Seminal proteins but not sperm induce morphological changes in the Drosophila melanogaster female reproductive tract during sperm storage

In most insects, sperm transferred by the male to the female during mating are stored within the female reproductive tract for subsequent use in fertilization. In Drosophila melanogaster, male accessory gland proteins (Acps) within the seminal fluid are required for efficient accumulation of sperm in the female's sperm storage organs. To determine the events within the female reproductive tract that occur during sperm storage, and the role that Acps and sperm play in these events, we identified morphological changes that take place during sperm storage in females mated to wild-type, Acp-deficient or sperm-deficient males. A reproducible set of morphological changes occurs in a wild-type mating. These were categorized into 10 stereotypic stages. Sperm are not needed for progression through these stages in females, but receipt of Acps is essential for progression beyond the first few stages of morphological change. Furthermore, females that received small quantities of Acps reached slightly later stages than females that received no Acps. Our results suggest that timely morphological changes in the female reproductive tract, possibly muscular in nature, may be needed for successful sperm storage, and that Acps from the male are needed in order for these changes to occur.     

Male diet and age influence to inhibit female remating in Ceratitis capitata (Diptera: Tephritidae)

This study evaluated the influence of age and adult nutritional status of Ceratitis capitata males on their ability to inhibit female remating. Their roles and that of copula duration on the amount of sperm transferred to female spermathecae were also analysed. After emergence, adults were kept in separate groups according to their diets (either high protein – 6.5 g of brewer’s yeast, or low protein – 3.5 g of brewer’s yeast) and their age at the time of use in experiments (4, 8, 12, 16 and 20 days old). The results demonstrated that: (i) male age was not a factor that influenced remating when females mated with well‐nourished males; however, the youngest males (4 days old) in the low‐protein group were less efficient in preventing female remating; (ii) 12‐ and 16‐day‐old males fed on a high‐protein diet transferred and produced more sperm than males of other groups; (iii) there was no correlation between copula duration and the amount of sperm transferred to the female; the longest copula durations were observed among low‐protein‐diet/20‐day‐old males. These results suggest that age and nutrition influence the ability of the males to inhibit female remating. The sterile insect technique is most effective when females do not remate after coupling with sterile males, and therefore, the capacity to inhibit female remating is an important characteristic of males released in the field.     

Protein and carbohydrate intake influence sperm number and fertility in male cockroaches,but not sperm viability

It is commonly assumed that because males produce many, tiny sperm, they are cheap to produce. Recent work, however, suggests that sperm production is not cost-free. If sperm are costly to produce, sperm number and/or viability should be influenced by diet, and this has been documented in numerous species. Yet few studies have examined the exact nutrients responsible for mediating these effects. Here, we quantify the effects of protein (P) and carbohydrate (C) intake on sperm number and viability in the cockroach Nauphoeta cinerea, as well as the consequences for male fertility. We found the intake of P and C influenced sperm number, being maximized at a high intake of diets with a P : C ratio of 1 : 2, but not sperm viability. The nutritional landscapes for male fertility and sperm number were closely aligned, suggesting that sperm number is the major determinant of male fertility in N. cinerea. Under dietary choice, males regulate nutrient intake at a P : C ratio of 1 : 4.95, which is midway between the ratios needed to maximize sperm production and pre-copulatory attractiveness in this species. This raises the possibility that males regulate nutrient intake to balance the trade-off between pre- and post-copulatory traits in this species.     

Why does inbreeding reduce male paternity? Effects on sexually selected traits

Mating with relatives has often been shown to negatively affect offspring fitness (inbreeding depression). There is considerable evidence for inbreeding depression due to effects on naturally selected traits, particularly those expressed early in life, but there is less evidence of it for sexually selected traits. This is surprising because sexually selected traits are expected to exhibit strong inbreeding depression. Here, we experimentally created inbred and outbred male mosquitofish (Gambusia holbrooki). Inbred males were the offspring of matings between full siblings. We then investigated how inbreeding influenced a number of sexually selected male traits, specifically: attractiveness, sperm number and velocity, as well as sperm competitiveness based on a male's share of paternity. We found no inbreeding depression for male attractiveness or sperm traits. There was, however, evidence that lower heterozygosity decreased paternity due to reduced sperm competitiveness. Our results add to the growing evidence that competitive interactions exacerbate the negative effects of the increased homozygosity that arises when there is inbreeding.     

