The efficacy of two synthesized sea lamprey sex pheromone components as a trap lure when placed in direct competition with natural male odors

The female sea lamprey (Petromyzon marinus), a devastating invasive fish of the Laurentian Great Lakes, locates potential mates by tracking a sex pheromone emitted by nesting males. We tested whether combinations of two putative components of the sex pheromone, 3-keto-petromyzonol sulfate (3kPZS) and 3-keto-allocholic acid (3kACA), were sufficiently attractive to function as a trap-bait when placed in direct competition with male odors. Ovulating females successfully located point sources of 3kPZS both in the presence and absence of a competing odor emitted by mature males placed upstream. However, 3kPZS was not able to retain females in the vicinity of a trap longer than two minutes, and retention time was reduced by 57% when competing male odors were present. 3kACA failed to elicit a response on its own and did not improve attraction to, or retention near, a source of 3kPZS. Application of an incomplete pheromone in trapping-for-control scenarios will require devices configured to minimize the effort necessary to enter a trap, features to offset the probable decrease in trap retention, and deployment into favorable habitats where competition with spawning males is minimal.     

Evidence for a receiver bias underlying female preference for a male mating pheromone in sea lamprey

Receiver bias models suggest that a male sexual signal became exaggerated to match a pre-existing sensory, perceptual or cognitive disposition of the female. Accordingly, these models predict that females of related taxa possessing the ancestral state of signalling evolved preference for the male trait in a non-sexual context. We postulated that female preference for the male-released bile alcohol mating pheromone, 3 keto petromyzonol sulfate (3kPZS), of the sea lamprey (Petromyzon marinus) evolved as a result of a receiver bias. In particular, we propose that migratory silver lamprey (Ichthyomyzon unicuspis), a basal member of the Petromyzontidae, evolved a preference for 3kPZS released by stream-resident larvae as a means of identifying productive habitat for offspring. Larval silver lamprey released 3kPZS at rates sufficient to be detected by migratory lampreys. Females responded to 3kPZS by exhibiting upstream movement behaviours relevant in a migratory context, but did not exhibit proximate behaviours important to mate search and spawning. Male silver lamprey did not release 3kPZS at rates sufficient to be detected by females in natural high-volume stream environments. We infer that female silver lamprey cue onto 3kPZS excreted by stream-resident larvae as a mechanism to locate habitat conducive to offspring survival and that males do not signal with 3kPZS. We suggest that this female preference for a male signal in a non-sexual context represents a bias leading to the sexual signalling observed in sea lamprey.     

Pheromones of the male sea lamprey,Petromyzon marinus L.: structural studies on a new compound, 3-keto allocholic acid,and 3-keto petromyzonol sulfate

This study reports the results of chemical and chromatographic studies which establish the presence of 3-keto allocholic acid (3kACA) in water extracts from spermiating male sea lamprey, Petromyzon marinus. This is the second compound to be isolated and identified from these extracts. The first was 3-keto petromyzonol sulfate (3kPZS), which was shown to act as strong pheromonal attractant for ovulated females. Some new characterization data on 3kPZS (utilizing an only recently available synthetic preparation of the compound) is also included. The possibility that a mixture of 3kACA and 3kPZS might be a more potent pheromonal attractant than either compound alone is discussed.     

Mixtures of Two Bile Alcohol Sulfates Function as a Proximity Pheromone in Sea Lamprey

Unique mixtures of pheromone components are commonly identified in insects, and have been shown to increase attractiveness towards conspecifics when reconstructed at the natural ratio released by the signaler. In previous field studies of pheromones that attract female sea lamprey (Petromyzon marinus, L.), putative components of the male-released mating pheromone included the newly described bile alcohol 3,12-diketo-4,6-petromyzonene-24-sulfate (DkPES) and the well characterized 3-keto petromyzonol sulfate (3kPZS). Here, we show chemical evidence that unequivocally confirms the elucidated structure of DkPES, electrophysiological evidence that each component is independently detected by the olfactory epithelium, and behavioral evidence that mature female sea lamprey prefer artificial nests activated with a mixture that reconstructs the male-released component ratio of 30:1 (3kPZS:DkPES, molar:molar). In addition, we characterize search behavior (sinuosity of swim paths) of females approaching ratio treatment sources. These results suggest unique pheromone ratios may underlie reproductive isolating mechanisms in vertebrates, as well as provide utility in pheromone-integrated control of invasive sea lamprey in the Great Lakes.     

