Neurobiology of egg-laying behavior in Drosophila: neural control of the female reproductive system.

Egg-laying is one of the key aspects of female reproductive behavior in insects. Egg-laying has been studied since the dawn of Drosophila melanogaster as a model organism. The female's internal state, hormones, and external factors, such as nutrition, light, and social environment, affect egg-laying output. However, only recently, neurobiological features of egg-laying behavior have been studied in detail. fruitless and doublesex, two key players in the sex determination pathway, have become focal points in identifying neurons of reproductive significance in both central and peripheral nervous systems. The reproductive tract and external terminalia house sensory neurons that carry the sensory information of egg maturation, mating and egg-laying. These sensory signals include the presence of male accessory gland products and mechanical stimuli. The abdominal neuromere houses neurons that receive information from the reproductive tract, including sex peptide abdominal ganglion neurons (SAGs), and send their information to the brain. In the brain, neuronal groups like aDNs and pC1 clusters modulate egg-laying decision-making, and other neurons like oviINs and oviDNs are necessary for egg-laying itself. Lastly, motor neurons involved in egg-laying, which are mostly octopaminergic, reside in the abdominal neuromere and orchestrate the muscle movements required for laying the egg. Egg-laying neuronal control is important in various evolutionary processes like cryptic female choice, and using different Drosophila species can provide intriguing avenues for the future of the field.

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.

A red flag: an experimental analysis of the function of pink colouration in males of Calopteryx haemorrhoidalis asturica (Odonata: Calopterygidae).

Males display phenotypic characteristics that may be associated with their quality, allowing non-random mating and post-copulatory female choice. In the damselfly Calopteryx haemorrhoidalis asturica, males have a conspicuous pink colouration in the underside of abdominal segments 8-10, which they exhibit during pre- and post-copulatory courtship. We hypothesized that this colouration functions to increase male mating success and/or to elicit females to oviposit. We estimated mating and oviposition success of 27 males, and on the following day, treated males had their segments 8-10 painted black and control males the seventh segment. We recorded the number of male-male fights and courtships, whether the courtship ended in copulation, and whether the female remained in the territory and laid eggs. Our results indicate that the mating success of male C. h. asturica was not significantly affected by the removal of the pink colouration of the abdominal tip, but this colouration clearly affected their success in enticing females to oviposit. Courtship frequency, fat content and muscle mass were positively correlated to male mating rate, and the number of aggressive encounters was negatively correlated. Our study yields experimental evidence for the function of pink colouration of male C. h. asturica, in the context of post-copulatory sexual selection.

The surprising complexity and diversity of sperm storage structures across Galliformes.

In internal fertilisers, the precise timing of ovulation with the arrival of sperm at the site of fertilisation is essential for fertilisation success. In birds, mating is often not synchronised with ovulation, but instead females utilise specialised sperm storage tubules (SSTs) in the reproductive tract, which can ensure sperm are always available for fertilisation at the time of ovulation, whilst simultaneously providing a mechanism of post-copulatory sexual selection. Despite the clear importance of SSTs for fertilisation success, we know little about the mechanisms involved in sperm acceptance, storage, and release. Furthermore, most research has been conducted on only a small number of species, based on which SSTs are usually assumed to look and function in the same way across all species. Here, we conduct a comparative exploration of SST morphology across 26 species of Galliformes. We show that SSTs, and the surrounding tissue, can vary significantly in morphology across species. We provide observational evidence that Galliformes exhibit at least 5 distinct categories of tubule types, including distinctive coiled and multi-branched tubules, and describe 2 additional features of the surrounding tissue. We suggest functional explanations for variation in tubule morphology and propose next steps for future research. Our findings indicate that SSTs are likely to be far more variable than has previously been assumed, with potentially important consequences for our understanding of sperm storage in birds and post-copulatory sexual selection in general.

Female Mate Choice in Wild Kenyan Blue Monkeys (Cercopithecus mitis).