Juvenile hormone regulation of male accessory gland activity in the red flour beetle, Tribolium castaneum

Male accessory gland proteins (Acps) act as key modulators of reproductive success in insects by influencing the female reproductive physiology and behavior. We used custom microarrays and identified 112 genes that were highly expressed in male accessory glands (MAG) in the red flour beetle, Tribolium castaneum. Out of these 112 identified genes, 59 of them contained sequences coding for signal peptide and cleavage site and the remaining 53 contained transmembrane domains. The expression of 14 of these genes in the MAG but not in other tissues of male or female was confirmed by quantitative real-time PCR. In virgin males, juvenile hormone (JH) levels increased from second day post adult emergence (PAE), remained high on third day PAE and declined on fourth day PAE. The ecdysteroid titers were high soon after adult emergence but declined to minimal levels from 1 to 5 days PAE. Feeding of juvenile hormone analog, hydroprene, but not the ecdysteroid analog, RH-2485, showed an increase in size of MAGs, as well as an increase in total RNA and protein content of MAG. Hydroprene treatment also increased the expression of Acp genes in the MAG. RNAi-mediated knock-down in the expression of JHAMT gene decreased the size of MAGs and expression of Acps. JH deficiency influenced male reproductive fitness as evidenced by a less vigor in mating behavior, poor sperm transfer, low egg and the progeny production by females mated with the JH deficient males. These data suggest a critical role for JH in the regulation of male reproduction especially through MAG secretions.     

Influence of nutrition on male development and reproduction in Tribolium castaneum

Tribolium beetles have evolved over several thousand years to colonize and exploit various food products that vary widely in their nutritional quality. Here Tribolium castaneum was used as a model organism to explore the effect of nutritional quality on male development and reproduction. The results showed, when tested across different qualities of nutritional diets, Tribolium males developed faster and their body size was larger on a high-quality diet, and there were significant correlations between male developmental traits. However, Tribolium males fed different nutritional diets did not show significant variation in olfactory attractiveness, mating rate, insemination rate, sperm defense (P1), sperm offense (P2), and reproductive success within a population context (RSPC). Moreover, there was no significant correlation of male reproductive performances except RSPC with developmental traits, and except for P2 and RSPC, no significant correlations between male reproductive performances. Therefore, although male developmental performance was significantly influenced by diet quality, reproductive performance was not. We discussed these findings and their sexual selection implications in light of its habitat.     

The Acp26Aa seminal fluid protein is a modulator of early egg hatchability in Drosophila melanogaster

Drosophila melanogaster male accessory gland proteins (Acps) that are transferred in the ejaculate with sperm mediate post-mating competition for fertilizations between males. The actions of Acps include effects on oviposition and ovulation, receptivity and sperm storage. Two Acps that modulate egg production are Acp26Aa (ovulin) and Acp70A (the sex peptide). Acp26Aa acts specifically on the process of ovulation (the release of mature eggs from the ovaries), which is initiated 1.5 h after mating. In contrast, sperm storage can take as long as 6-9 h to complete. Initial ovulations after matings by virgin females will therefore occur before all sperm are fully stored and the extra eggs initially laid as a result of Acp26Aa transfer are expected to be inefficiently fertilized. Acp26Aa-mediated release of existing eggs should not cause a significant energetic cost or lead to a decrease in female lifespan assuming, as seems likely, that the energetic cost of egg laying comes from de novo egg synthesis (oogenesis) rather than from ovulation. We tested these predictions using Acp26Aa(1) mutant males that lack Acp26Aa but are normal for other Acps and Acp26Aa(2) males that transfer a truncated but fully functional Acp26Aa protein. Females mating with Acp26Aa(2) (truncation) males that received functional Acp26Aa produced significantly more eggs following their first matings than did mates of Acp26Aa(1) (null) males. However, as predicted above, these extra eggs, which were laid as a result of Acp26Aa transfer to virgin females, showed significantly lower egg hatchability. Control experiments indicated that this lower hatchability was due to lower rates of fertilization at early post-mating times. There was no drop in egg hatchability in subsequent non-virgin matings. In addition, as predicted above, females that did or did not receive Acp26Aa did not differ in survival, lifetime fecundity or lifetime progeny, indicating that Acp26Aa transfer does not represent a significant energetic cost for females and does not contribute to the survival cost of mating. Acp26Aa appears to remove a block to oogenesis by causing the clearing out of existing mature eggs and, thus, indirectly allowing oogenesis to be initiated immediately after mating. The results show that subtle processes coordinate the stimulation of egg production and sperm storage in mating pairs.     

