Mating Events Research Articles

Reproductive ecology of the critically endangered pancake tortoise (Malacochersus tornieri) in the wild

Reproduction of the pancake tortoise in the wild has remained poorly known over the decades. This study fills the knowledge gap by investigating the reproductive ecology of the species in its natural habitats in Kenya. Data were collected using the time‐constrained search‐and‐seize method, group‐level scan sampling, radio tracking, and camera‐trapping. Our observations revealed a total of 19 mating events that occurred between December and April, predominantly during the rainy seasons. Mating duration averaged 15.2 ± 2.4 min. Male–male aggression was observed during the mating season, with the dominant male keeping off all other males and mating with the resident females. Nest preparation events occurred between April and June. Nine nesting events were recorded, but in only five of them was an egg deposited. The nests were dug in loose soil, appeared almost circular, and measured 8.4 ± 0.9 cm wide and 7.0 ± 0.1 cm deep (n = 7). The average clutch size was one egg. The eggs were white, hard‐shelled, oval, and elongated measuring 4.4 ± 0.4 cm long, 2.7 ± 0.04 cm wide and weighed 17 ± 0.6 g (n = 3). Incubation period lasted 177 ± 5.7 days (n = 2), and hatching coincided with the onset of the short rainy season in November, aligned with the availability of abundant food for the tortoises. The hatchlings were minimally wider than long, having an average straight carapace length of 4.5 ± 0.6 cm, an average width of 4.5 ± 0.4 cm, and on average weighed 14.7 ± 3.7g, (n = 9). They remained near the nest site for 4–5 days before relocating to tiny rock crevices away from the adult tortoise crevices. Egg and hatchling predation was high, with four of the total of six eggs, observed after they were laid, being destroyed. Three juveniles, out of nine, were also eaten by predators. These findings contribute useful information for the formulation of effective conservation and management strategies for this critically endangered species.

Genetic Links between Reproductive Traits and Amino Acid Pairwise Distances of Swine Leukocyte Antigen Alleles among Mating Partners in Microminipigs.

Previously, we found that a greater dissimilarity in swine leukocyte antigen (SLA) class I and class II alleles between mating partners resulted in increased farrowing rates in a highly inbred population of Microminipigs (MMPs). In this follow-up study, we have analyzed the effects of dissimilarity in SLA alleles between mating partners for seven different reproductive traits, including litter size and the number of stillborn and live or dead weaned piglets. We determined the relationships among reproductive traits within each mating event and the amino acid distances of SLA alleles as markers of diversity between mating partners. Our results indicate that mating partners with greater amino acid pairwise genetic distances in the SLA-1 class I gene or DQB1 class II gene alleles were associated with significantly larger litter sizes and higher numbers of live piglets at birth and weaning. Also, partners with greater pairwise distances in the SLA-2 class I gene alleles exhibited fewer pre-weaning deaths. These findings suggest that the dissimilarity in SLA class I and class II alleles between mating partners may affect not only farrowing rates but also other key reproductive traits such as litter size and improved piglet survival rates. Consequently, SLA alleles could serve as valuable genetic markers for selecting mating partners in breeding programs and for conducting epistatic studies on various reproductive traits in MMPs.

Fake news? The impact of information mismatch on mating behaviour.

Multiple cues are often used for mate choice in complex environments, potentially entailing mismatches in the information conveyed by different sources. We address the consequences of this information mismatch for receivers using the spider mite Tetranychus urticae, in which virgin females are highly valuable mates compared to mated females, given first male sperm precedence. Accordingly, males are known to prefer virgins and distinguish them using cues from the females themselves and that they leave on the substrate. Whereas cues from females are highly reliable, those left on the substrate may not reflect the real female mating status if females move and/or mate. Here, we tested the consequences of such mismatch by exposing males to mated or virgin females on patches previously impregnated with cues deposited by females of either mating status. Male mating attempts were solely affected by substrate cues while female acceptance and the number of mating events were independently affected by both cues. Copulation duration, in contrast, depended mainly on the mating status of the female, with the number of copulations and the total time spent mating being intermediate in environments with mismatched information. We also show that males incur mating costs, reflected in reduced survival in environments with virgin cues. These results suggest that substrate cues left by females are instrumental for males to find their mates. However, in environments with mismatched information, males may pay survival costs without the associated benefit of mating with virgins, or they may lose opportunities to mate with virgins by responding to substrate cues from mated females. The benefit of using multiple cues will then hinge upon the frequency of information mismatch, which itself should vary with the dynamics of populations.

