Dibutyl phthalate released by solitary female locusts mediates sexual communication at low density.

Sex pheromones play a crucial role in mate location and reproductive success. Insects face challenges in finding mates in low-density environments. The population dynamics of locusts vary greatly, ranging from solitary individuals to high-density swarms, leading to multiple-trait divergence between solitary and gregarious phases. However, differences in sexual communication between solitary and gregarious locusts have not been sufficiently explored. Herein, we found that solitary locusts but not gregarious ones heavily rely on a single compound, dibutyl phthalate (DBP), for sexual communication. DBP is abundantly released by solitary female locusts and elicits strong attraction of male solitary and gregarious locusts. Solitary adult males display much higher electrophysiological responses to DBP than adult females. Additionally, LmigOr13 was identified as the DBP-specific odorant receptor expressed in neurons housed in basiconic sensilla. Male LmigOr13-/- mutants generated by CRISPR/Cas9 have low electrophysiological responses and behavioral attraction to DBP in both laboratory and field cage experiments. Notably, the attractiveness of DBP to male locusts becomes more evident at lower population densities imposed by controlling the cage size. This finding sheds light on the utilization of a sex pheromone to promote reproductive success in extremely low-density conditions and provides important insights into alternative approaches for population monitoring of locusts.

Tunable control of insect pheromone biosynthesis in Nicotiana benthamiana.

Previous work has demonstrated that plants can be used as production platforms for molecules used in health, medicine, and agriculture. Production has been exemplified in both stable transgenic plants and using transient expression strategies. In particular, species of Nicotiana have been engineered to produce a range of useful molecules, including insect sex pheromones, which are valued for species-specific control of agricultural pests. To date, most studies have relied on strong constitutive expression of all pathway genes. However, work in microbes has demonstrated that yields can be improved by controlling and balancing gene expression. Synthetic regulatory elements that provide control over the timing and levels of gene expression are therefore useful for maximizing yields from heterologous biosynthetic pathways. In this study, we demonstrate the use of pathway engineering and synthetic genetic elements for controlling the timing and levels of production of Lepidopteran sex pheromones in Nicotiana benthamiana. We demonstrate that copper can be used as a low-cost molecule for tightly regulated inducible expression. Further, we show how construct architecture influences relative gene expression and, consequently, product yields in multigene constructs. We compare a number of synthetic orthogonal regulatory elements and demonstrate maximal yields from constructs in which expression is mediated by dCas9-based synthetic transcriptional activators. The approaches demonstrated here provide new insights into the heterologous reconstruction of metabolic pathways in plants.

Functional differentiation of two general odorant-binding proteins to sex pheromones in Spodoptera frugiperda.

A precise chemosensory system can help insects complete various important behavioral responses by accurately identifying different external odorants. Therefore, deeply understanding the mechanism of insect recognition of important odorants will help us develop efficient and environmentally-friendly behavioral inhibitors. Spodoptera frugiperda is a polyphagous pest that feeds on >350 different host plants worldwide and also harms maize production in China. However, the molecular mechanism of the first step for males to use odorant-binding proteins (OBPs) to recognize sex pheromones remains unclear. Here, we obtained 50 OBPs from the S. frugiperda genome, and the expression level of SfruGOBP1 in females was significantly higher than that in males, whereas SfruGOBP2 displayed male-biased expression. Fluorescence competitive binding assays showed that only SfruGOBP2 showed binding affinities for the four sex pheromones of female S. frugiperda. Subsequently, we identified some key amino acid residues that can participate in the interaction between SfruGOBP2 and sex pheromones using molecular docking and site-directed mutagenesis methods. These findings will help us explore the interaction mechanism between GOBPs and sex pheromones in moths, and provide important target genes for developing new mating inhibitors of S. frugiperda in the future.

Sex Pheromone Receptor-Derived Peptide Biosensor for Efficient Monitoring of the Cotton Bollworm Helicoverpa armigera.