Isolation and identification of petromyzestrosterol, a polyhydroxysteroid from sexually mature male sea lamprey (Petromyzon marinus L.)

Petromyzestrosterol (1), a novel polyhydroxylated steroid, was identified from water conditioned with sexually mature male sea lamprey (Petromyzon marinus), a jawless vertebrate animal. Along with this novel steroid, two known steroids, 7α,12α,24-trihydroxy-5α-cholan-3-one-24-sulfate (3k PZS) and 7α,12α,24-trihydroxy-5α-cholan-24-sulfate (PZS), were isolated. Structures of these compounds were unequivocally established by spectroscopic analyses and by comparison with spectra of known compounds. Electro-olfactogram recordings (EOG) showed that 1 at nanomolar concentrations was stimulatory for the olfactory epithelium of adult females. 3k PZS, known to function as a male sex pheromone, was more stimulatory than 1 for the female olfactory epithelia. The concentration-response curve of 3k PZS was exponential in shape with steep slopes between 10(-10) and 10(-6) mol L(-1). The concentration-response curve for 1 was shallower than that for 3k PZS.     

Phylogenetic distribution of a male pheromone that may exploit a nonsexual preference in lampreys

Pheromones are among the most important sexual signals used by organisms throughout the animal kingdom. However, few are identified in vertebrates, leaving the evolutionary mechanisms underlying vertebrate pheromones poorly understood. Pre‐existing biases in receivers' perceptual systems shape visual and auditory signalling systems, but studies on how receiver biases influence the evolution of pheromone communication remain sparse. The lamprey Petromyzon marinus uses a relatively well‐understood suite of pheromones and offers a unique opportunity to study the evolution of vertebrate pheromone communication. Previous studies indicate that male signalling with the mating pheromone 3‐keto petromyzonol sulphate (3kPZS) may exploit a nonsexual attraction to juvenile‐released 3kPZS that guides migration into productive rearing habitat. Here, we infer the distribution of male signalling with 3kPZS using a phylogenetic comparison comprising six of 10 genera and two of three families. Our results indicate that only P. marinus and Ichthyomyzon castaneus release 3kPZS at high rates. Olfactory and behavioural assays with P. marinus, I. castaneus and a subset of three other species that do not use 3kPZS as a sexual signal indicate that male signalling might have driven the evolution of female adaptations to detect 3kPZS with specific olfactory mechanisms and respond to 3kPZS with targeted attraction relevant during mate search. We postulate that 3kPZS communication evolved independently in I. castaneus and P. marinus, but cannot eliminate the alternative that other species lost 3kPZS communication. Regardless, our results represent a rare macroevolutionary investigation of a vertebrate pheromone and provide insight into the evolutionary mechanisms underlying pheromone communication.     

Electrophysiological evidence for detection and discrimination of pheromonal bile acids by the olfactory epithelium of female sea lampreys (<Emphasis Type="Italic">Petromyzon marinus</Emphasis>)