Observation of mating behavior using marked flies of black soldier fly (Hermetia illucens) under sunlight condition

Abstract In recent years, various studies have investigated the growth of the larval stage of the black soldier fly (BSF). Nevertheless, the biology and reproductive behaviors of the adult is still largely unknown. Expanding the understanding of this area of research will assist the optimization of breeding-systems of BSF. In this present study, the mating behavior of BSF adults under summer sunlight conditions was evaluated. The number of couplings were evaluated based on a variety of environmental parameters that may have affected the copula: temperature (°C), humidity (RH) and light intensity (lux). 1000 g of pupae were put inside a cage to obtain one hundred adults to put in each three experimental units. Each specimen was then marked with an individual code in the dorsal portion of the thorax using fine-tipped acrylic markers and for each fly the body length parameters were taken. Individuals of interest were subsequently marked; in the first experiment only females, in the second only male, and in the third both sexes. During the trials observations were carried out from 9.30 am to 5.30 pm. During this time the key behaviors evaluated are as follows: mating, mating failure, struggles, deposition, failure to lay eggs, and multiple mating. The rate recorded in the Male and Female trials was 16.5%, but the peak of copulas changed with the trials. Mating events demonstrated strong positive correlation between light intensity (0.73) and humidity (0.64), whereas a negative correlation was seen between temperature and matings (−0.59). A weakly negative correlation was seen between deposition number and light intensity (−0.34) and humidity (−0.41), while positively correlated with the temperature (0.47). Multiple mating events were seen 2 times for females and up to 4 times for male.

Genetic modules for α-factor pheromone controlled growth regulation of Saccharomyces cerevisiae.

Saccharomyces cerevisiae is a commonly used microorganism in the biotechnological industry. For the industrial heterologous production of compounds, it is of great advantage to work with growth-controllable yeast strains. In our work, we utilized the natural pheromone system of S. cerevisiae and generated a set of different strains possessing an α-pheromone controllable growth behavior. Naturally, the α-factor pheromone is involved in communication between haploid S. cerevisiae cells. Perception of the pheromone initiates several cellular changes, enabling the cells to prepare for an upcoming mating event. We exploited this natural pheromone response system and developed two different plasmid-based modules, in which the target genes, MET15 and FAR1, are under control of the α-factor sensitive FIG1 promoter for a controlled expression in S. cerevisiae. Whereas expression of MET15 led to a growth induction, FAR1 expression inhibited growth. The utilization of low copy number or high copy number plasmids for target gene expression and different concentrations of α-factor allow a finely adjustable control of yeast growth rate.

Impact of age on the reproductive output of Hermetia illucens (Diptera: Stratiomyidae).

The current study examined the impact of adult black soldier fly (Hermetia illucens (L.)), age on reproductive output. Four treatments consisting of an equal sex ratio of the following combinations were used: 2-day-old males with 2-day-old females (YMYF); 6-day-old males with 2-day-old females (OMYF); 2-day-old males with 6-day-old females (YMOF); 6-day-old males with 6-day-old females (OMOF). Adult age was a significant factor for the overall number of mating events observed, with YMYF having 2.2× more than YMOF and 2.7× more than OMOF. Time was a significant factor in the number of mating events observed, with 97.8% observed on days 2-6. Age was not a significant factor for the total number of oviposition events, but the inclusion of time as a factor was significant, with 91.5% of all events occurring on days 2-6. Treatment and trial significantly interacted with egg production. This response can be attributed to treatments containing old females, which had a 50% variation in egg output across trials. Treatments containing young females were consistent in terms of egg production. As anticipated, due to oviposition events, 94.8% of total eggs were oviposited on days 2-6. Fecundity and egg hatch were also significantly impacted by female age, with a 50% reduction in both cases across trials occurring in old females. Young females paired with old males had a 20% reduction in egg hatch, but this difference was not statistically significant.

