F prostaglandins function as potent olfactory stimulants that comprise the postovulatory female sex pheromone in goldfish

This study establishes that ovulated female goldfish release F type prostaglandins (PGFs) to the water where they stimulate male spawning behavior and comprise the goldfish postovulatory pheromone. We first demonstrated that ovulated and prostaglandin-injected female goldfish release immunoreactive PGFs to the water. Next, using electro-olfactogram recording (EOG), we determined that waterborne prostaglandins function as potent olfactory stimulants for mature male goldfish. Prostaglandin F2 alpha (PGF2 alpha) and its metabolite 15-keto-prostaglandin F2 alpha (15K-PGF2 alpha) were the most potent prostaglandins; the former had a detection threshold of 10(-10) M and the latter a detection threshold of 10(-12) M. Studies of prostaglandin-injected fish indicated that PGF metabolites are an important component of the pheromone. Cross-adaptation experiments using the EOG demonstrated that goldfish have separate olfactory receptor sites for PGF2 alpha and 15K-PGF2 alpha that are independent from those that detect other olfactory stimulants. Finally, we established that male goldfish exposed to low concentrations of waterborne PGFs exhibit reproductive behaviors similar to those elicited by exposure to the odor of ovulated fish. Together with our recent discovery that a steroidal maturational hormone functions as a preovulatory "priming" pheromone for goldfish, these findings suggest that hormones and their metabolites may commonly serve as reproductive pheromones in fish.     

Hormonal Regulation of Female Reproductive Behavior in Fish

SYNOPSIS. Fundamentally different mechanisms regulate femalesexual behavior in the ovoviviparous guppy and the oviparousgoldfish. In the female guppy, ovarian estrogen evidently synchronizescycles of sexual receptivity with endogenous cycles of ovarianmaturation and also increases female attractivity at the timeof maximum receptivity by stimulating the release of a sexualpheromone. In the goldfish, it appears that prostaglandin, releasedfrom the ovary or oviduct in conjunction with ovulation andthe presence of ovulated eggs, acts on the brain to stimulatespawning behavior. In contrast to the situation in the guppy,steroid treatments alone (in the absence of ovulated eggs) failto stimulate spawning behavior in the goldfish. It isproposedthat endocrine mechanisms regulating female sexual behaviorin the teleosts and in other vertebrates are less related tophylogeny than to the mode of reproduction employed. In thegoldfish and several other externally fertilizing teleosts,where sexual behavior involves oviposition, female sexual behaviorapparently is synchronized with ovulation by mechanisms whichrespond to elevated plasma prostaglandin as an indicator ofthe presence of ovulated eggs. In internally fertilizing species(guppy, reptiles, birds, mammals), where sexual behavior andfertilizationare temporally dissociated, female sexual behavior is synchronizedwith ovulation by mechanisms which anticipate either an imminentspontaneous ovulation, or the potential for reflex ovulation,by responding to increases in plasma estrogen associated withfolliculardevelopment.     

The three steroidal components of the goldfish preovulatory pheromone signal evoke different behaviors in males

The goldfish sex pheromone system is the best understood among the teleost fishes. Pheromones in this species are unspecialized hormonal products, which are released in ratios that vary with reproductive status. This study examined behavioral responses of male goldfish to three steroidal components of the female preovulatory pheromone: 17,20beta-dihydroxy-4-pregnen-3-one (1720betaP); 17,20beta-dihydroxy-4-pregnen-3-one-20-sulfate (1720betaP-S); and androstenedione (AD). Males were observed during exposure to nanomolar concentrations of each steroid over a 2-h period. We observed chasing, nudging (courtship behaviors) and pushing (an aggressive behavior). Each steroid elicited a different set of behaviors. 1720betaP, which is released by ovulatory females, elicited a low level of chasing and nudging that persisted throughout the experiment. Exposure to 1720betaP-S, which is released primarily by ovulatory females, triggered a large increase in nudging and chasing that lasted for only 5 min. In contrast, AD, which is released by females early in the ovulatory cycle and by mature males, elicited increases in aggressive behavior. 1720betaP and 1720betaP-S both caused increases in GtH-II release while AD did not. These results demonstrate that goldfish can discriminate components found in the female pheromone blend, suggesting that goldfish, and likely other fish species, may employ blends of hormonal products as pheromones.     