Adult nutrition,but not inbreeding,affects male primary sexual traits in the leaf‐footed cactus bug Narnia femorata (Hemiptera: Coreidae)

Adverse conditions may be the norm rather than the exception in natural populations. Many populations experience poor nutrition on a seasonal basis. Further, brief interludes of inbreeding can be common as population density fluctuates and because of habitat fragmentation. Here, we investigated the effects of poor nutrition and inbreeding on traits that can be very important to reproductive success and fitness in males: testes mass, sperm concentration, and sperm viability. Our study species was Narnia femorata, a species introduced to north‐central Florida in the 1950s. This species encounters regular, seasonal changes in diet that can have profound phenotypic effects on morphology and behavior. We generated inbred and outbred individuals through a single generation of full‐sibling mating or outcrossing, respectively. All juveniles were provided a natural, high‐quality diet of Opuntia humifusa cactus cladode with fruit until they reached adulthood. New adult males were put on a high‐ or low‐quality diet for at least 21 days before measurements were taken. As expected, the low‐quality diet led to significantly decreased testes mass in both inbred and outbred males, although there were surprisingly no detectable effects on sperm traits. We did not find evidence that inbreeding affected testes mass, sperm concentration, and sperm viability. Our results highlight the immediate and overwhelming effects of nutrition on testes mass, while suggesting that a single generation of inbreeding might not be detrimental for primary sexual traits in this particular population.     

On the evolution of heightened condition dependence of male sexual displays

The maintenance of genetic variation in male sexual display traits in the face of strong directional sexual selection from female preferences is an ongoing evolutionary conundrum. Condition dependence and the genic capture hypothesis are often cited as theoretical resolutions to this problem, yet little is known about the ability of condition dependence itself to evolve. We set out to test how a suite of cuticular hydrocarbons (CHCs) used in sexual displays are affected by adult diet and the potential for any condition-dependent response to evolve in a laboratory-adapted population of the Australian fruit fly Drosophila serrata. We performed a dietary manipulation within a half-sib breeding design, raising adult males either with or without access to live yeast, a manipulation that had previously shown strong effects on female fitness. Diet had strong phenotypic effects, with males from the different diets producing different CHC blends. The blend of CHCs under sexual selection showed a degree of elevated condition dependence. Regardless of the heightened sensitivity of favoured CHC blends to diet and the presence of genetic variance for the traits, we were unable to detect any genetic variance in the reaction norms for the male dietary response. Our results suggest that there is limited opportunity for males to evolve further condition dependence in response to yeast availability in this population.     

The effects of male social environment on sperm phenotype and genome integrity

Sperm function and quality are primary determinants of male reproductive performance and hence fitness. The presence of rival males has been shown to affect ejaculate and sperm traits in a wide range of taxa. However, male physiological conditions may not only affect sperm phenotypic traits but also their genetic and epigenetic signatures, affecting the fitness of the resulting offspring. We investigated the effects of male‐male competition on sperm quality using TUNEL assays and geometric morphometrics in the zebrafish, Danio rerio. We found that the sperm produced by males exposed to high male–male competition had smaller heads but larger midpiece and flagellum than sperm produced by males under low competition. Head and flagella also appeared less sensitive to the osmotic stress induced by activation with water. In addition, more sperm showed signals of DNA damage in ejaculates of males under high competition. These findings suggest that the presence of a rival male may have positive effects on sperm phenotypic traits but negative effects on sperm DNA integrity. Overall, males facing the presence of rival males may produce faster swimming and more competitive sperm but this may come at a cost for the next generation.     