Novel behavioral observations and body scarring for the bluntnose sixgill shark (Hexanchus griseus) offer clues to reproductive patterns and potential mating events

Novel behavioral observations and body scarring for the bluntnose sixgill shark (Hexanchus griseus) offer clues to reproductive patterns and potential mating events

Experience matters: genetic variation affects male reproductive success across sequential mating events in Drosophila melanogaster.

The cost of reproduction is well studied in females but only recently have the costs of mating been investigated in males. Research suggests that males allocate resources between subsequent mating events, resulting in differential success across mating bouts. Selection should favor allocation strategies that match the likelihood of successive matings. The complexity of the system, however, suggests that one fixed strategy is unlikely to be universally favored and thus I predict that genetic variation for different allocation strategies will be segregating in natural populations. To test this, I measured several components of reproductive performance in eight inbred genotypes of Drosophila melanogaster across three sequential mating events. As predicted, there was genetic variation for how previous experience affected a male's reproductive performance for both the proportion of matings that produced offspring and the proportion of offspring sired (P1). Some genotypes had the highest success in their first matings and declined in successive matings while other genotypes did best in later matings. Mating experience had consistent effects across genotypes on fertility and induced refractoriness to remating. On average, virgin matings produced the highest fertility and third matings most effectively induced refractoriness. Genotype also had a significant effect on fertility. These results have important implications for understanding how selection may be acting on males when there is variation in the likelihood of multiple mating events and could affect the evolution of male allocation strategies in the face of perceived competitors.

Sperm storage organs change with female age and mating history in the Golden Silk spider Trichonephila clavipes (Araneae: Araneidae)

AbstractFemale sperm storage organs in arthropods are used as taxonomic characters since it is assumed that they do not change after maturation. However, in the Golden Silk spider, Trichonephila clavipes, the shape and sclerotization of the spermathecae change with a mating event. Moreover, mating in T. clavipes consists of several hundred copulations, but the male transfers all sperm during the first insertion of each pedipalp. Given this puzzling mating behaviour, we hypothesize that multiple copulations serve purposes other than sperm transfer and uptake, such as further altering the morphology of the storage site and/or transferring substances other than sperm. We compared virgin female T. clavipes with females after two copulations, one for each spermatheca, and after ad‐libitum mating. Each treatment consisted of two sub‐treatments in which the state of the sperm storage organs was inspected after a short (1–3 days) or a long (11 days) time interval after the female's final moult. The size of the spermathecae increased twofold in all mating treatments compared to virgin females. The thickness of the spermathecal wall increased significantly with time in all treatments similarly. The spermathecal epithelium, which releases secretion into the lumen of the spermathecae, seems most active in virgin females during the time when mating would normally occur and turns less active after mating and with age. The spermathecae contain secretion produced by the female before mating; and after mating, sperm and another secretion transferred by the male. The numerous sperm‐less matings in the ad‐libitum treatment resulted in male secretion in the copulatory ducts of the female genitalia, likely impeding sperm transfer by subsequent males. Out study shows that female genitalia can change considerably after having reached maturity and suggests that sperm‐less matings evolved in the context of sperm competition.

Evolutionary trade-offs between testes size and parenting in Neotropical glassfrogs.

