Generalization of courtship learning in Drosophila is mediated by cis-vaccenyl acetate

Reproductive behavior in Drosophila has both stereotyped and plastic components that are driven by age- and sex-specific chemical cues. Males who unsuccessfully court virgin females subsequently avoid females that are of the same age as the trainer. In contrast, males trained with mature mated females associate volatile appetitive and aversive pheromonal cues and learn to suppress courtship of all females. Here we show that the volatile aversive pheromone that leads to generalized learning with mated females is (Z)-11-octadecenyl acetate (cis-vaccenyl acetate, cVA). cVA is a major component of the male cuticular hydrocarbon profile, but it is not found on virgin females. During copulation, cVA is transferred to the female in ejaculate along with sperm and peptides that decrease her sexual receptivity. When males sense cVA (either synthetic or from mated female or male extracts) in the context of female pheromone, they develop a generalized suppression of courtship. The effects of cVA on initial courtship of virgin females can be blocked by expression of tetanus toxin in Or65a, but not Or67d neurons, demonstrating that the aversive effects of this pheromone are mediated by a specific class of olfactory neuron. These findings suggest that transfer of cVA to females during mating may be part of the male's strategy to suppress reproduction by competing males.     

Copulation Song in Drosophila: Do Females Sing to Change Male Ejaculate Allocation and Incite Postcopulatory Mate Choice?

Drosophila males sing a courtship song to achieve copulations with females. Females were recently found to sing a distinct song during copulation, which depends on male seminal fluid transfer and delays female remating. Here, it is hypothesized that female copulation song is a signal directed at the copulating male and changes ejaculate allocation. This may alter female remating and sperm usage, and thereby affect postcopulatory mate choice. Mechanisms of how female copulation song is elicited, how males respond to copulation song, and how remating is modulated, are considered. The potential adaptive value of female signaling during copulation is discussed with reference to vertebrate copulation calls and their proposed function in eliciting mate guarding. Female copulation song may be widespread within the Drosophila genus. This newly discovered behavior opens many interesting avenues for future research, including investigation of how sexually dimorphic neuronal circuits mediate communication between nervous system and reproductive organs.     

Females of the Cellar Spider Discriminate Against Previous Mates

Mate choice for novel partners should evolve when remating with males of varying genetic quality provides females with fitness‐enhancing benefits. We investigated sequential mate choice for same or novel mating partners in females of the cellar spider Pholcus phalangioides (Pholcidae) to understand what drives female remating in this system. Pholcus phalangioides females are moderately polyandrous and show reluctance to remating, but double‐mated females benefit from a higher oviposition probability compared to single‐mated females. We exposed mated females to either their former (same male) or a novel mating partner and assessed mating success together with courtship and copulatory behaviours in both sexes. We found clear evidence for mate discrimination: females experienced three‐fold higher remating probabilities with novel males, being more often aggressive towards former males and accepting novel males faster in the second than in the first mating trial. The preference for novel males suggests that remating is driven by benefits derived from multiple partners. The low remating rates and the strong last male sperm precedence in this system suggest that mating with novel partners that represent alternative genotypes may be a means for selecting against a former mate of lower quality.     

Esterase-6 polymorphism inDrosophila melanogaster:Effects of temperature and methyl malonate on genotypic trajectories in polymorphic populations set up with highly inbred lines