The cotton bollworm, Helicoverpa armigera (H. armigera), causes damage to a wide range of cultivated crops and is one of the pests with the greatest economic importance for global agriculture. Currently, the detection of H. armigera is based on manual sampling. A low limit of detection (LOD), convenient, and real-time monitoring method is urgently needed for its early warning and efficient management. Here, we characterized the amino acid sequence in the sex pheromone receptors (SPRs) recognizing the pheromone components of H. armigera by three-dimensional (3D) modeling and molecular docking. Next, sex pheromone receptor-derived peptides (SPRPs) were synthesized and conjugated to nanotubes by chemical connection. The modified nanotubes were used to fabricate a sensor capable of real-time monitoring of gaseous sex pheromone compounds with a low LOD (∼10 ppb for Z11-16:Ald) and selectivity, and the sensor was able to detect a single live H. armigera. Furthermore, the developed biosensor allowed direct monitoring of the pheromone release dynamics by female H. armigera and showed that the release was instantly reduced in response to light. Here, we report the first demonstration of a biosensing method for detecting gaseous sex pheromones and live H. armigera. The findings show the great potential of the SPRP sensor for broad applications in insect biology study and infestation monitoring.

Expression Profiles and Binding Properties of the Chemosensory Protein PxylCSP11 from the Diamondback Moth, Plutella xylostella (Lepidoptera: Plutellidae).

The diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae) is one of the most destructive pests to cruciferous plants worldwide. The oligophagous moth primarily utilizes its host volatiles for foraging and oviposition. Chemosensory proteins (CSPs) are soluble carrier proteins with low molecular weight, which recognize and transport various semiochemicals in insect chemoreception. At present, there is limited information on the recognition of host volatiles by CSPs of P. xylostella. Here, we investigated expression patterns and binding characteristics of PxylCSP11 in P. xylostella. The open reading frame of PxylCSP11 was 369-bp encoding 122 amino acids. PxylCSP11 possessed four conserved cysteines, which was consistent with the typical characteristic of CSPs. PxylCSP11 was highly expressed in antennae, and the expression level of PxylCSP11 in male antennae was higher than that in female antennae. Fluorescence competitive binding assays showed that PxylCSP11 had strong binding abilities to several ligands, including volatiles of cruciferous plants, and (Z)-11-hexadecenyl acetate (Z11-16:Ac), a major sex pheromone of P. xylostella. Our results suggest that PxylCSP11 may play an important role in host recognition and spouse location in P. xylostella.

The Effect of Pheromone Synthesis and Gland Retraction on Translocation and Dynamics of Pheromone Release in the Moth Chloridea virescens.

Most species of moths use a female-produced sex pheromone to bring mates together. Typically, sex pheromone is synthesized in a specialized gland and released during the behavior of "calling", in which the ovipositor and gland are extruded, allowing pheromone to evaporate. Although there has been much study on how a gland makes specific pheromone components, we know relatively little about how it actually functions with regard to synthesis, storage and release. In this paper, we investigated three aspects of gland function in the noctuid moth Chloridea virescens (Fabricius): (i) whether translocation of pheromone from site of synthesis to release is dependent on calling or ovipositor movement, (ii) whether pheromone synthesis rate limits release and (iii) how intermittent calling (observed in this and other species) might affect the dynamics of release rate. Firstly, by manipulating the gland to simulate calling (extruded) or non-calling (retracted), we showed that pheromone translocation occurred regardless of whether the gland was retracted or extruded. Secondly, by manipulating pheromone production, we found that females that produced more pheromone had higher release rates. It was especially noticeable that females had a higher release rate at the start of calling, which dropped rapidly and leveled off over time. Together, these data suggest that intermittent calling in C. virescens (and other species) may function to allow females to replenish pheromone stores on the gland surface between calling bouts, so that brief, high release rates occur at the start of a calling bout; thus, potentially increasing a female's chances of attracting a mate.

Key Amino Acid Residues Influencing Binding Affinities of Pheromone-Binding Protein from Athetis lepigone to Two Sex Pheromones.