In males, large testes size signifies high sperm production and is commonly linked to heightened sperm competition levels. It may also evolve as a response to an elevated risk of sperm depletion due to multiple mating or large clutch sizes. Conversely, weapons, mate or clutch guarding may allow individuals to monopolize mating events and preclude sperm competition, thereby reducing the selection of large testes. Herein, we examined how paternal care, sexual size dimorphism (SSD), weaponry and female fecundity are linked to testes size in glassfrogs. We found that paternal care was associated with a reduction in relative testes size, suggesting an evolutionary trade-off between testes size and parenting. Although females were slightly larger than males and species with paternal care tended to have larger clutches, there was no significant relationship between SSD, clutch size and relative testes size. These findings suggest that the evolution of testes size in glassfrogs is influenced by sperm competition risk, rather than sperm depletion risk. We infer that clutch guarding precludes the risk of fertilization by other males and consequently diminishes selective pressure for larger testes. Our study highlights the prominent role of paternal care in the evolution of testes size in species with external fertilization.

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.

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.

Let's talk about sex: Mechanisms of neural sexual differentiation in Bilateria.

In multicellular organisms, sexed gonads have evolved that facilitate release of sperm versus eggs, and bilaterian animals purposefully combine their gametes via mating behaviors. Distinct neural circuits have evolved that control these physically different mating events for animals producing eggs from ovaries versus sperm from testis. In this review, we will describe the developmental mechanisms that sexually differentiate neural circuits across three major clades of bilaterian animals-Ecdysozoa, Deuterosomia, and Lophotrochozoa. While many of the mechanisms inducing somatic and neuronal sex differentiation across these diverse organisms are clade-specific rather than evolutionarily conserved, we develop a common framework for considering the developmental logic of these events and the types of neuronal differences that produce sex-differentiated behaviors. This article is categorized under: Congenital Diseases > Stem Cells and Development Neurological Diseases > Stem Cells and Development.

Drop dead! Female mate avoidance in an explosively breeding frog.

Males' and females' reproductive strategies may differ, potentially leading to sexual conflict. Increased efforts by males (harassment, forced copulation, intimidation) to gain access to females could even negatively affect female survival and thus lead to reproductive failure for both individuals. In anurans, a higher mortality risk of mating females has been reported in explosive breeding species. During these mating events, several males cling to a female, which are mostly unable to get rid of the unwanted males. This can lead to the female's death. From the literature, it seems that females of explosive breeding frogs have no means to reject unwanted males. Here we describe female mate avoidance behaviours in the European common frog. We observed three female avoidance behaviours, namely 'rotation', 'release call(s)' and tonic immobility (death feigning). These behaviours were significantly associated with smaller female body size, and smaller females were more successful in escaping amplexus. Tonic immobility as a tactic to avoid mating or male harassment has only been observed in a handful of species and only in one other amphibian. Our observations show that females in explosive breeding frogs may not be as passive and helpless as previously thought.

Personality traits differentially affect components of reproductive success in a Neotropical poison frog.

Individual reproductive success has several components, including the acquisition of mating partners, offspring production, and offspring survival until adulthood. While the effects of certain personality traits-such as boldness or aggressiveness-on single components of reproductive success are well studied, we know little about the composite and multifaceted effects behavioural traits can have on all the aspects of reproductive success. Behavioural traits positively linked to one component of reproductive success might not be beneficial for other components, and these effects may differ between sexes. We investigated the influence of boldness, aggressiveness, and exploration on the number of mating partners, mating events, and offspring surviving until adulthood in males and females of the Neotropical poison frog Allobates femoralis. Behavioural traits had different-even opposite-effects on distinct components of reproductive success in both males and females. For example, males who displayed high levels of aggressiveness and exploration (or low levels of aggressiveness and exploration) managed to attract high number of mating partners, while males with low levels of boldness, low levels of aggressiveness, and high levels of exploration had the most offspring surviving until adulthood. Our results therefore suggest correlational selection favouring particular combinations of behavioural traits.

It is buzziness time: rearing, mating, and overwintering Bombus vosnesenskii (Hymenoptera: Apidae).