It is generally difficult to identify possible effects of selection at a specific locus because of the heterogeneity of the genetic background. Geographical patterns ofEst-6 gene frequencies suggest that there is selection at this locus but selection on loci closely linked to it cannot be excluded. Differences in catalytic properties between allozymes have been shownin vitro; further, several laboratory studies have shown apparent fitness differences between allozymes. Our study used inbred lines highly homogeneous in the genetic background. Four populations were set up fromEst-6s andEst-6F homozygous females inseminated by males of the same genotype at each combination of three factors: temperature (18 and 25°C); methyl malonate (presence or absence); input gene frequencies [p(S) = 0.2 and 0.8]. The populations were sampled periodically for about 28 generations. Methyl malonate was chosen to exert pressure in the enzymatic function of esterase-6. Statistical analyses show that: there are no sex differences; gene frequencies change from input values to those of the first sampling, when only individuals of the first generation are present at 18^oC or individuals of the second generation just begin to appear at 25°C; gene frequencies do not change thereafter and Hardy-Weinberg equilibrium is established. The changes in gene frequencies observed in the first generations suggest thatEst-6 can under certain conditions be a target of selection. Such conditions may not, however, occur in natural populations.     

No Effect of Male Courtship Intensity on Female Remating in the Butterfly <Emphasis Type="Italic">Pieris napi</Emphasis>

In Pieris napi, female fitness increases with number of matings, but wild females mate at an unexpectedly low rate. From a sexual conflict perspective this could be because males manipulate female remating, or alternatively, because wild females experience costs associated with remating which are not applicable under laboratory conditions. To get an indication which sex controls remating and/or the different sexes’ relative costs and benefits of remating, we here test whether female mating frequency is affected by male courtship intensity. We found no effect on female mating frequency or lifespan. This indicates that (i) females control remating and their optimal mating frequency is lower compared to males, or (ii) males can manipulate female remating. We argue that both these alternatives may apply simultaneously to P. napiand that they are inseparable.     

Genetics of a tissue esterase polymorphism (Est-6) in the rabbit (Oryctolagus cuniculus)

Genetic analysis of a polymorphic tissue esterase revealed a new locus (Est-6) with two alleles (Est-6 ^a andEst-6 ^b) on linkage group VI of the rabbit.Est-6 is closely linked to theEst-1,2,4 cluster. Esterase ofEst-6 is found in many organs, particularly in liver and small intestine, but not in erythrocytes and serum.Est-6 esterase hydrolyzes -naphthyl acetate and butyrate, naphthol AS-D acetate, indoxyl acetate, and butyrate as well as 5-bromoindoxyl acetate,N-acetyl-l-alanine--naphthyl ester but not 4-methylumbelliferyl acetate and fluorescein diacetate. The enzyme is inhibited by bis-p-nitrophenyl phosphate and eserine but not byp-chloromercuribenzoate. It was classified as a carboxylesterase (EC 3.1.1.1). Based on chromosomal localization, tissue distribution, substrate specificity, inhibitor sensitivity, and range ofpI's, rabbitEst-6 is assumed to be homologous with mouseEs-7.The contribution of Dr. O. von Deimling (No. 59) was supported by the Deutsche Forschungsgemeinschaft (De 315/2-2).     

Modification of Male Courtship Motivation by Olfactory Habituation via the GABAA Receptor in Drosophila melanogaster

A male-specific component, 11-cis-vaccenyl acetate (cVA) works as an anti-aphrodisiac pheromone in Drosophila melanogaster. The presence of cVA on a male suppresses the courtship motivation of other males and contributes to suppression of male-male homosexual courtship, while the absence of cVA on a female stimulates the sexual motivation of nearby males and enhances the male-female interaction. However, little is known how a male distinguishes the presence or absence of cVA on a target fly from either self-produced cVA or secondhand cVA from other males in the vicinity. In this study, we demonstrate that male flies have keen sensitivity to cVA; therefore, the presence of another male in the area reduces courtship toward a female. This reduced level of sexual motivation, however, could be overcome by pretest odor exposure via olfactory habituation to cVA. Real-time imaging of cVA-responsive sensory neurons using the neural activity sensor revealed that prolonged exposure to cVA decreased the levels of cVA responses in the primary olfactory center. Pharmacological and genetic screening revealed that signal transduction via GABA_A receptors contributed to this olfactory habituation. We also found that the habituation experience increased the copulation success of wild-type males in a group. In contrast, transgenic males, in which GABA input in a small subset of local neurons was blocked by RNAi, failed to acquire the sexual advantage conferred by habituation. Thus, we illustrate a novel phenomenon in which olfactory habituation positively affects sexual capability in a competitive environment.     