Building a Beetle: How Larval Environment Leads to Adult Performance in a Horned Beetle

The link between the expression of the signals used by male animals in contests with the traits which determine success in those contests is poorly understood. This is particularly true in holometabolous insects such as horned beetles where signal expression is determined during metamorphosis and is fixed during adulthood, whereas performance is influenced by post-eclosion feeding. We used path analysis to investigate the relationships between larval and adult nutrition, horn and body size and fitness-related traits such as strength and testes mass in the horned beetle Euoniticellus intermedius. In males weight gain post-eclosion had a central role in determining both testes mass and strength. Weight gain was unaffected by adult nutrition but was strongly correlated with by horn length, itself determined by larval resource availability, indicating strong indirect effects of larval nutrition on the adult beetle’s ability to assimilate food and grow tissues. Female strength was predicted by a simple path diagram where strength was determined by eclosion weight, itself determined by larval nutrition: weight gain post-eclosion was not a predictor of strength in this sex. Based on earlier findings we discuss the insulin-like signalling pathway as a possible mechanism by which larval nutrition could affect adult weight gain and thence traits such as strength.     

The effect of developmental nutrition on life span and fecundity depends on the adult reproductive environment in Drosophila melanogaster

Both developmental nutrition and adult nutrition affect life‐history traits; however, little is known about whether the effect of developmental nutrition depends on the adult environment experienced. We used the fruit fly to determine whether life‐history traits, particularly life span and fecundity, are affected by developmental nutrition, and whether this depends on the extent to which the adult environment allows females to realize their full reproductive potential. We raised flies on three different developmental food levels containing increasing amounts of yeast and sugar: poor, control, and rich. We found that development on poor or rich larval food resulted in several life‐history phenotypes indicative of suboptimal conditions, including increased developmental time, and, for poor food, decreased adult weight. However, development on poor larval food actually increased adult virgin life span. In addition, we manipulated the reproductive potential of the adult environment by adding yeast or yeast and a male. This manipulation interacted with larval food to determine adult fecundity. Specifically, under two adult conditions, flies raised on poor larval food had higher reproduction at certain ages – when singly mated this occurred early in life and when continuously mated with yeast this occurred during midlife. We show that poor larval food is not necessarily detrimental to key adult life‐history traits, but does exert an adult environment‐dependent effect, especially by affecting virgin life span and altering adult patterns of reproductive investment. Our findings are relevant because (1) they may explain differences between published studies on nutritional effects on life‐history traits; (2) they indicate that optimal nutritional conditions are likely to be different for larvae and adults, potentially reflecting evolutionary history; and (3) they urge for the incorporation of developmental nutritional conditions into the central life‐history concept of resource acquisition and allocation.     

Male contributions to egg production: the role of accessory gland products and sperm in Drosophila melanogaster

Drosophila melanogatser seminal fluid components, accessory gland proteins (Acps) and sperm, induce females to deposit high numbers of fertilized eggs for about 11 days. This high and sustained level of egg deposition requires that oogenesis be stimulated to provide the necessary mature oocytes. To investigate the relative timing and contributions of Acps and sperm in the egg-production process, we examined the rates of oogenic progression and egg deposition in females mated to genetically altered males that have seminal fluid deficient in Acps and/or sperm, and subjected these data to path analysis. We found that Acps and sperm are complementary stimuli necessary for inducing high rates of oogenic progression and rapid egg deposition. While egg deposition and oogenic progression can be induced by Acps alone, both Acps and sperm are required for maximum stimulation of oogenic progression and egg deposition immediately after mating.     