A releaser pheromone that attracts males in the urine of mature female masu salmon

A releaser pheromone of masu salmon Oncorhynchus masou was investigated using Y-maze behavioural experiments. During the reproductive season, urine of mature females contains a releaser pheromone which acts as a sex attractant for spermiating mature male parr. The releaser pheromone in mature female urine is one or more low molecular weight substances (less than 10 000) soluble in ether under basic conditions. The attractant was not present in either the coelomic fluid of ovulated females nor in neutral or acidic extracts of female urine which contain free steroids and F-type prostaglandins, respectively.     

Effects of indomethacin and prostaglandins on the spawning behaviour of female goldfish

In goldfish, injection of ovulated eggs (from donor females) through the ovipore and into the ovarian lumen of females with vitellogenic oocytes induces spawning behaviour within several hours. Intraperitoneal (i.p.) injection of indomethacin (IM), 10 μg/g, either 10 h prior to, or coincident with, injection of ovulated eggs, completely inhibits the onset of spawning behaviour. IM injection similarly terminates ongoing spawning behaviour induced by egg injection. PGF_2α (5 μg/g; i.p. injection) restores spawning behaviour of egg-injected, IM-blocked fish; at the same dosage, PGE₂ is marginally effective and PGE₁ is without effect. As PGF_2α and PGE₂ also induce spawning behaviour in females which have not been injected with ovulated eggs, it is suggested that distension of the oviduct following ovulation or egg injection results in the release of PG which then acts in some way to induce spawning behaviour. The ability of PG to induce spawning behaviour is eliminated by hypophysectomy and restored by treatment with salmon gonadotropin: no steroid treatment was effective in restoring PG-induced spawning in fish which had been hypophysectomized for 3–4 months. The possible mode of action of PG in inducing spawning behaviour in female goldfish is discussed.     

Pheromone production and mating behaviour by allatectomized males of the cockroach, Nauphoeta cinerea

Males of Nauphoeta cinerea produce a volatile pheromone which attracts the female for mating. Allatectomy of males either 5 days prior to or within 12 hr following the imaginal ecdysis does not impair pheromone production, pheromone release, nor any observable aspect of mating behaviour. It is proposed that in N. cinerea, and in other cockroach species where the male releases a volatile pheromone to attract the female, pheromone production is not controlled by the corpora allata, and pheromone release is under direct motor control.     

Production and perception of pheromones by the beetle Tribolium confusum

Adults of Tribolium confusum secrete two pheromones. The first, produced by the male, is attractive to both sexes and the second, produced by the female, is attractive to the male only. Pheromone production and perception was studied in relation to habituation, beetle age, time of day and previous mating. A living source of each pheromone habituates the responding beetles, the male pheromone habituating more strongly; female pheromone habituates only in the absence of the male pheromone. Habituation to one pheromone was always accompanied by an enhanced response to the other.Five days after emergence, production of male pheromone reaches a peak that is maintained. Production of female pheromone peaks after 3 days. Both sexes are responsive to male pheromone immediately upon eclosion, males reaching maximum response at 14 days, females at 8 days. Males are also responsive to female pheromone upon eclosion reaching maximum response at 8 days; female response to female pheromone is imperceptible. Males but not females display a 24 hr rhythm in pheromone production. Mated beetles did not differ significantly from unmated beetles in their ability to perceive pheromones. Alteration in male pheromone production after mating was detected by females but not males; this pheromone may, therefore, act as both a sex and aggregation pheromone.     

Sound production in Scolytidae: Female sonic stimulus of male pheromone release in two Dendroctonus beetles

The hypothesis that female sonic stimulus may evoke male pheromone release in a behavioural interaction analogous to the known male sonic stimulus of female pheromone release, was confirmed in Dendroctonus pseudotsugae, and also in D. brevicomis. In both species known male-produced substances collected from males stimulated by recorded female stridulation were identified by coupled gas chromatography/mass spectrometry. In a second test with D. pseudotsugae, male pheromone release during recorded female stridulation was evident in the change of male stridulation from the simple attractant chirp to the interrupted chirp, which is known to result from a medium concentration of 3,2-MCH. Also, the D. pseudotsugae male attractant chirp was synthesised with an electronic pulse generator and used to evoke pheromone release. It is concluded that the antiaggregative pheromone of this species is released by each sex at the sonic stimulus of the other sex.     

