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.     

Multiple functions of a multi-component mating pheromone in sea lamprey Petromyzon marinus

The role of the C24 sulphate in the mating pheromone component, 7α,12α,24-trihydroxy-5α-cholan-3-one 24-sulphate (3kPZS), to specifically induce upstream movement in ovulated female sea lampreys Petromyzon marinus was investigated. 7α,12α-dihydroxy-5α-cholan-3-one 24-oic acid (3kACA), a structurally similar bile acid released by spermiated males, but lacking the C24 sulphate ester, was tested in bioassays at concentrations between 10(-11) and 10(-14) molar (M). 3kACA did not induce upstream movement in females or additional reproductive behaviours. In contrast, spermiated male washings induced upstream movement, prolonged retention on a nest and induced an array of nesting behaviours. Differential extraction and elution by solid-phase extraction resins showed that components other than 3kPZS + 3kACA are necessary to retain females on nests and induce nest cleaning behaviours. All pheromone components, including components in addition to 3kPZS + 3kACA that retain females and induce nest cleaning behaviours were released from the anterior region of the males, as had been reported for 3kPZS. It is concluded that the sea lamprey male mating pheromone has multiple functions and is composed of multiple components.     

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.     

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.     

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.     

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.     

Petromyzonol sulfate and its derivatives: the chemoattractants of the sea lamprey

Petromyzonol sulfate (PZS) and 3 keto-PZS are bile alocohol derivatives that serve as chemoattractants during the life cycle of sea lamprey (Petromyzon marinus). The sulfonate moiety is crucial perhaps conferring the required solubility for the pheromone that is released into the streams and for the specificity to bind to its receptor. During the life cycle of lamprey, larvae produce copious amounts of 5 alpha-cholan-PZS, and trace amounts of allocholic acid (ACA), which attracts adults to the same breeding ground. Later the spermeating males produce 3keto-PZS, and trace amounts of 3-keto-ACA, which attracts the ovulating females, signaling both its reproductive status and its nesting location for successful reproduction. In both stages, a mixture of components serves as pheromone plume, similar to insects. The receptors for the migratory and the reproductive pheromones need to be molecularly cloned and characterized in order to understand the molecular biology of olfaction in the sea lamprey.     

Synthesis and olfactory activity of unnatural, sulfated 5β-bile acid derivatives in the sea lamprey (Petromyzon marinus)

A variety of unnatural bile acid derivatives (9a-9f) was synthesized and used to examine the specificity with which the sea lamprey (Petromyzon marinus) olfactory system detects these compounds. These compounds are analogs of petromyzonol sulfate (PS, 1), a component of the sea lamprey migratory pheromone. Both the stereochemical configuration at C5 (i.e., 5α vs. 5β) and the extent and sites of oxygenation (hydroxylation or ketonization) of the bile acid derived steroid skeleton were evaluated by screening the compounds for olfactory activity using electro-olfactogram recording. 5β-Petromyzonol sulfate (9a) elicited a considerable olfactory response at sub-nanomolar concentration. In addition, less oxygenated systems (i.e., 9b-9e) elicited olfactory responses, albeit with less potency. The sea lamprey sex pheromone mimic 9f (5β-3-ketopetromyzonol sulfate) was also examined and found to produce a much lower olfactory response. Mixture studies conducted with 9a and PS (1) suggest that stimulation is occurring via similar modes of activation, demonstrating a relative lack of specificity for recognition of the allo-configuration (i.e., 5α) in sea lamprey olfaction. This attribute could facilitate design of pheromone analogs to control this invasive species.     