Male Investments in High Quality Sperm Improve Fertilization Success,but May Have Negative Impact on Offspring Fitness in Whitefish

Many ejaculate traits show remarkable variation in relation to male social status. Males in disfavoured (subordinate) mating positions often invest heavily on sperm motility but may have less available resources on traits (e.g., secondary sexual ornaments) that improve the probability of gaining matings. Although higher investments in sperm motility can increase the relative fertilization success of subordinate males, it is unclear whether status-dependent differences in sperm traits could have any consequences for offspring fitness. We tested this possibility in whitefish (Coregonus lavaretus L.) by experimentally fertilizing the eggs of 24 females with the sperm of either highly-ornamented (large breeding tubercles, dominant) or less-ornamented (small tubercles, subordinate) males (split-clutch breeding design). In comparison to highly-ornamented individuals, less-ornamented males had higher sperm motility, which fertilized the eggs more efficiently, but produced embryos with impaired hatching success. Also offspring size and body condition were lower among less-ornamented males. Furthermore, sperm motility was positively associated with the fertilization success and offspring size, but only in highly-ornamented males. Together our results indicate that male investments on highly motile (fertile) sperm is not necessarily advantageous during later offspring ontogeny and that male status-dependent differences in sperm phenotype may have important effects on offspring fitness in different life-history stages.     

Male seminal fluid proteins are essential for sperm storage in Drosophila melanogaster.

The seminal fluid that is transferred along with sperm during mating acts in many ways to maximize a male's reproductive success. Here, we use transgenic Drosophila melanogaster males deficient in the seminal fluid proteins derived from the accessory gland (Acps) to investigate the role of these proteins in the fate of sperm transferred to females during mating. Competitive PCR assays were used to show that while Acps contribute to the efficiency of sperm transfer, they are not essential for the transfer of sperm to the female. In contrast, we found that Acps are essential for storage of sperm by females. Direct counts of stored sperm showed that 10% of normal levels are stored by females whose mates transfer little or no Acps along with sperm.     

Genes regulated by mating, sperm, or seminal proteins in mated female Drosophila melanogaster

In Drosophila melanogaster, sperm and accessory gland proteins ("Acps," a major component of seminal fluid) transferred by males during mating trigger many physiological and behavioral changes in females (reviewed in ). Determining the genetic changes triggered in females by male-derived molecules and cells is a crucial first step in understanding female responses to mating and the female's role in postcopulatory processes such as sperm competition, cryptic female choice, and sexually antagonistic coevolution. We used oligonucleotide microarrays to compare gene expression in D. melanogaster females that were either virgin, mated to normal males, mated to males lacking sperm, or mated to males lacking both sperm and Acps. Expression of up to 1783 genes changed as a result of mating, most less than 2-fold. Of these, 549 genes were regulated by the receipt of sperm and 160 as a result of Acps that females received from their mates. The remaining genes whose expression levels changed were modulated by nonsperm/non-Acp aspects of mating. The mating-dependent genes that we have identified contribute to many biological processes including metabolism, immune defense, and protein modification.     

One size fits all? Determinants of sperm transfer in a highly dimorphic orb‐web spider

The evolutionary significance of widespread hypo‐allometric scaling of genital traits in combination with rapid interspecific genital trait divergence has been of key interest to evolutionary biologists for many years and remains poorly understood. Here, we provide a detailed assessment of quantitative genital trait variation in males and females of the sexually highly dimorphic and cannibalistic orb‐weaving spider Argiope aurantia. We then test how this trait variation relates to sperm transfer success. In particular, we test specific predictions of the one‐size‐fits‐all and lock‐and‐key hypotheses for the evolution of genital characters. We use video‐taped staged matings in a controlled environment with subsequent morphological microdissections and sperm count analyses. We find little support for the prediction of the one‐size‐fits‐all hypothesis for the evolution of hypo‐allometric scaling of genital traits, namely that intermediate trait dimensions confer highest sperm transfer success. Likewise, our findings do not support the prediction of the lock‐and‐key hypothesis that a tight fit of male and female genital traits mediates highest sperm transfer success. We do, however, detect directional effects of a number of male and female genital characters on sperm transfer, suggesting that genital trait dimensions are commonly under selection in nature. Importantly, even though females are much larger than males, spermatheca size limits the number of sperm transferred, contradicting a previous hypothesis about the evolutionary consequences of genital size dimorphism in extremely size‐dimorphic taxa. We also find strong positive effects of male body size and copulation duration on the probability of sperm transfer and the number of sperm transferred, with implications for the evolution of extreme sexual size dimorphism and sexual cannibalism in orb weavers.     