INHIBITION OF FEMALE DROSOPHILA MELANOGASTER REMATING BY A SEMINAL FLUID PROTEIN (ESTERASE 6)

Recent experiments have indicated that EST 6 transferred to D. melanogaster females during copulation mediates a large decrease in their attractiveness within the first six hours after mating. This decrease in attractiveness could increase the male's fitness if it were associated with less frequent remating by their mates, because a large proportion of the first male's sperm is lost if a female remates within one day. Here, I confirm a strong EST 6 influence on the receptivity of mated females; females with EST 6⁺ first mates remate significantly less frequently between 6 and 18 hours after mating than females that have not received EST 6 in the ejaculate. However, the previously observed effect of EST 6 on the attractiveness of mated females could not be substantiated. Transfer of EST 6 did not lead to a significant difference in attractiveness between EST 6°-and EST 6⁺-mated females at any time within the first 10 hours after mating for females from either of two strains.     

Females Mated without Sperm Transfer Maintain High Sexual Receptivity in the Wolf Spider Schizocosa malitiosa

The mating of the Neotropical lycosid Schizocosa malitiosa is long and complex, involving intense genital stimulation and copulatory courtship. This suggests functions other than just insemination. Previous data indicated that mated females of this species are less sexually receptive than virgins. We hypothesise that copulatory characteristics presented by males during prolonged copulations could be responsible for subsequent female sexual reluctance, and may be selected by cryptic female choice. Our objective was to examine the influence of copulatory behaviour on subsequent female sexual receptivity in S. malitiosa, isolating it from the effects of sperm transfer per se. For this purpose, we obtained males without sperm in their copulatory organs (palpal bulbs), and prevented them from charging their palps by sealing their genital pores immediately after their last moult (treated males). Virgin females were separated into three groups: (i) females exposed once to normal males, (ii) females exposed twice to normal males, and (iii) females exposed first to treated and second to normal males. The results showed that, 3 d after their first mating, females first mated with untreated males were frequently refractory to remating, whereas all those first mated with treated males were receptive. Copulations performed by treated males showed some differences from those performed by normal males, but maintained the basic behavioural pattern with abundant sexual stimulation. The presence of sperm fluids in the female receptacles appears to be the most likely factor generating female remating reluctance. Males may manipulate female responses using receptivity inhibiting substances in their sperm, like those described for insects. Females would first ensure sperm supply, becoming more choosy afterwards. All females, whether mated once or twice, generated similar numbers of progeny, indicating no relationship between number of matings and number of spiderlings.     

Mating with sperm-depleted males does not increase female mating frequency in the parasitoid Lariophagus distinguendus

Sexual conflicts due to divergent male and female interests in reproduction are common in parasitic Hymenoptera. The majority of parasitoid females are monandrous, whereas males are able to mate repeatedly. Thus, accepting only a single mate might be costly when females mate with a sperm‐depleted male, which may not transfer a sufficient amount of sperm. In the present study, we investigated the reproductive performance in the parasitoid Lariophagus distinguendus Först. (Hymenoptera: Pteromalidae) and studied whether mating with experimentally sperm‐depleted males increases the tendency of females to remate. Males were able to mate with up to 17 females offered in rapid succession within a 10‐h test period. The resulting female offspring, as an indirect measure of sperm transfer, remained constant during the first six matings and then decreased successively with increasing number of copulations by the males. Experimentally sperm‐depleted males continued to mate even if they transferred only small amounts or no sperm at all. Unlike males, the majority of females mated only once during a 192‐h test period. A second copulation was observed only in a few cases (maximum 16%). The frequency of remating was not influenced by the mating status of the first male the females had copulated with, suggesting that these events are not controlled by sperm deficiency of the females. Furthermore, we investigated male courtship behaviour towards mated females. Male courtship intensity towards mated females decreased with increasing time. However, females that had mated with an experimentally sperm‐depleted male did not elicit stronger or longer‐lasting behavioural responses in courting males than those that had mated with a virgin male. As the observed behaviours in L. distinguendus are known to be elicited by a courtship pheromone, these results suggest that females no longer invest in pheromone biosynthesis after mating (as indicated by ceasing behavioural responses of courting males), irrespective of whether they have received a sufficient amount of sperm or not. We discuss the results with respect to a possible mating strategy of sperm‐depleted males.     