Athetis lepigone is a polyphagous pest found around the world that feeds on maize, wheat, and various other important crops. Although it exhibits a degree of resistance to various chemical insecticides, an effective pest-control method has not yet been developed. The sex pheromone communication system plays an essential role in the mating and reproduction of moths, in which pheromone-binding proteins (PBPs) are crucial genes. In this study, we cloned and purified the protein AlepPBP1 using an E. coli expression system and found it had a higher binding affinity to two sex pheromones of A. lepigone, namely, Z7-12:Ac and Z9-14:Ac (with Ki 0.77 ± 0.10 and 1.10 ± 0.20 µM, respectively), than to other plant volatiles. The binding-mode analysis of protein conformation with equilibrium stabilization was obtained using molecular dynamics (MD) simulation and indicated that hydrophobic interactions involving several nonpolar residues were the main driving force for the binding affinity of AlepPBP1 with sex pheromones. Computational alanine scanning (CAS) was performed to further identify key amino acid residues and validate their binding contributions. Each key residue, including Phe36, Trp37, Val52, and Phe118, was subsequently mutated into alanine using site-directed mutagenesis. Binding assays showed that the efficient binding abilities to Z7-12:Ac (F36A, W37A, and F118A) and Z9-14:Ac (F36A, W37A, V52A, and F118A) were almost lost in the mutated proteins. Our results demonstrated that these key amino acid residues are crucial for determining the binding ability of AlepPBP1 to sex pheromones. These findings provide a basis for the use of AlepPBP1 in the studies as a specific target for the development of novel behavioral antagonists with marked inhibition or mating-disruption abilities using computer-aided drug design (CADD).

DNA methylation patterns in the tobacco budworm, Chloridea virescens.

DNA methylation is an important epigenetic modification that is prone to stochastic variation and is responsive to environmental factors. Yet changes in DNA methylation could persist across generations and thus play an important role in evolution. In this study, we used methylation-sensitive amplified fragment length polymorphisms (MS-AFLP) to evaluate whether DNA methylation could contribute to the evolution of the sexual communication signal in the noctuid moth Chloridea virescens. We found that most DNA methylation was consistent across tissues, although some methylation sites were specifically found in pheromone glands. We also found significant DNA methylation differences among families and two pheromone phenotype selection lines, and these differences correlated with genetic variation. Most DNA methylation patterns were inherited, although some sites were subject to spontaneous de novo DNA methylation across generations. Thus, DNA methylation likely plays a role in a wide range of processes in moths. Together, our results present an important initial step towards understanding the potential role of DNA methylation in the evolution of sexual communication signals in moths.

Attraction and Electrophysiological Response to Identified Rectal Gland Volatiles in Bactrocera frauenfeldi (Schiner).

Bactrocera frauenfeldi (Schiner) (Diptera: Tephritidae) is a polyphagous fruit fly pest species that is endemic to Papua New Guinea and has become established in several Pacific Islands and Australia. Despite its economic importance for many crops and the key role of chemical-mediated sexual communication in the reproductive biology of tephritid fruit flies, as well as the potential application of pheromones as attractants, there have been no studies investigating the identity or activity of rectal gland secretions or emission profiles of this species. The present study (1) identifies the chemical profile of volatile compounds produced in rectal glands and released by B. frauenfeldi, (2) investigates which of the volatile compounds elicit an electroantennographic or electropalpographic response, and (3) investigates the potential function of glandular emissions as mate-attracting sex pheromones. Rectal gland extracts and headspace collections from sexually mature males and females of B. frauenfeldi were analysed by gas chromatography-mass spectrometry. Male rectal glands contained (E,E)-2-ethyl-8-methyl-1,7-dioxaspiro [5.5]undecane as a major component and (E,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane as a moderate component. Minor components included palmitoleic acid, palmitic acid, and ethyl oleate. In contrast, female rectal glands contained (E,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane and ethyl laurate as major components, ethyl myristate and ethyl palmitoleate as moderate components, and 18 minor compounds including amides, esters, and spiroacetals. Although fewer compounds were detected from the headspace collections of both males and females than from the gland extractions, most of the abundant chemicals in the rectal gland extracts were also detected in the headspace collections. Gas chromatography coupled electroantennographic detection found responses to (E,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane from the antennae of both male and female B. frauenfeldi. Responses to (E,E)-2-ethyl-8-methyl-1,7-dioxaspiro[5.5]undecane were elicited from the antennae of females but not males. The two spiroacetals also elicited electropalpographic responses from both male and female B. frauenfeldi. Ethyl caprate and methyl laurate, found in female rectal glands, elicited responses in female antennae and palps, respectively. Y-maze bioassays showed that females were attracted to the volatiles from male rectal glands but males were not. Neither males nor females were attracted to the volatiles from female rectal glands. Our findings suggest (E,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane and (E,E)-2-ethyl-8-methyl-1,7-dioxaspiro[5.5]undecane as components of a sex-attracting pheromone in B. frauenfeldi.