Female mate choice may drive sexual selection, but discerning whether female behaviors reflect free expression of choice or responses to constraints can be difficult. We investigated the efficacy of female choice in wild blue monkeys using 10 years of behavior and paternity data (N = 178 male-female dyads). Although blue monkeys live modally in one-male polygynous groups, where male-biased intersexual power is expected, females can access multiple potential mates during seasonal male influxes and occasional intergroup encounters. Additionally, extra-group males sire offspring. We examined female resistance rates to male-initiated sexual interactions, and unsolicited proceptive behavior that females directed to males (corrected for male availability). Females seldom resisted male solicitation, but initiated sexual interactions more than males. Females generally preferred residents. Those who preferred non-residents tended to have residents with longer tenures, but neither female parity nor rank influenced the tendency to prefer non-residents vs. residents. The male most solicited by a particular female fathered that female's infant 82% of the time; odds of siring were 26 times higher for most vs. nonpreferred males. Female preference predicted paternity even more strongly among non-resident males, with odds of siring 33 times higher for most vs. nonpreferred non-residents. Neither female rank nor parity influenced her likelihood of having her preferred partner as sire. Paternity by preferred males did not affect infant survival. While we cannot fully discount the effect of male-male competition on paternity, these results suggest that blue monkey females can exercise choice successfully, even in a polygynous mating system.

Population density effects on gamete traits and fertilisation dynamics under varying sperm environments in mussels.

Gamete traits can vary widely among species, populations and individuals, influencing fertilisation dynamics and overall reproductive fitness. Sexual selection can play an important role in determining the evolution of gamete traits with local environmental conditions determining the strength and direction of sexual selection. Here, we test for signatures of post-mating selection on gamete traits in relation to population density, and possible interactive effects of population density and sperm concentration on sperm motility and fertilisation rates among natural populations of mussels. Our study shows that males from high-density populations produce smaller sperm compared with males from low-density populations, but we detected no effect of population density on egg size. Our results also reveal that females from low-density populations tended to exhibit lower fertilisation rates across a range of sperm concentrations, although this became less important as sperm concentration increased. Variances in fertilisation success were higher for females than males and the effect of gamete compatibility between males and females increases as sperm concentrations increase. These results suggest that local population density can influence gamete traits and fertilisation dynamics but also highlight the importance of phenotypic plasticity in governing sperm-egg interactions in a highly dynamic selective environment.

Long-sperm precedence and other cryptic female choices in Drosophila melanogaster.

Females that mate multiply make postmating choices about which sperm fertilize their eggs (cryptic female choice); however, the male characteristics they use to make such choices remain unclear. In this study, we sought to understand female sperm use patterns by evaluating whether Drosophila melanogaster females adjust sperm use (second male paternity) in response to four main factors: male genotype, male courtship effort, male pheromone alteration, and male postmating reproductive morphology. Our experiment was replicated across four different D. melanogaster lines, in a full factorial design, including a pheromone manipulation in which second males were perfumed to resemble heterospecific (D. yakuba) males. We found that females prefer longer sperm-regardless of mating order-in almost all contexts; this observed pattern of 'long-sperm precedence' is consistent with female postmating choice of high-fitness male traits. Nonetheless, we also found that this general preference can be plastically altered by females in response to effects including perfuming treatment; this differential female sperm use is between otherwise identical males, and therefore solely female-mediated. Furthermore, our finding that females exercise choice using diverse criteria suggests a possible mechanism for the maintenance of variation in sexually selected male traits.

Ultraviolet signaling in a butterfly is preferred by females and conveys male genetic quality.