Bombus vosnesenskii Radowszkowski, 1862 is one of three bumble bee species commercially available for pollination services in North America; however, little is documented about B. vosnesenskii colony life cycle or the establishment of ex situ rearing, mating, and overwintering practices. In this study, we documented nest success, colony size, and gyne production; recorded the duration of mating events; assessed overwintering survival of mated gynes; and evaluated second-generation nest success for colonies established from low- and high-elevation wild-caught B. vosnesenskii gynes. Of the 125 gynes installed, 62.4% produced brood cells (nest initiation) and 43.2% had at least 1 worker eclose (nest establishment). High-elevation B. vosnesenskii gynes had significantly higher nest initiation and establishment success than low-elevation gynes. However, low-elevation colonies were significantly larger with queens producing more gynes on average. Mating was recorded for 200 low-elevation and 37 high-elevation gynes, resulting in a mean duration of 62 and 51 min, respectively. Mated gynes were then placed into cold storage for 54 days to simulate overwintering, which resulted in 59.1% of low-elevation gynes surviving and 91.9% of high-elevation gynes surviving. For second-generation low-elevation gynes, 26.4% initiated nesting and 14.3% established nesting. Second-generation high-elevation gynes did not initiate nesting despite CO2 narcosis treatments. Overall, these results increase our understanding of B. vosnesenskii nesting, mating, and overwintering biology from 2 elevations. Furthermore, this study provides information on successful husbandry practices that can be used by researchers and conservationists to address knowledge gaps and enhance the captive rearing of bumble bees.

Male exposure to guava volatiles affects female remating behavior in Anastrepha fraterculus

AbstractPlant‐derived volatiles can have positive or negative effects on insects. Exposure to volatiles of guava (Psidium guajava L.) (Myrtaceae) enhances male mating success in the South American fruit fly, Anastrepha fraterculus (Wiedemann) (Diptera: Tephritidae), an important pest of various fruits. These volatiles can therefore be used as a post‐teneral, pre‐release supplement in sterile insect technique (SIT) programs. In SIT, mass‐reared sterile males compete for and mate with wild females, in order to reduce populations of insect pests. However, the success of SIT will be diminished if males are not able to inhibit females from remating with other (potentially wild) males. Here, we investigated the remating propensity of A. fraterculus females mated with males exposed to guava volatiles or non‐exposed (control) in choice and no‐choice tests. In addition, we tested whether exposure to guava volatiles affected male reproductive organ size (testes, accessory glands, and seminal vesicles) and cuticular hydrocarbon (CHC) profile. Females mated with guava‐exposed males were more likely to remate than females mated with control males, but there was no difference in the length of the refractory period (i.e., the time between the first and second mating event). Reproductive organ size did not differ between exposed and non‐exposed males, but exposed males had a higher quantity of waxes than non‐exposed males, and CHC profiles differed. The results are discussed in the context of SIT programs for the control of this insect pest.

The effect of the addition of a companion forage to a perennial ryegrass sward on ewe and lamb performance

Ireland's competitive advantage in sheepmeat production is based on the efficient and cost effective production and utilisation of pasture. The objective of this study was to assess the influence of binary mixtures of perennial ryegrass (Lolium perenne L.) plus a companion forage on ewe and lamb performance at pasture. Five sward mixtures were compared, namely; i) perennial ryegrass (Lolium perenne L.;PRG), ii) PRG plus white clover (Trifolium repens L.;PRG + WC), iii) PRG plus red clover (Trifolium pratense L.;PRG + RC), iv) PRG plus plantain (Plantago lanceolate L.;PRG + Plan) and v) PRG plus chicory (Chicorium intybus L.;PRG + Chic). Five farmlets with one sward treatment assigned to each were established which 23 ewes plus their lambs grazed from March to December for the years 2018 to 2021 inclusive. Farmlets were rotationally grazed, stocked at 11.5 ewes/ha and received 130 kg nitrogen/ha/year. Lambs were weaned on average at 15 weeks of age. Post-weaning a leader-follower grazing system was implemented with lambs grazing ahead of the ewes. Post weaning, lambs were weighed fortnightly and drafted at a live weight of 42 kg in June and increasing by 1 kg per month thereafter to produce a target carcass weight of 20 kg. Results show that lamb lifetime average daily gain (ADG) was greater for lambs grazing all binary sward mixtures relative to grazing PRG. Relative to PRG, lamb lifetime ADG was increased by between 17 g/day (PRG + Plan; P < 0.01) to 31 g/day (PRG + RC, and PRG + Chic; P < 0.001) and subsequently the number of days required to reach slaughter (DTS) was reduced by between 15.8 days (PRG + Plan; P < 0.01) to 28.6 days (PRG + RC; P < 0.001) with the addition of a companion forage. As a result of lower DTS, the average total number of anthelmintic treatments administered per lamb was significantly reduced for lambs grazing on all of the binary sward mixtures relative to PRG (P < 0.001). Scanned litter size and ewe milk yield were similar across all sward types as were ewe body weight (BW) and body condition score (BCS) from mating up until 6 weeks post-lambing (P > 0.05). Of the ewes retained from one production year to the next, those grazing PRG + WC, PRG + RC and PRG + Plan had a greater BW than those grazing PRG at the second mating event (P < 0.05), whilst, PRG + RC, PRG + Plan and PRG + Chic ewes had a greater BCS than PRG ewes (P < 0.05). These results suggest the suitability of binary sward mixtures under an intensive sheep grazing system to improve ewe and lamb performance at pasture.