Urine of ovulated female masu salmon attracts immature male parr treated with methyltestosterone

Methyltestosterone-treated immature male masu salmon Oncorhynchus masou parr were attracted to both the urine of ovulated females and the ether soluble basic substances extracted from the urine, but not to immature female urine. It is suggested that the male response to the sex attractant (releaser pheromone) in the urine is under the control of androgens.     

Pheromone-Induced Spawning of Pacific Herring

A spawning pheromone in the milt (semen) and testes of the Pacific herring,Clupea harengus pallasi,is thought to facilitate school spawning of this species. We found that responsiveness to the spawning pheromone was variable among ripe fish (milt-producing or ovulated). Measurement of five principle reproductive steroids in the free form and five steroids in conjugated forms in the plasma of male fish early in the spawning season (newly ripe fish) showed that elevated plasma levels of 3α,17α-dihydroxy-5β-pregnan-20-one and 17α-hydroxyprogesterone coincided with responsiveness to the spawning pheromone in these fish; levels of other steroids did not differ. In contrast, responsiveness to the pheromone by female fish later in the spawning season (ripe-and-holding fish) coincided with lower levels of glucuronated 17α,20β-dihydroxyprogesterone and a lower gonadosomatic index. We suggest that these differences indicate a more advanced mature reproductive state in the responsive individuals among both the newly ripe male and the ripe-and-holding female fish. We found no differences in the level of cortisol in the blood of the herring that could be correlated with differences in pheromonal responsiveness. We conclude that differences in responsiveness to the spawning pheromone coincide to some extent with levels of reproductive maturation but probably not with recent stress.     

Nippostrongylus brasiliensis: factors influencing movement of males toward a female pheromone

Single, responding males of Nippostrongylus brasiliensis exhibited a dose-dependent movement toward a pheromone source that was derived from incubation or homogenization of female helminths in Tyrode's solution. Increases in female homogenate beyond an optimum concentration resulted in reduced male movement toward the pheromone source. Crowding of male N. brasiliensis prior to their exposure to female pheromone gradients diminished the males' response. However, crowding of females had no effect on response to the pheromone. The locomotory responses of males exposed to various mixed male and female pheromone sources reaffirmed earlier suggestions of a pheromone feedback system in which males influence the pheromone released from females.     

Large and persistent effect of a female steroid pheromone on ejaculate size in goldfish Carassius auratus

This study determined if ejaculate size in male goldfish Carassius auratus is increased by the female preovulatory steroid pheromone 4‐pregnen‐17,20β‐diol‐3‐one (17,20βP), which previously has been shown to affect male behaviour and to increase sperm motility and stripped sperm number, and also to increase paternity in competitive spawning and competitive in vitro fertilization. Experimental males were exposed overnight to 17,20βP whereas control males were not. The morning following exposure, each male was placed with a reproductively active female and, after one to 20 spawning acts, aquarium water was sampled to quantify released sperm. Although exposure to 17,20βP induced a five‐fold difference in the number of sperm that could be stripped, the median number of sperm in first ejaculates of pheromone‐exposed males was >60 sixty times that of control males, a pheromonal effect on ejaculate size that persisted for at least 20 spawning acts. The magnitude of the pheromone effect on ejaculate size indicates that it is a critical component of C. auratus sperm allocation, and that examining this effect in concert with other factors (e.g. presence of competitors, male and female size and frequency of spawning) will reveal the contribution of the preovulatory pheromone to male fitness in this promiscuous species.     

Male moth songs tempt females to accept mating: the role of acoustic and pheromonal communication in the reproductive behaviour of Aphomia sociella

Background

Members of the subfamily Galleriinae have adapted to different selective environmental pressures by devising a unique mating process. Galleriinae males initiate mating by attracting females with either chemical or acoustic signals (or a combination of both modalities). Six compounds considered candidates for the sex pheromone have recently been identified in the wing gland extracts of Aphomia sociella males. Prior to the present study, acoustic communication had not been investigated. Signals mediating female attraction were likewise unknown.