Indicator models of sexual selection posit that females choose males on the basis of traits that reveal male genetic quality and thereby enjoy increased offspring production. Here, we report that females of the butterfly Eurema hecabe receive indirect benefits from choosing males based on their ultraviolet (UV) wing coloration, a heritable and condition-dependent trait in this species. We first used a large laboratory-bred pedigree to demonstrate a per-family association between inbreeding and male UV trait value. Females exerted choice for UV-bright males within this protocol, and the average male UV trait value increased over six consecutive generations, presumably due to such selection and despite an increasing rate of pedigree-wide inbreeding. We then experimentally imposed a standard strength of inbreeding upon lines of divergent male UV trait values. Inbreeding depressed the siring performance of low UV treatment males more severely and resulted in a marginal reduction of their UV brightness, which rebounded sharply following subsequent outcrossing. These findings are consistent with the ornament-based signaling of genetic quality as a function of underlying individual-level mutational load.

Paternity bias and cryptic female choice in chickens.

Sperm competition and cryptic female choice (CFC) are 2 significant mechanisms of postcopulatory sexual selection that greatly impact fertilization success in various species. Despite extensive research has conducted on sperm competition and the evolution of sperm traits in internal fertilization, our understanding of the female preferences in selecting sperm is still limited. Here, we aimed to investigate the characteristics of CFC in chickens by utilizing artificial insemination with mixed semen to control for variations in male fertilization success caused by female perception of male quality and mating order. Our results revealed that the offspring from multiple-mated females exhibited mixed paternity. Although the males had an equal number of viable sperm, 1 male consistently exhibited a 15% higher success rate on average, regardless of whether the insemination was performed with fresh or diluted semen. This result suggested that this male demonstrates superior performance in sperm competition, and exhibited a potential advantage in fertilization success. While the dominant male generally made a greater genetic contribution to most offspring, the degree of this advantage varied greatly, ranging from 11.11 to 75%. Furthermore, our study provided evidence of female preferences influenced the precedence of sperm from certain males over others. Interestingly, this bias is not consistently observed among all individuals, as offspring derived from some females were predominantly sired by an overall disadvantaged male while others were predominantly by a different disadvantaged male. Overall, these results underscored the complex processes involved in sperm selection and emphasized the importance of females in sexual selection theory.

Sex ratio affects sexual selection against mutant alleles in a locus-specific way.

Higher male:female operational sex ratio (OSR) is often assumed to lead to stronger sexual selection on males. Yet, this premise has been directly tested by very few studies, with mixed outcomes. We investigated how OSR affects the strength of sexual selection against two deleterious alleles, a natural ebony mutant and a transgenic GFP insertion, in Drosophila melanogaster. To this end, we estimated the relative paternity share of homozygous mutant males competing against wild-type males under different OSRs (1:2, 1:1, 2:1). We also manipulated the mating pool density (18, 36, or 54 individuals) and assessed paternity over three consecutive days, during which the nature of sexual interaction changed. The strength of sexual selection against the ebony mutant increased with OSR, became weaker after the first day, and was little affected by density. In contrast, sexual selection against the GFP transgene was markedly affected by density: at the highest density, it increased with OSR, but at lower densities, it was strongest at 1:1 OSR, remaining strong throughout the experiment. Thus, while OSR can strongly affect the strength of sexual selection against "bad genes," it does not necessarily increase monotonically with male:female OSR. Furthermore, the pattern of relationship between OSR and the strength of sexual selection can be locus-specific, likely reflecting the specific phenotypic effects of the mutation.

The evolution of female-biased genital diversity in bedbugs (Cimicidae).