Synchronous firing of antennal-lobe projection neurons encodes the behaviorally effective ratio of sex-pheromone components in male Manduca sexta

Olfactory stimuli that are essential to an animal’s survival and reproduction are often complex mixtures of volatile organic compounds in characteristic proportions. Here, we investigated how these proportions are encoded in the primary olfactory processing center, the antennal lobe, of male Manduca sexta moths. Two key components of the female’s sex pheromone, present in an approximately 2:1 ratio, are processed in each of two neighboring glomeruli in the macroglomerular complex (MGC) of males of this species. In wind-tunnel flight experiments, males exhibited behavioral selectivity for ratios approximating the ratio released by conspecific females. The ratio between components was poorly represented, however, in the firing-rate output of uniglomerular MGC projection neurons (PNs). PN firing rate was mostly insensitive to the ratio between components, and individual PNs did not exhibit a preference for a particular ratio. Recording simultaneously from pairs of PNs in the same glomerulus, we found that the natural ratio between components elicited the most synchronous spikes, and altering the proportion of either component decreased the proportion of synchronous spikes. The degree of synchronous firing between PNs in the same glomerulus thus selectively encodes the natural ratio that most effectively evokes the natural behavioral response to pheromone.     

Mixture of new sulfated steroids functions as a migratory pheromone in the sea lamprey

The sea lamprey is an ancient, parasitic fish that invaded the Great Lakes a century ago, where it triggered the collapse of many fisheries. Like many fishes, this species relies on chemical cues to mediate key aspects of its life, including migration and reproduction. Here we report the discovery of a multicomponent steroidal pheromone that is released by stream-dwelling larval lamprey and guides adults to spawning streams. We isolated three compounds with pheromonal activity (in submilligram quantities from 8,000 l of larval holding water) and deduced their structures. The most important compound contains an unprecedented 1-(3-aminopropyl)pyrrolidin-2-one subunit and is related to squalamine, an antibiotic produced by sharks. We verified its structure by chemical synthesis; it attracts adult lamprey at very low (subpicomolar) concentrations. The second component is another new sulfated steroid and the third is petromyzonol sulfate, a known lamprey-specific bile acid derivative. This mixture is the first migratory pheromone identified in a vertebrate and is being investigated for use in lamprey control.     

Sex pheromone analysis and effective attraction of males of the cabbage webworm,Hellula undalis,inhabiting the Mekong Delta of Vietnam

Hellula undalis is a harmful insect pest of green mustard in the Mekong Delta of Vietnam. In order to establish a tool for a sustainable pest control program, the sex pheromone of H. undalis inhabiting the Mekong Delta was examined. GC-EAD and GC–MS analyses of pheromone gland extracts from the virgin females elucidated three new components, (Z)-11-tetradecenyl acetate (Z11-14:OAc), (Z)-11-hexadecenal (Z11-16:Ald), and (11E,13E)-11,13-hexadecadien-1-ol, in addition to the known pheromone component (11E,13E)-11,13-hexadecadienal (E11,E13-16:Ald). Double bond positions of the two monoenyl components were determined by GC–MS analysis of the pheromone extract treated with dimethyl disulfide. On the other hand, GC–MS analysis of the female body extract detected the unsaturated hydrocarbon (3Z,6Z,9Z)-3,6,9-tricosatriene (Z3,Z6,Z9-23:H). Field examinations of their synthetic compounds indicated the significant role of E11,E13-16:Ald as a major component and a clear synergistic effect of the two monoenyl compounds as a minor component. Although the 3:3:7 mixture of Z11-14:OAc, E11-16:Ald, and E11,E13-16:Ald captured the largest number of males among the tested mixtures, the activity was still quite a bit lower than that of virgin females. However, the 3:3:7:1 mixture, which was prepared by adding a small amount of Z3,Z6,Z9-23:H to the 3:3:7 ternary lure, succeeded in attracting males more powerfully than the females did. This strong synergistic effect was not observed when the triene was added to unmixed E11,E13-16:Ald, indicating important roles of not only the triene but also the two monoenyl compounds as natural pheromone components.     

A critical review of the discovery and application of a migratory pheromone in an invasive fish, the sea lamprey Petromyzon marinus L.