Female remating in butterflies: interaction between female genotype and nonfertile sperm

Female mating rate is fundamental to evolutionary biology as it determines the pattern of sexual selection and sexual conflict. Despite its importance, the genetic basis for female remating rate is largely unknown and has only been demonstrated in one species. In paternally investing species there is often a conflict between the sexes over female mating rate, as females remate to obtain male nutrient donations and males try to prevent female remating to ensure high fertilization success. Butterflies produce two types of sperm: fertilizing, eupyrene sperm, and large numbers of nonfertile, apyrene sperm. The function of apyrene sperm in the polyandrous, paternally investing green‐veined white butterfly, Pieris napi, is to fill the female’s sperm storage organ thereby reducing her receptivity. However, there is large variation in number of apyrene sperm stored. Here, I examine the genetic basis to this variation, and if variation in number of apyrene sperm stored is related to females’ remating rate. The number of apyrene sperm stored at the time of remating has a genetic component and is correlated with female remating tendency, whereas no such relationship is found for fertilizing sperm. The duration of the nonreceptivity period in P. napi also has a genetic component and is inversely related to the degree of polyandry. Sexual conflict over female remating rate appears to be present in this species, with males using their apyrene sperm to exploit a female system designed to monitor sperm in storage. Ejaculates with a high proportion of nonfertile sperm may have evolved to induce females to store more of these sperm, thereby reducing remating. As a counter‐adaptation, females have evolved a better detection system to regain control over their remating rate. Sexually antagonistic co‐evolution of apyrene sperm number and female sperm storage may be responsible for ejaculates with predominantly nonfertile sperm in this butterfly.     

ENVIRONMENTAL EFFECTS ON REMATING IN DROSOPHILA MELANOGASTER

The effects of density and food on remating were investigated using Drosophila melanogaster. The frequency of remating was unaffected by density for some combinations of fly strains but was reduced at low relative densities for other combinations. Until females had used most of their stored sperm, remating was less likely when food was absent or contact with food was prevented. Food availability had little effect on the incidence of remating once stored sperm were depleted and had no effect on initial virgin mating frequency. This study indicates that environmental factors can have a substantial direct influence on the frequency of remating in populations of Drosophila melanogaster.     

Esterase-6 allozymes: Biochemical studies of two common and one rare variant inDrosophila melanogaster

The biochemical properties of three allozymes coded by theEst-6 locus, two common forms (EST-6^S and EST-6^F) and one rare form (EST-6^VF), were studied. The results show the existence of differences in isoelectric point, activity, activation energy, K_m, and temperature coefficient among the three variants, especially between the two common forms and the one rare form. The specific activity of the rare enzymatic variant seems to be less affected by temperature variation. The possible significance of these findings in relation to the mechanism of reproduction is briefly discussed.     

