sex. For example, when grandparents experienced rosemary essential oil in the embryonic stage, they produced more tolerant female grand offspring with a better ability to cope with the same essential oil (increased adult longevity and egg number). However, male grandoffspring were more sensitive (had a higher mortality percentage and less copulation success). Grandparental effects of exposure to mint essential oil diminished female grand offspring longevity and improved male copulation behavior parameters such as increased copulation duration and decreased rejection by females. In all, grandparental effects were different in male and female grand offspring based on the essential oil type experienced by F0.