Rapid genitalia evolution is believed to be mainly driven by sexual selection. Recently, noncopulatory genital functions have been suggested to exert stronger selection pressure on female genitalia than copulatory functions. In bedbugs (Cimicidae), the impact of the copulatory function can be isolated from the noncopulatory impact. Unlike in other taxa, female copulatory organs have no function in egg-laying or waste-product expulsion. Males perform traumatic mating by piercing the female integument, thereby imposing antagonistic selection on females and suspending selection to morphologically match female genitalia. We found the location of the copulatory organ evolved rapidly, changing twice between dorsal and ventral sides, and several times along the anteroposterior and the left-right axes. Male genital length and shape varied much less, did not appear to follow the positional changes seen in females, and showed no evidence for coevolution. Female genitalia position evolved 1.5 times faster than male genital length and shape and showed little neutral or geographic signals. Instead, we propose that nonmorphological male traits, such as mating behavior, may drive female genitalia morphology in this taxon. Models of genitalia evolution may benefit from considering morphological genital responses to nonmorphological stimuli, such as male mating behavior or copulatory position.

Contrasting the form and strength of pre- and postcopulatory sexual selection in a flatworm.

Sexual traits may be selected during multiple consecutive episodes of selection, occurring before, during, or after copulation. The overall strength and form of selection acting on traits may thus be determined by how selection (co-)varies along different episodes. However, it is challenging to measure pre- and postcopulatory phenotypic traits alongside variation in fitness components at each different episode. Here, we used a transgenic line of the transparent flatworm Macrostomum lignano expressing green fluorescent protein (GFP) in all cell types, including sperm cells, enabling in vivo sperm tracking. We assessed the mating success, sperm-transfer efficiency, and sperm fertilizing efficiency of GFP(+) focal worms in which we measured 13 morphological traits. We found linear selection on sperm production rate arising from pre- and postcopulatory components and on copulatory organ shape arising from sperm fertilizing efficiency. We further found nonlinear (mostly concave) selection on combinations of copulatory organ and sperm morphology traits arising mostly from sperm-transfer efficiency and sperm fertilizing efficiency. Our study provides a fine-scale quantification of sexual selection, showing that both the form and strength of selection can change across fitness components. Quantifying how sexual selection builds up along episodes of selection allows us to better understand the evolution of sexually selected traits.

Paternal environment effects are driven by female reproductive fluid but not sperm age in an external fertilizer.

Sperm ageing after ejaculation can generate paternal environment effects that impact offspring fitness. In many species, female reproductive fluids (FRFs), i.e. ancillary fluids released by eggs or within the female reproductive tract, may protect sperm from ageing and can additionally interact with sperm to influence offspring viability. This raises the intriguing prospect that FRFs may alleviate paternal effects associated with sperm ageing. Here, we test this novel hypothesis using the broadcast spawning mussel, Mytilus galloprovincialis. We show that incubating sperm in FRF prior to fertilization increases offspring viability, and that these effects occur independently of sperm age. Our results provide novel evidence that FRFs allow females to selectively bias fertilization toward higher quality sperm within an ejaculate, which in turn yields more viable offspring. We consider this FRF-mediated paternal effect in the context of female physiological control over fertilization and the transgenerational effects of female-regulated haploid selection.

Consistency in responses to conspecific advertisement calls with various signal-to-noise ratios in both sexes of the Anhui tree frog.

Environmental noise has a significant negative impact on acoustic communication in most situations, as it influences the production, transmission, and reception of acoustic signals. However, how animals respond to conspecific sounds when there is interference from environmental noise, and whether males and females display convergent behavioral responses in the face of noise masking remain poorly understood. In this study, we investigated the effects of conspecific male advertisement calls with different signal-to-noise ratios on male-male competition and female choice in the Anhui tree frog Rhacophorus zhoukaiyae using playback and phonotaxis experiments, respectively. The results showed that (1) female Anhui tree frogs preferentially selected the conspecific calls with higher SNR compared to calls with lower SNR; (2) males preferentially responded vocally to the conspecific calls with higher SNR compared to calls with lower SNR; and (3) males' competitive strategies were flexible in the face of noise interference. These results suggest that preferences of both sexes converge in outcome, and that male competitive strategies may depend on predictable female preferences. This study will provide an important basis for further research on decision-making in animals.

Bite force, body size, and octopamine mediate mating interactions in the house cricket (Acheta domesticus).