The sea lamprey Petromyzon marinus , an ancient and parasitic agnathan fish native to the North Atlantic, invaded the Laurentian Great Lakes approximately a century ago triggering its fisheries to collapse. Presently, this species is held in check through a toxicant-based control programme directed by a bi-national treaty organization that seeks to develop a more broadly based integrated pest management (IPM) strategy. After a long and difficult search, the most active components of a migratory pheromone for the sea lamprey were identified and synthesized. This pheromone has remarkable potency and considerable potential to enhance various control strategies that fall within an IPM scheme. The pheromone is comprised of a mixture of at least three sulphated steroids: petromyzonamine disulphate (PADS), petromyzosterol disulphate (PSDS) and petromyzonol sulphate (PS). A steroidal sex pheromone appears to have similar promise. These discoveries, which are prompting development of the first pheromonally guided pest management programme for an invasive fish, have revealed significant scientific and policy challenges. Among the former are the needs to acquire long-term and significant support for the science, to synthesize sufficient quantities of cues, and to deploy and measure pheromonal components in natural waters. Among the latter are the needs to regulate this new type of 'pesticide', protect these technologies from exploitation and promote future research on this and other invasive fish species. Lessons from these experiences may be applicable to the study of other invasive fishes.     

Within-population variability in a moth sex pheromone blend: genetic basis and behavioural consequences

Evolutionary diversification of sexual communication systems in moths is perplexing because signal and response are under stabilizing selection in many species, and this is expected to constrain evolutionary change. In the moth Heliothis virescens, we consistently found high phenotypic variability in the female sex pheromone blend within each of four geographically distant populations. Here, we assess the heritability, genetic basis and behavioural consequences of this variation. Artificial selection with field-collected moths dramatically increased the relative amount of the saturated compound 16:Ald and decreased its unsaturated counterpart Z11–16:Ald, the major sex pheromone component (high line). In a cross between the high- and low-selected lines, one quantitative trait locus (QTL) explained 11–21% of the phenotypic variance in the 16:Ald/Z11–16:Ald ratio. Because changes in activity of desaturase enzymes could affect this ratio, we measured their expression levels in pheromone glands and mapped desaturase genes onto our linkage map. A delta-11-desaturase had lower expression in females producing less Z11–16:Ald; however, this gene mapped to a different chromosome than the QTL. A model in which the QTL is a trans-acting repressor of delta-11 desaturase expression explains many features of the data. Selection favouring heterozygotes which produce more unsaturated components could maintain a polymorphism at this locus.     

Site of production of an oviposition-deterring pheromone component in Rhagoletis pomonella flies

Using behavioural and electrophysiological assay techniques, we identified the posterior half of the midgut as being a principal site of production of a major component of the oviposition-deterring, fruit-marking pheromone of female Rhagoletis pomonella flies. Following secretion into, and accumulation in, the gut lumen, this component is released, together with other gut contents, in the marking trail deposited during dragging of the ovipositor on the fruit surface after egg-laying, as well as in the faeces. Other components of the pheromone may be produced elsewhere.     

Sex pheromone blend of Manduca sexta: Responses of central olfactory interneurons to antennal stimulation in male moths

Recently, chemical analysis of solvent rinses of the external surfaces of pheromone glands from female Manduca sexta revealed a blend of 12 aldehydes, including the previously identified sex pheromone component, (E,Z)-10,12-hexadecadienal (bombykal). Previous electrophysiological studies showed that olfactory (deutocerebral) interneurons in the antennal lobes of males exhibited a wide range of responsiveness to pheromonal stimulation of the ipsilateral antenna. These experiments were performed with crude extracts of pheromone glands as well as two synthetic compounds: the major pheromone component, bombykal, and (E,Z)-11,13-pentadecadienal, a mimic of a second component of the female's pheromone blend. Using intracellular methods, we have now reexamined similar olfactory interneurons, using each of the 12 chemically identified components as well as synthetic blends of various combinations of them. Eight of the 12 components isolated from female glands elicited some form of response in olfactory interneurons in males. In accordance with biochemical and behavioral data, the most potent are bombykal and two trienals, (E,E,E)- and (E,E,Z)-10,12,14-hexadecatrienal. We also conclude that the C₁₅ dienal is selective for one of the trienal receptors on the antenna, but is much less potent than the natural trienal stimulant.     