Electro-olfactograms were used to determine sensitivity and specificity of olfactory organs of female sea lampreys (Petromyzon marinus) to four bile acids: 3-keto petromyzonol sulfate and 3-keto allocholic acid from spermiating males and petromyzonol sulfate and allocholic acid from larvae. Spermiating male bile acids are thought to function as a mating pheromone and larval bile acids as a migratory pheromone. The response threshold was 10^–12 mol l^–1 for 3-keto petromyzonol sulfate and 10^–10 mol l^–1 for the other bile acids. At concentrations above 10^–9 mol l^–1, the sulfated bile acids showed almost identical potency, as did the non-sulfated bile acids. The two sulfated bile acids were more potent than the two non-sulfated ones. In addition, 3-keto petromyzonol sulfate and water conditioned with spermiating males induced similar concentration-response curves and response thresholds. Cross-adaptation experiments demonstrated that the sulfated and non-sulfated bile acids represent different odors to the olfactory epithelium of females. Further exploration revealed that 3-keto petromyzonol sulfate represents a different odor than petromyzonol sulfate, while 3-keto allocholic acid and allocholic acid represent the same odor. Results indicate that male-specific bile acids are potent and specific stimulants to the female olfactory organ, supporting the previous hypothesis that these bile acids function as a pheromone.Abbreviations 3kACA 3-keto allocholic acid - 3kPZS 3-keto petromyzonol sulfate - ACA allocholic acid - ANOVA analysis of variance - ELISA enzyme-linked immunosorbent assay - EOG electro-olfactogram - PIR percent initial response - PZS petromyzonol sulfate - SMW spermiating male washings     

Rhythmicity of sex pheromone content in female Heliothis virescens: impact of mating

Abstract Pheromone production in virgin females of Heliothis virescens (F.) (Lepidoptera: Noctuidae) peaked between the fourth and seventh hours of scotophase on the second, third, fourth and fifth days following eclosion. The highest peak (186 ng) occurred on day 3 after eclosion. Z-11-Hexadecenal comprised the highest proportion of seven components in the pheromone glands. Disproportionately higher amounts of hexadecanal and Z-11-hexadecenol occurred during photo-phase and other periods when low quantities of total pheromone were recorded.
Mating suppressed pheromone production which remained low until 48 h after mating. Coupling females with males mated three times previously or with 6-day-old males was less effective in causing a drop in pheromone content which peaked again 24 h after mating. This suggests the transfer of a male factor, a pheromonostatic factor, that suppresses pheromone production in mated females and that the factor from older and previously mated males is less effective.     

Male sea lampreys,Petromyzon marinus L., excrete a sex pheromone from gill epithelia

During the period when they are producing sperm, male sea lampreys (Petromyzon marinus L.) release a sex pheromone 7alpha, 12alpha, 24-trihydroxy-5alpha-cholan-3-one-24-sulfate (3 keto-petromyzonol sulfate, 3ketoPZS) that induces search and preference behaviors in ovulating females. In this study, we conducted a series of experiments to demonstrate that release of this pheromone into water takes place exclusively through the gills. In a behavioral maze, water conditioned with the anterior region of spermiating males induced an increase of search and preference behaviors in ovulating females. Similar behavior was not elicited by water conditioned by the posterior region. The anterior region washings and whole-body washings from spermiating males also elicited large and virtually identical electro-olfactogram responses from female sea lampreys, while the posterior washings produced negligible responses. Further, mass spectrometry and immunoassay confirmed that virtually all the 3ketoPZS released into water was through the gills. Immunocytochemistry revealed some gill epithelial cells and hepatocytes from spermiating males contained dense immunoreactive 3ketoPZS, but not those from prespermiating males. These results demonstrate that 3ketoPZS is released through the gill epithelia and suggest that this pheromone or its precursor may be produced in the liver.     

Male housefly (Musca domestica L.) genital system as a source of mating pheromone

Male volatile mating pheromone is released by male houseflies placed in close proximity to females or in contact with female hydrocarbon extract. The pheromone induces the aggregation of virgin females in the vicinity of the male. Preparations of macerated testicular and ejaculatory duct tissue exert a similar effect, and these organs are, apparently, the source of the pheromone. Such preparations are not attractive to males or mated females. The pheromone also appears to induce female receptivity, since females that are exposed to emanations from male system preparations show a quicker rate of pairing with males. The pheromone effect results from the testes and ejaculatory ducts acting together, as preparations of each of these alone are inactive. Pheromone activity is age dependent and the greatest female reaction is observed when tissues of 6–10-day old males are used.     