Mating interactions are rife with conflict because the evolutionary interests of males and females seldom coincide. Intersexual conflict affects sexual selection, yet the proximate factors underlying male coercive ability and female resistance are poorly understood. Male combat outcomes are often influenced by bite force, with superior biters being more likely to achieve victory over poorer biters in a range of species, including crickets. If good performers also achieve mating success through sexual coercion, then bite force might play a role in intersexual conflict as well. We tested the capacity of bite force to influence mating interactions in house crickets both directly by measuring bite forces of males and females and by altering male bite capacity through neuropharmacological manipulation. In addition, the invertebrate neurotransmitter octopamine both mediates aggression and underlies motivation to bite in male house crickets. By blocking octopamine receptors through the application of an antagonist, epinastine, we tested the effects of reduced bite force on male mating success. Our results show that male bite capacity, in combination with body size, influences both the likelihood and the outcomes of mating interactions, whereas treatment of males with epinastine eliminates motivation to mate. Our results suggest a functional role for bite force in affecting both sexual conflict and sexual selection and expand our knowledge of the influence of biogenic amines on reproductive behaviour.

Mismatch between calf paternity and observed copulations between male and female reindeer: Multiple mating in a polygynous ungulate?

In polygynous systems, such as that exhibited by reindeer Rangifer tarandus, mate choice can be difficult to disentangle from male intrasexual competition because male behavior may constrain female choice. Multiple mating may provide an avenue for female mate choice, though it is difficult to identify using behavioral estimators alone. Molecular techniques address this issue by affording ecologists an opportunity to reassess mating systems from a genetic perspective. We assessed the frequency and possible explanations for multiple mating in reindeer using a genetic approach to determine the success of observed copulations in a semi-domesticated herd in Kaamanen, Finland. Behavioral and genetic data were synthesized with population characteristics over a 7-year period to test the hypothesis that, if present, polyandry in reindeer is driven by sexual harassment from sub-dominant males. We observed multiple mating in 42% of females, with as many as 60% exhibiting multiple mating in certain years. We found no evidence that multiple mating resulted from sexual harassment by sub-dominant males, suggesting that it is likely a deliberate strategy among females. Conversion rate of copulations into paternities varied with male size, with smaller males more likely to experience mismatch than larger males. Female preference for larger males persisted despite the occurrence of multiple mating, possibly suggesting a mechanism for cryptic post-copulatory selection. We suggest further research to delineate the possible influence of cryptic post-copulatory selection and multiple mating to defend against infertility in exhausted males.

Exposure to sublethal concentration of flupyradifurone alters sexual behavior and cuticular hydrocarbon profile in Heriades truncorum, an oligolectic solitary bee.

The aboveground oligolectic bee, Heriades truncorum, is a particularly good model for studying the impact of pesticides on sexual communication, since some aspects of its mating behavior have previously been described. We have tested (1) the interference of the pesticide flupyradifurone on male precopulatory behavior and male mating partner preferences, (2) the way that the pesticide interferes in male quality assessment by the female, and (3) the effects of the pesticide on the chemical compounds in the female cuticle. We exposed bees of both sexes to a sublethal concentration of flupyradifurone. Various behaviors were registered in a mating arena with two females (one unexposed and one exposed) and one male (either unexposed or exposed). Unexposed males were quicker to attempt to mate. Treatment also impacted precopulatory behavior and male quality assessment by females. Males approached unexposed females more quickly than insecticide-exposed ones. Females exposed to insecticide produced lower amounts of some cuticular hydrocarbons (sex pheromone candidates) and appeared less choosy than unexposed females. Our findings suggest that insecticide exposure affects sexual communication, playing a role both in male preference and in male quality assessment by the female.

Sperm of more colourful males are better adapted to ovarian fluids in lake char (Salmonidae).