Effect of probiotic adult diets on fitness components of sterile male Mediterranean fruit flies (Diptera: Tephritidae) under laboratory and field cage conditions

The aim of the current study was to investigate the effect of probiotic adult diets, i.e., adult diets containing viable symbiotic intestinal bacteria, on the pheromone-calling activity, mating success, life expectancy, and survival of mass-reared male Mediterranean fruit flies, Ceratitis capitata (Wiedemann), as an avenue for improving the field performance of sterile males in release programs to eradicate, suppress, or prevent spread of wild populations. The effect of inoculation of two standard adult diets (sugar-yeast granulate [SY] and sugar agar [s]) and two experimental formulations (yeast-reduced granulate [Sy] and yeast-enhanced sugar agar [sy]) with Enterobacter agglomerans and Klebsiella pneumoniae (typically occurring in the gut of wild flies) on the different fitness components was assessed in the laboratory and on field-caged host trees. We found that, in the laboratory, males reared on the probiotic yeast-enhanced agar, sy, had a significant mating advantage over competitors fed the standard s agar (probiotic and control) or noninoculated sy agar; no effect of probiotic enrichment (or lowering the yeast content) was found with the granular diets. Mating test results obtained in the field were inconsistent with laboratory data in that no differences in the numbers of matings were observed between males reared on any of the probiotic and control agar diets (or the SY granulate), whereas males feeding on the probiotic modified granulate, Sy, scored significantly more matings than their control competitors. The pheromone-calling activity of males maintained on the granular diets was not affected by probiotic enrichment on any of the seven observation days. Agar-fed males, however, "called" more frequently on days 6 and 7 (but not on days 1-5) when their diet contained the probiotic load. Laboratory survival of granulate-fed males was found to be significantly prolonged with probiotic inoculation and lowering the yeast content of the standard SY granulate (but not with probiotic inoculation of sy). Similarly, males reared on the probiotic and control modified agars (sy) survived significantly longer than those feeding on the standard s agars (inoculated and control). Again, the results obtained in the field were inconsistent, because no differences between treated and control males were found for any of the diets. The findings are discussed in the light of other published studies on adult nutrition and behavioral ecology in C. capitata.     

Strategic Ejaculation in the Black Scavenger Fly Sepsis cynipsea Revisited: Copula Duration as a Function of Sperm Depletion and Body Size

The massive numbers of sperm males transfer during a single mating are physiologically costly and the amount of sperm that can be stored is limited. Therefore, males can perform only a finite number of successive copulations without loss of fertility, and males should allocate sperm prudently. We investigated sperm availability and depletion in male black scavenger flies, Sepsis cynipsea (Diptera: Sepsidae), asking whether males adjust copula duration according to nutrition, their sperm stores, their own and their partner’s body size, as predicted by theory. We created a gradient of sperm limitation by restricting dung (their protein resource as adults) and subjecting males to a varying number of copulations. While male fertility did not depend on access to fresh dung (contrary to females), it did decline after three copulations, and more so when males were small. Larger females tended to lay more unfertilized eggs after copulating with previously mated males. However, copula duration was not influenced by a male’s number of previous copulations, and therefore apparently not by his current sperm stores. Nevertheless, copula duration varied with male size, with small males copulating longer, and with female size, as copulations lasted longer with larger females, suggesting that males are investing more sperm in larger, more fecund females. While male copula adjustments to their own nutrition and body size may be simple (proximate) physiological responses, responses to female size indicate more strategic and sophisticated sperm‐allocation strategies than previously thought.