Characterization of Esterases in a Brazilian Population of Zaprionus Indianus (Diptera: Drosophilidae)

The aim of this study was to characterize esterases in Zaprionus indianus, a drosophilid recently introduced into Brazil. A further aim was study the variation of activity of esterases in the presence of inhibitors and their expression according to sex, sexual activity and age of individual flies. Polymorphisms were detected in two esterase loci (Est-2 and Est-3) and monomorphisms in four others (Est-1, Est-4, Est-5 and Est-6). Biochemical tests using α- and β-naphthyl acetate and the inhibitors malathion, eserine sulphate and PMSF allowed us to classify EST-2 and EST-5 as β-esterases, both carboxyl-esterases, and EST-1, EST-3, EST-4 and EST-6 as α-esterases. EST-1 and EST-3 were classified as carboxyl-esterases and EST-4 and EST-6 as cholinesterases. EST-5 activity was more pronounced in males and EST-2 was restricted to them or to recently copulated females. EST-4, rarely detected, was not characterized. Based on their biochemical characteristics possible roles for these enzymes are suggested.     

Genetic mapping,distribution, and properties of an aconitase isozyme in Anopheles albimanus (Diptera:Culicidae)

Electrophoretic analysis of the developmental stages and tissues of Anopheles albimanus showed that qualitatively similar allozymes of aconitase (Acon-2) occur at all stages, and the enzyme is widespread in every larval and adult tissues. Relative heat stabilities of the allozymes were investigated by electrophoresis of heated aqueous extracts and by heating the enzyme in situ in acrylamide gels after electrophoretic separation in Tris-citrate and Tris-maleate buffer systems. The pupal aconitase in the crude extract is more stable to heat than the larval and adult enzyme. The presence of citrate ions in the gel increased the stability of aconitase to heat. Studies of substrate specificities indicated that cis-aconitic acid is the best substrate but citric acid can also serve as a substrate. Zymograms developed with isocitric acid as a substrate showed no aconitase electromorphs and produced only isocitrate dehydrogenase bands. Aconitase has a pH optimum of 8.0 and this enzyme is completely inhibited if treated in situ with ethylenediaminetetra-acetic acid (EDTA), p-chloromercuribenzoate (PCMB), and urea at concentrations higher than 5mm, 5×10^–5 m, and 2 m, respectively. Acon-2¹⁰⁰ and Acon-2¹⁰⁵ do not respond differently to the above treatments. Genetic crosses involving a holandric translocation, pericentric inversions, visible mutants, and allozyme markers were analyzed to map the aconitase (Acon-2) locus on the left arm of chromosome 3. The gene sequence (and map distances) on 3L is centromere—esterase-8 (Est-8)—2—esterase-4 (Est-4)—25—esterase-2 (Est-2)—9—Acon-2—5—phosphoglucomutase (Pgm)—7—esterase-6 (Est-6).     

Female remating and sperm competition patterns in a terrestrial crustacean

In most species, both sexes may mate with more than one partner during their life. In terrestrial isopods (woodlice) female remating can occur within a reproductive season (immediate remating) or after a period of sexual rest and sperm storage, that is in a subsequent reproductive season (delayed remating). The pattern of sperm precedence is unknown in both cases. These two female remating patterns may shape male-male competition in different ways. To elucidate both patterns of female remating and sperm precedence, we used an albinism mutation in Armadillidium vulgare to track paternity in laboratory experiments. Males had low remating success after immediate remating attempts, mainly because of the female's refractory behaviour. However, refractory behaviour seemed to be lost after female sexual rest: delayed remating attempts were as successful as first mating attempts with virgin females. In both immediate and delayed remating, competing males had similar fertilization success, but varied in sperm precedence. We hypothesize that males might induce the refractory mating behaviour in females to ensure their paternity. This could be a strategy that evolved in woodlice after the loss of precopulatory mate guarding during adaptation to the terrestrial environment. We discuss the consequences of these findings for woodlice optimal mating strategies. Copyright 2002 The Association for the Study of Animal Behaviour. Published by Elsevier Science Ltd. All rights reserved.     