Characterization of a Novel Bile Alcohol Sulfate Released by Sexually Mature Male Sea Lamprey (Petromyzon marinus)

A sulphate-conjugated bile alcohol, 3,12-diketo-4,6-petromyzonene-24-sulfate (DKPES), was identified using bioassay-guided fractionation from water conditioned with sexually mature male sea lamprey (Petromyzon marinus). The structure and relative stereochemistry of DKPES was established using spectroscopic data. The electro-olfactogram (EOG) response threshold of DKPES was 10⁻⁷ Molar (M) and that of 3-keto petromyzonol sulfate (3 KPZS; a known component of the male sea lamprey sex pheromone) was 10⁻¹⁰ M. Behavioural studies indicated that DKPES can be detected at low concentrations by attracting sexually mature females to nests when combined with 3 KPZS. Nests baited with a mixture of DKPES and 3 KPZS (ratio 1∶29.8) attracted equal numbers of sexually mature females compared to an adjacent nest baited with 3 KPZS alone. When DKPES and 3 KPZS mixtures were applied at ratios of 2∶29.8 and 10∶29.8, the proportion of sexually mature females that entered baited nests increased to 73% and 70%, respectively. None of the sexually mature females released were attracted to nests baited with DKPES alone. These results indicated that DKPES is a component of the sex pheromone released by sexually mature male sea lamprey, and is the second biologically active compound identified from this pheromone. DKPES represents the first example that a minor component of a vertebrate pheromone can be combined with a major component to elicit critical sexual behaviors. DKPES holds considerable promise for increasing the effectiveness of pheromone-baited trapping as a means of sea lamprey control in the Laurentian Great Lakes.     

Control of attractivity and receptivity in female red-sided garter snakes

Female red-sided garter snakes emerge from their hibernacula in the spring attractive and receptive to males. Attractivity is communicated by a pheromone released through the female's skin and is a consequence of ovarian recrudescence the previous summer. Receptivity, on the other hand, is stimulated by ovarian estrogen secretion during emergence itself. Mating renders females both unattractive and unreceptive. Another "mating" pheromone of male origin is important in making females unattractive after mating. To investigate the role of cloacal stimulation in the loss of attractivity and receptivity we injected a local anesthetic (lidocaine or tetracaine) in the cloacal region of females before mating. This does not prevent mating, although it blocks neural transmission of copulatory sensory stimuli. The time course of transition from attractive and receptive states was then observed. Females treated with local anesthetic as well as control females were unattractive within 15 min of mating. However, when retested 2-3 and 24 h after mating, a significantly higher proportion of treated females regained their attractivity, while mated control females remained unattractive. This restorative effect was transient, though, as treated females retested 48 h after mating were as unattractive as the controls. Both anesthetized and control females were unreceptive when tested following mating and did not regain receptivity with time. Last, the mating-induced surge in circulating concentrations of prostaglandin was diminished in females that received a local anesthetic prior to mating. Taken together these results indicate that the loss of attractivity and receptivity following mating in the red-sided garter snake is due to combined effects of a mating pheromone and a physiological, neurally mediated response to the sensation of stimuli associated with the act of mating.     

Ovarian steroid sulphate functions as priming pheromone in maleBarilius bendelisis (Ham.)

The study reveals that pre-ovulatory females of the fishBarilius bendelisis (Ham.) release sex steroids and their conjugates into the water and that a steroid sulphate of these compounds functions as a potent sex pheromone which stimulates milt production in conspecific males prior to spawning. Since males exposed to the purified sub-fraction III of the steroid sulphate fraction have increased milt volume and more spermatozoa with greater motility, the function of this priming pheromone appears to be to enhance male spawning success. High turbulence and faster water currents render the hillstream ecosystem extremely challenging for chemical communication. Therefore, ovulatory female fish secrete highly water soluble steroid sulphates for rapid pheromonal action in males. Inhibited milt volume in olfactory tract lesioned (OTL) males exposed to the steroid sulphate fraction and 17α,20β-dihydroxy-4-pregnen-3-one supports the concept that the pheromonally induced priming effect in male fish is mediated through olfactory pathways.     