Fish often spawn eggs with ovarian fluids that have been hypothesized to support the sperm of some males over others (cryptic female choice). Alternatively, sperm reactions to ovarian fluids could reveal male strategies. We used wild-caught lake char (Salvelinus umbla) to experimentally test whether sperm react differently to the presence of ovarian fluid, and whether any differential sperm reaction could be predicted by male breeding coloration, male inbreeding coefficients (based of 4150 SNPs) or the kinship coefficients between males and females. Male coloration was positively linked to body size and current health (based on lymphocytosis and thrombocytosis) but was a poor predictor of inbreeding or kinship coefficients. We found that sperm of more colourful males were faster in diluted ovarian fluids than in water only, while sperm of paler males were faster in water than in ovarian fluids. We then let equal numbers of sperm compete for fertilizations in the presence or absence of ovarian fluids and genetically assigned 1464 embryos (from 70 experimental trials) to their fathers. The presence of ovarian fluids significantly increased the success of the more colourful competitors. Sperm of less inbred competitors were more successful when tested in water only than in diluted ovarian fluids. The kinship coefficients had no significant effects on sperm traits or fertilization success in the presence of ovarian fluids, although parallel stress tests on embryos had revealed that females would profit more from mating with least related males rather than most coloured ones. We conclude that sperm of more colourful males are best adapted to ovarian fluids, and that the observed reaction norms suggest male strategies rather than cryptic female choice.

How sexual and natural selection interact and shape the evolution of nests and nesting behaviour in fishes.

Among ray-finned fishes that provide parental care, many spawn in constructed nests, ranging from bowls, burrows and ridges to nests made of algae or bubbles. Because a nest by definition is a construction that enhances the nest-builder's fitness by helping it meet the needs of the developing offspring, nest-building behaviour is naturally selected, as is a preference for spawning with mates that provide well-built nests. However, nest-building behaviour can also be sexually selected, when nest traits increase mating success, protect against sperm competition or nest take-overs by conspecifics. Here, we offer a systematic review, with examples of how competition for sites and location of fish nests relates to sexual selection. We examine direct and indirect benefits of mate choice linked to nest traits, and different types of nests, from a sexual selection perspective. Nest-related behaviours are often under both natural and sexual selection, and we disentangle examples where that is the case, with special attention to females. We highlight some taxa in which nest building is likely to be sexually selected, but lack of research has left them uninvestigated. Some of them are established aquarium species, making them particularly amenable for future research. Finally, we compare with arthropods, amphibians and birds. This article is part of the theme issue 'The evolutionary ecology of nests: a cross-taxon approach'.

Female reproductive fluid concentrations affect sperm performance of alternative male phenotypes in an external fertilizer.

There is growing evidence that the female reproductive fluid (FRF) plays an important role in cryptic female choice through its differential effect on the performance of sperm from different males. In a natural spawning event, the male(s) may release ejaculate closer or further away from the spawning female. If the relative spatial proximity of competing males reflects the female pre-mating preference towards those males, then favoured males will encounter higher concentrations of FRF than unpreferred males. Despite this being a common situation in many external fertilizers, whether different concentrations of FRF can differentially influence the sperm performance of distinct male phenotypes (favoured and unfavoured by the female) remains to be elucidated. Here, we tested this hypothesis using the grass goby (Zosterisessor ophiocephalus), a fish with distinct territorial-sneaker reproductive tactics and female pre-mating preference towards territorial males, that consequently mate in an advantaged position and whose sperm experience higher concentrations of FRF. Our findings revealed a differential concentration-dependent effect of FRF over sneaker and territorial sperm motility only at low concentrations (i.e. at the distance where sneakers typically ejaculate), with increasing FRF concentrations (i.e. close to the eggs) similarly boosting the sperm performance of both sneaker and territorial males. The ability to release sperm close to the eggs is a prerogative of territorials, but FRF can likewise advantage the sperm of those sneakers that are able to get closer, allowing flexibility in the direction of female post-mating choice.