How aggressive interactions with biomimetic agents optimize reproductive performances in mass-reared males of the Mediterranean fruit fly

Mass-rearing procedures of insect species, often used in biological control and Sterile Insect Technique, can reduce the insects competitiveness in foraging, dispersal, and mating. The evocation of certain behaviours responsible to induce specific neuroendocrine products may restore or improve the competitiveness of mass-reared individuals. Herein, we used a mass-reared strain of Ceratitis capitata as model organism. C. capitata is a polyphagous pest exhibiting territorial displays that are closely related to its reproductive performance. We tested if the behaviour of C. capitata males could be altered by hybrid aggressive interactions with a conspecific-mimicking robotic fly, leading to more competitive individuals in subsequent mating events. Aggressive interactions with the robotic fly had a notable effect on subsequent courtship and mating sequences of males that performed longer courtship displays compared to naïve individuals. Furthermore, previous interactions with the robotic fly produced a higher mating success of males. Reproductive performances of C. capitata males may be improved by specific octopaminergic neurones activated during previous aggressive interactions with the robotic fly. This study adds fundamental knowledge on the potential role of specific neuro-behavioural processes in the ecology of tephritid species and paves the way to innovative biotechnological control methods based on robotics and bionics.

Characterizing Physical Interactions between Male and Female Mosquitoes (Aedes aegypti) in Relation to Female Receptivity and Insemination Outcomes Using a Hydrophobic Fluorescent Dye.

Aedes aegypti, the yellow fever mosquito, presents a major threat to human health across the globe as a vector of disease-causing pathogens. Females of this species generally mate only once. From this single mating event, the female stores sufficient sperm to fertilize the multiple clutches of eggs produced during her lifetime. Mating causes dramatic changes in the female's behavior and physiology, including a lifetime suppression of her mating receptivity. Female rejection behaviors include male avoidance, abdominal twisting, wing-flicking, kicking, and not opening vaginal plates or extruding the ovipositor. Many of these events occur on a scale that is too miniscule or fast to see by eye, so high-resolution videography has been used to observe these behaviors instead. However, videography can be labor intensive, require specialized equipment, and often requires restrained animals. We used an efficient, low-cost method to record physical contact between males and females during attempted and successful mating, determined by recording spermathecal filling after dissection. A hydrophobic oil-based fluorescent dye can be applied to the abdominal tip of one animal and can be subsequently transferred to the genitalia of animals of the opposite sex when genital contact occurs. Our data indicate that male mosquitoes make high levels of contact with both receptive and unreceptive females and that males attempt to mate with more females than they successfully inseminate. Female mosquitoes with disrupted remating suppression mate with and produce offspring from multiple males, transferring dye to each. These data suggest that physical copulatory interactions occur independently of the female's receptivity to mate and that many of these interactions represent unsuccessful mating attempts that do not result in insemination.