Methodology/Principal Findings

Observations of A. sociella mating behaviour and recordings of male acoustic signals confirmed that males initiate the mating process. During calling behaviour (stationary wing fanning and pheromone release), males disperse pheromone from their wing glands. When a female approaches, males cease calling and begin to produce ultrasonic songs as part of the courtship behaviour. Replaying of recorded courting songs to virgin females and a comparison of the mating efficiency of intact males with males lacking tegullae proved that male ultrasonic signals stimulate females to accept mating. Greenhouse experiments with isolated pheromone glands confirmed that the male sex pheromone mediates long-range female attraction.

Conclusion/Significance

Female attraction in A. sociella is chemically mediated, but ultrasonic communication is also employed during courtship. Male ultrasonic songs stimulate female sexual display and significantly affect mating efficiency. Considerable inter-individual differences in song structure exist. These could play a role in female mate selection provided that the female''s ear is able to discern them. The A. sociella mating strategy described above is unique within the subfamily Galleriinae.     

Mating Effect on Sex Pheromone Production of the Oriental tobacco budworm,Helicoverpa assulta

This study was undertaken to clarify the suppression phenomenon of sex pheromone production after mating and its relationship to the physiological mechanism in adult females of Helicoverpa assulta, and determine the mating factor from males causing depletion of sex pheromonc production. Sex pheromone production of H. assulta females was mostly terminated in 3 hours after mating. Mated females maintained with a low titer of sex pheromone until 3 days when it started to increase again, which showed a characteristic of species mating more than once. The mated female again produced pheromone upon injection of pheromone biosynthesis activating neuropeptide (PBAN) or extracts of brain-suboesophageal ganglion complexes (Br-Sg) of mated female, which were shown similar pheromonotropic activities as compared with virgin females. These results indicated that the mating did not inhibit the receptivity of pheromone gland itself and PBAN biosynthesis in suboesophageal ganglion of the mated females. And it seems to support that the depletion of sex pheromone production is responsible for blocking of PBAN release from head. To investigate the mating factor from adult males, when extracts of reproductive organs of male were injected into hemocoel of virgin females evoking depletion of sex pheromone production as shown in mated female. The results suggest that a chemical substance(s) from the male reproductive organs could be responsible for the loss of sex pheromone biosynthesis in H. assulta.     

Electrophysiological basis for the behavioural response of male and female Trichoplusia ni to synthetic female pheromone

Electroantennogram (EAG) studies demonstrated that antennae of both male and female Trichoplusia ni have: (1) receptor-neurones sensitive to female pheromone, (2) a low response threshold, (3) an identical mean-percentage EAG curve over a broad concentration range of pheromone, and (4) a similar absolute recovery interval from adaptation to pheromonal stimulation. These factors suggest that antennae of male and female T. ni have homologous and homogeneous acceptor sites for the female pheromone. Pheromonal stimulation of female antennae elicited EAGs with only 25% of the amplitude of those elicited in males.     

Responses of Second-Instar Male Nymphs of Four Mealybug Species (Hemiptera: Pseudococcidae) to Conspecific and Heterospecific Female Sex Pheromones

The response of the late second-instar male nymphs of the mealybug species (Hemiptera, Pseudococcidae), Planococcus citri (Risso), Planococcus ficus (Signoret), Pseudococcus cryptus Hempel Nipaecoccus viridis (Newstead), to their conspecific and heterospecific female pheromone was studied. Males that exhibited the typical appearance of late-second-instar nymphs were tested. The male behavior was monitored soon after their exposure to the tested female sex pheromone in glass Petri dish arenas. Male nymph behavior toward the pheromone source was characterized based on their aggregation on the disks in the arena. Males of all four tested mealybug species were attracted to their conspecific female pheromone. By contrast, almost no interceptions of male nymphs with disks impregnated with a heterospecific female pheromone were observed. The mode of attraction of each of male nymphs of P. ficus, among most of the tested individuals (>80%), to the conspecific female sex pheromone, (S)-lavandulyl senecioate and or (S)-lavandulyl isovalerate, was the same as the mode of attraction latter on as adult. We suggest that by being attracted to the conspecific pheromone these males may direct themselves to a suitable pupation site near conspecific non-sibling mature females, thus preventing inbreeding. The repellency of heterospecific sex pheromone to males that are looking for a pupation site suggests that the latter try to avoid close contact with heterospecific females.     