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.     

SPLAT-OrB reveals competitive attraction as a mechanism of mating disruption in oriental beetle (Coleoptera: Scarabaeidae)

This study compared the efficacy of SPLAT-OrB, a new pheromone formulation for oriental beetle mating disruption that can be mechanically applied, with hand-applied plastic dispensers in commercial blueberry fields. Both formulations were tested at 2.5 and 5 g of the major sex pheromone component (Z)-7-tetradecen-2-one per hectare, and evaluated by measuring trap shut-down, mating success of caged females, and the number of grubs in sentinel blueberry pots baited with tethered females. All pheromone-treated plots had fewer male captures in traps and lower mating success of caged females compared with untreated plots. SPLAT-OrB, and plastic dispensers at the higher rate, also reduced the number of grubs in sentinel pots. To understand the mechanism of mating disruption in oriental beetle, males were observed approaching the pheromone sources in disrupted plots. In addition, male oriental beetle captures were quantified in plots treated with varying SPLAT-OrB dollop densities per ha. Consistent with predictions for competitive attraction, field observations revealed males approaching the pheromone source and male captures decreasing concavely with increasing dollop density. In a mark-release-recapture study, male oriental beetles responded to SPLAT-OrB dollops and plastic dispensers at least 60 m from the source. Additionally, SPLAT-OrB emitted pheromone that was attractive to male oriental beetles for >5 wk; however, emission rates and attraction dropped rapidly during the first 3-4 wk. This study demonstrates the feasibility of using SPLAT-OrB for oriental beetle mating disruption as an alternative to hand-applied plastic dispensers, and conclusively reveals that a principal mechanism is the competitive attraction of males.     

Vertical distribution of codling moth adults in pheromone-treated and untreated plots

The vertical distribution of codling moth,Cydia pomonella (L.) within pheromone-treated and untreated apple and pear orchard canopies was determined using tethered virgin females, unbaited sticky traps, and blacklight observation of released moths. Mating of virgin females tethered at various heights in untreated orchard canopies increased with placement height from 1–4 m. Application of pheromone dispensers for mating disruption at 2 and 4 m above the ground greatly decreased mating. Greatest capture of males and females on unbaited sticky traps occurred at mid- and upper-canopy heights. Total capture of males and females in pheromone-treated plots was not statistically different than in untreated plots. The percentage of mated females captured on sticky traps did not vary with trap height or pheromone treatment. Released moths marked with flourescent powder and observed at dark with a blacklight indicated that moths are primarily distributed high in the canopy. However, males shifted to a position lower in the canopy when pheromone dispensers were placed 2.1 m above the ground. Results suggest that pheromone dispensers be placed in the upper canopy for optimal disruption of codling moth mating.     

Synthesis of homoursodeoxycholic acid and [11,12-3H]homoursodeoxycholic acid

Homoursodeoxycholic acid and [11,12-³H]homoursodeoxycholic acid were synthesized from ursodeoxycholic acid and homocholic acid, respectively. Ursodeoxycholic acid (Ia) was converted to 3α,7β-diformoxy-5β-cholan-24-oic acid (Ib) using formic acid. Reaction of the diformoxy derivative (Ib) with thionyl chloride yielded the acid chloride (II) which was treated with diazomethane to produce 3α,7β-diformoxy-25-diazo-25-homo-5β-cholan-24-one (III). Homoursodeoxycholic acid (IV) was formed from the diazoketone (III) by means of the Wolff rearrangement of the Arndt-Eistert synthesis.N-Bromosuccinimide oxidation of homocholic acid (V), which was prepared from cholic acid by the same procedure described above, afforded 3α,12α-dihydroxy-7-oxo-25-homo-5β-cholan-25-oic acid (VI). Reduction of the 7-ketohomodeoxycholic acid (VI) with sodium in 1-propanol gave 3α,7β,12α-trihydroxy-25-homo-5β-cholan-25-oic acid (VII). The methyl ester of 7-epihomocholic acid (VII) was partially acetylated to give methyl 3α,7β-diacetoxy-12α-hydroxy-25-homo-5β-cholan-25-oate (VIII) using a mixture of acetic anhydride, pyridine and benzene. Dehydration of the diacetoxy derivative (VIII) with phosphorus oxychloride yielded methyl 3α,7β-diacetoxy-25-homo-5β-chol-11-en-25-oate (IX). Reduction of the unsaturated ester (IX) with tritium gas in the presence of platinum oxide catalyst followed by alkaline hydrolysis gave [11,12-³H]homoursodeoxycholic acid.     