Oviposition and refractory periods of the small water strider Microvelia horvathi (Heteroptera: Veliidae) after a single mating event

Multiple mating can be costly, but also beneficial for females. When the costs outweigh the benefits, mated females can be reluctant to remate for some time. Conversely, females will be likely to remate when they need to replenish sperm and/or receive nutrients contained in the accessory ejaculate substances. Water striders have been commonly used in mating studies as many show a vigorous struggle between the sexes. Generally, water strider females frequently mate with many males and lay many eggs during mating period. However, little is known about their oviposition and refractory periods after an initial mating event. In this study, we revealed that single-mated females of the small water strider, Microvelia horvathi, could lay eggs for 40 days, reflecting approximately half of their potential oviposition periods. It suggested that M. horvathi females do not need to mate to replenish sperm for some time once they mate. Our results also showed that no female remated after 1 or 3 days of an initial mating event. The proportion of female remating increased with time after the initial mating, however, remating levels reached 26.7% even after 14 days. In this sense, any significant cost of mating outweighs the benefit, which might be one of the factors lowering their remating levels.     

SEX RATIO DRIVE PROMOTES SEXUAL CONFLICT AND SEXUAL COEVOLUTION IN THE FLY DROSOPHILA PSEUDOOBSCURA

Selfish genetic elements occur in all living organisms and often cause reduced fertility and sperm competitive ability in males. In the fruit fly Drosophila pseudoobscura, the presence of a sex‐ratio distorting X‐chromosome meiotic driver Sex Ratio (SR) has been shown to promote the evolution of increased female remating rates in laboratory populations. This is favored because it promotes sperm competition, which decreases the risk to females of producing highly female‐biased broods and to their offspring of inheriting the selfish gene. Here, we show that non‐SR males in these SR populations evolved an increased ability to suppress female remating in response to the higher female remating rates, indicating male–female coevolution. This occurred even though SR was rare in the populations. This was further supported by a correlation between females’ remating propensity and males’ ability to suppress female remating across populations. Thus SR can generate sexual conflict over female remating rate between females and the noncarrier males that make up the majority of the males, promoting evolution of increased ability of males to suppress female remating.     

Courtship rate signals fertility in an externally fertilizing fish

Sperm limitation is widespread across many animal species. Several mechanisms of sperm allocation have been proposed, including optimal allocation according to clutch size and equal allocation across females. However, considerably less effort has been directed at investigating the behavioural signals associated with sperm limitation in males, which may include mating rate and the intensity of courtship. We investigated whether multiple successive spawnings affect individual male fertilization success, mating rates and courtship rates in Japanese medaka (Oryzias latipes). Across an average of 17 spawning events per male, fertilization success decreased from 83.7 per cent for the first spawning to 40 per cent for the last spawning while courtship rate decreased from 3.4 to 1.5 min⁻¹. Females appeared to respond to male sperm depletion by reducing clutch size. Our results suggest that male Japanese medaka are sperm-limited, and that courtship rate may be an honest indication of fertilization ability.     

Receptivity of female remating and sperm number in the sperm storage organ in the bean bug,Riptortus clavatus (Heteroptera: Alydidae)

This study examines the relationship between the number of sperm in the seminal receptacle (spermatheca) and the receptivity of female remating in the bean bugRiptortus clavatus Thunberg. On the 21 st day after the first mating when receptivity to remating was > 70%, females receptive to remating had significantly fewer sperm ( < 40 on average) in the spermathecae than females reluctant to do (about 150 on average). However, averages of the number of eggs laid by receptive and reluctant females within 21 days were almost same. The proportion of fertilized eggs for receptive females at 15–21 days after copulation was significantly lower than that for reluctant females. Spermatozoa transferred from a male to a female’s spermatheca were detected 5 min after copulation and then increased continuously to about 500 with the first hour. When copulation durations were manipulated artificially, the shorter the copulation period (=females had less sperm in their spermathecae), the higher the remating rate became. Females may perceive the number of sperm in their seminal receptacles and then determine whether they copulate or not. These results support the hypothesis that females mate multiply in order to replenish inadequate sperm supplies to fertilize all eggs produced.