HPLC and ELISA analyses of larval bile acids from Pacific and western brook lampreys

Comparative studies were performed on two native lamprey species, Pacific lamprey (Lampetra tridentata) and western brook lamprey (Lampetra richardsoni) from the Pacific coast along with sea lamprey (Petromyzon marinus) from the Great Lakes, to investigate their bile acid production and release. HPLC and ELISA analyses of the gall bladders and liver extract revealed that the major bile acid compound from Pacific and western brook larval lampreys was petromyzonol sulfate (PZS), previously identified as a migratory pheromone in larval sea lamprey. An ELISA for PZS has been developed in a working range of 20 pg-10 ng per well. The tissue concentrations of PZS in gall bladder were 127.40, 145.86, and 276.96 micro g/g body mass in sea lamprey, Pacific lamprey, and western brook lamprey, respectively. Releasing rates for PZS in the three species were measured using ELISA to find that western brook and sea lamprey released PZS 20 times higher than Pacific lamprey did. Further studies are required to determine whether PZS is a chemical cue in Pacific and western brook lampreys.     

Female sex pheromone of Athetis lepigone (Lepidoptera: Noctuidae): Identification and field evaluation

Athetis lepigone has been recorded in many countries in Europe and Asia, but it had never been documented as an agricultural pest until 2005. For the purpose of using the sex pheromone to control this pest, we conducted a study to identify the sex pheromone of A. lepigone by gas chromatography with an electroantennographic detector (GC‐EAD) and GC coupled with mass spectrometry (GC/MS) analyses. Three pheromone candidates were detected by GC‐EAD analysis in the extracts of the female sex pheromone gland, and two candidates were identified as (Z)‐7‐dodecenyl acetate (Z7‐12:OAc) and (Z)‐9‐tetradecenyl acetate (Z9‐14:OAc) in a ratio of 1:5 by mass spectral analysis of natural pheromone components and dimethyl disulphide adducts. In the field male trapping test, the traps baited with the binary blend captured high number of males, while traps with single component hardly caught males, indicating that the two components are essential for the male attractiveness. In addition, the optimum ratios of Z7‐12:OAc and Z9‐14:OAc were determined as 3:7–7:3, and the best doses for the binary blend (at ratio of 3:7 between Z7‐12:OAc and Z9‐14:OAc) were 0.25–0.5 mg/trap, based on the number of male catches. The identification of a highly attractive sex pheromone will help in developing efficient strategies for monitoring and control of A. lepigone.     

Effects of different sex pheromone compositions and host plants on the mating behavior of two Grapholita species

The two congener species Grapholita molesta and Grapholita dimorpha share two major sex pheromone components: cis-8-dodecenyl acetate (Z8-12Ac) and trans-8-dodecenyl acetate (E8-12Ac). In fact, commercial sex pheromone lures composed of only these two major components attract the males of both species. In this study, we aimed to determine the reproductive isolation components of these two species by analyzing the effects of the minor sex pheromone components and host plants. First, different ratios of the two major sex pheromone components were greatly favored by either male species. Sex pheromone gland extracts of G. dimorpha contained a lesser proportion of Z8-12Ac than that of G. molesta. In the three (apple, pear, and peach) orchards investigated in this study, a larger number of G. molesta males were attracted to the 95:5 pheromone mixture (Z8-12Ac and E8-12Ac, respectively), while a larger number of G. dimorpha males were attracted to the 85:15 mixture. Second, there was a significant variation in male attractions in different host plants. G. molesta males were more attracted to the sex pheromone lure in the apple orchards than that in the pear and peach orchards. In contrast, G. dimorpha males were more attracted to the lures in the pear and peach orchards than that in the apple orchard. Third, the minor sex pheromone components were important for reproductive isolation. Among the four minor components tested, addition of (Z)-8-dodecenol (Z8-12OH) to the major sex pheromone components significantly suppressed male attraction in G. dimorpha and slightly elevated male attraction in G. molesta. The discriminating effect of Z8-12OH was further validated using male electroantennogram analysis. These results suggest that reproductive isolation between two congeners can be achieved by variations in the minor sex pheromone components and in the host plants, as well as by changes in the ratio of the two major components.     

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.