Nippostrongylus brasiliensis: Female incubation,release of pheromone,fractionation of incubates

The release of pheromone by female Nippostrongylus brasiliensis as a crude incubate was linear for the first 2 hr, but declined after this period. Pheromone release in solution increased with temperature elevations until a 37 C incubation temperature was reached. Higher temperatures of incubation apparently caused diminished pheromone release. Gel filtration of female pheromone that was prepared as a crude incubate revealed biologically active elutions at K_av, 0.64 and 1.0. The timed release of female pheromone activity at these two elution regions coincided additively with the production of activity as a crude incubate. Each individual Chromatographic fraction from females accounted for about 50% of the pheromone activity of the crude, nonfractionated incubate, based on the male's bioassay response. Recombination of the K_av 0.64 and 1.0 elutions enabled recovery of pheromone activity that was similar to crude incubate. The gel filtration elution of 260-nm absorbance from male- or female-produced incubate was qualitatively similar, but a range of quantitative differences were evident. The fractionation of incubate from several female densities revealed only quantitative differences.     

Induction of developmental abnormalities in larval goldfish, Carassius auratus L., under cool incubation conditions

Compared with incubation at a constant 22° C, exposure of goldfish embryos and larvae to 13° C, under a variety of thermal protocols, caused increased frequencies of abnormal development and, in some cases, reduced survival to hatching. The low-temperature incubation conditions were particularly deleterious when eggs were incubated at 13° C from the outset, regardless of the temperature at which the donor female ovulated and the eggs were fertilized. Significantly higher frequencies of developmental abnormalities were also noted when embryos were transferred from 22°C to 13°C at 6, 24, 128 and, in one case, 175 h after fertilization. In three of five experiments, subjecting embryos and larvae to diel fluctuations between 22 and 13° C, with a 5-h hold at the lower temperature, caused an increase in development abnormalities. These results demonstrate that the thermal requirements of goldfish embryos and larvae necessitate a delay in ovulation and spawning until water is sufficiently warm. Developmental abnormalities can be induced by exposure to cool (13° C) conditions, at least up to the time that swimbladder inflation occurs.     

Mechanisms of pheromone inactivation in the gut of Periplaneta americana

The sex attractant of the cockroach, Periplaneta americana, has been shown in earlier work to be largely inactivated by dissected midgut from males and from mated females. It is only slightly inactivated by the midgut from virgin females. In this paper, the sex pheromone inactivating system is further studied and shown to be active in late instar larvae of both sexes. In the male this pheromone inactivation is inhibited by piperonyl butoxide, a microsomal oxidase inhibitor. This compound appears to act on the midgut tissue directly. In the mated female, piperonyl butoxide has little effect. When the pheromone inactivating capacity is partitioned into soluble and tissue components, it appears that the soluble component is most active in the male, whereas the tissue component is most active in the female. Evidence from heat inactivation, trichloracetic acid precipitation, and the use of soy bean trypsin inhibitor, as well as the time course of the reaction, suggest that the factor or factors inactivating pheromone are proteins, probably enzymes. Evidence that at least part of the pheromone inactivating capacity is due to microsomal oxidases is considered. It is also observed that both pheromone and piperonyl butoxide absorb to membranes.     

Species specificity of methyl ketone profiles in the skin lipids of female garter snakes,genus Thamnophis

One of the relatively few vertebrate pheromones to be chemically identified, the female sex pheromone of the red-sided garter snake (Thamnophis sirtalis parietalis) is a series of saturated and monounsaturated methyl ketones contained within female skin lipids. During the breeding season, this pheromone is responsible for eliciting male courtship behaviors and males are able to utilize pheromonal variation to discriminate among females. While the pheromone system of the red-sided garter snake has been the subject of many studies, relatively little is known about the pheromone systems of other garter snakes. Through chemical analyses, we demonstrate that female skin lipids of the red-spotted garter snake (Thamnophis sirtalis concinnus), northwestern garter snake (Thamnophis ordinoides), and plains garter snake (Thamnophis radix) contain similar methyl ketones. The methyl ketone profiles of these snakes differ qualitatively from one another and from the methyl ketone profiles of red-sided garter snakes with differences particularly pronounced between sympatric species. Our results provide evidence that the use of methyl ketones in sexual signaling may be ubiquitous for Thamnophis species and suggest that these compounds could play a role in reproductive isolation between species in this genus.