Alternative reproductive tactics and courtship in the common goby

In the marine common goby Pomatoschistus microps , males either build nests and attract females to spawn or act as sneakers, forcing themselves into other males' nests during mating, to fertilize some of the eggs in the clutch. The reproductive tactics of small males were studied in the presence of surplus nest material and an excess of gravid females, while a larger, nestholding male spawned with a female. Of 24 small males, 13 only attempted to sneak, seven only courted females and/or spawned in their own nest, and four performed both behaviours. The small males who attracted females successfully were significantly larger than the ones who failed to do so. Females had been courting the small male more often when he subsequently spawned in his own nest, than when he did not get a female. The small males did not court the females very frequently, and their courtship rate did not depend on whether or not they attracted females. Instead, female courtship behaviour may be a cue for a male either to spawn as a nest holder or become a sneaker. The study pointed out the importance of female behaviour for male reproductive tactics, which could be more important in fish than is known currently.     

Seasonal variation in male alternative reproductive tactics

Genetic parentage analyses reveal considerable diversity in alternative reproductive behaviours (e.g. sneaking) in many taxa. However, little is known about whether these behaviours vary seasonally and between populations. Here, we investigate seasonal variation in male reproductive behaviours in a population of two‐spotted gobies (Gobiusculus flavescens) in Norway. Male two‐spotted gobies guard nests, attract females and care for fertilized eggs. We collected clutches and nest‐guarding males early and late in the breeding season in artificial nests and used microsatellite markers to reconstruct parentage from a subset of offspring from each nest. We hypothesized that mating, reproductive success and sneaking should be more prevalent early in the breeding season when competition for mates among males is predicted to be higher. However, parentage analyses revealed similar values of mating, reproductive success and high frequencies of successful sneaking early (30% of nests) and late (27% of nests) in the season. We also found that multiple females with eggs in the same nest were fertilized by one or more sneaker males, indicating that some males in this population engage in a satellite strategy. We contrast our results to previous work that demonstrates low levels of cuckoldry in a population in Sweden. Our results demonstrate marked stability in both the genetic mating system and male alternative reproductive tactics over the breeding season. However, sneaking rates may vary geographically within a species, likely due to local selection influencing ecological factors encountered at different locations.     

Electrophysiological investigations of sex pheromone reception and release in Bruchidius atrolineatus

ABSTRACT. Male antennal sensitivity to female sex pheromone in Bruchidius atrolineatus was investigated electrophysiologically (EAG). The existence of receptors for this pheromone was shown in the antennae of males. (No such receptors exist in the antennae of females.) Young (age < 24h) males gave an EAG of amplitude 25–30% of the response of older males. Diapausing males several days old gave a much smaller response than active males of the same age.
EAGs were used to test the production/release of pheromone by groups of twenty females. Before they are 24 h old, active females do not produce/release the pheromone; older females release it at an almost constant rate. Sexually diapausing females do not produce and/or release the sex pheromone.
In sexually active females, mating inhibits pheromone release almost immediately. There is a correlation between reproductive status of the females (development of ovaries, oogenesis) and the production or release of the pheromone. A corresponding correlation also exists in the males whose antennae show a very low sensitivity when they are young or when they are in reproductive diapause.