A Δ9 desaturase (SlitDes11) is associated with the biosynthesis of ester sex pheromone components in Spodoptera litura.

Sex pheromone biosynthesis in moths relies on the activity of multiple enzymes, including Δ9 desaturase, which plays an important role in catalyzing desaturation at the Δ9 position of the carbon chain. However, the physiological function of moth Δ9 desaturase has not been elucidated in vivo. In this study, we used the CRISPR/Cas9 system to knockout the Δ9 desaturase gene (SlitDes11) of Spodoptera litura to analyze its role in sex pheromone biosynthesis. First, through the direct injection of SlitDes11-single guide RNA (sgRNA)/Cas9 messenger RNA into newly laid eggs, gene editing was induced in around 30% of eggs 24 h after injection and was induced in 20.8% of the resulting adult moths. Second, using a sibling-crossing strategy, insects with mutant SlitDes11 (bearing a premature stop codon) were selected, and homozygous mutants were obtained in the G5 generation. Third, pheromone gland extracts of adult female homozygous SlitDes11 mutants were analyzed using Gas chromatography (GC). The results showed that titers of all three ester sex pheromone components; Z9, E11-14:Ac, Z9,E12-14:Ac, and Z9-14:Ac; were reduced by 62.40%, 78.50%, and 72.50%, respectively. This study provides the first direct evidence for the role of SlitDes11 in sex pheromone biosynthesis in S. litura, and indicates the gene could be as potential target to disrupt sexual communication in S. litura for developing a new pollution-free insecticide.

Wax Ester Composition of Songbird Preen Oil Varies Seasonally and Differs between Sexes, Ages, and Populations.

Chemical signaling has been well studied in invertebrates and mammals but less so in birds, due to the longstanding misconception that olfaction is unimportant or even non-existent in this taxon. However, recent findings suggest that olfaction plays an important role in avian mate choice and reproductive behavior, similar to other taxa. The leading candidate source for compounds involved in avian chemical communication is preen oil, a complex mixture secreted from the uropygial gland. Preen oil contains volatile compounds and their potential wax ester precursors, and may act as a reproductive chemosignal. Reproductive signals are generally sexually dimorphic, age-specific, seasonally variable, and may also vary geographically. We tested whether preen oil meets these expectations by using gas chromatography to examine the wax ester composition of preen oil in song sparrows (Melospiza melodia). We found that the wax ester composition of preen oil was significantly different between sexes, age classes, seasons, and populations. Collectively, our results suggest that song sparrow preen oil meets the criteria of a chemical cue that may influence mate choice and reproduction. Our findings in song sparrows, which are sexually monomorphic in plumage, also parallel patterns described for dark-eyed juncos (Junco hyemalis), a closely related songbird with sexually dimorphic plumage. Behavioral tests are needed to confirm that song sparrows attend to the cues present in preen oil, but our findings support the increasingly accepted idea that chemical communication is common and widespread in birds as it is in other taxa.

Identification and Field Testing of Volatile Components in the Sex Attractant Pheromone Blend of Female House Mice.

Recently, it was reported (i) that the sex pheromone blend of male house mice, Mus musculus, comprises not only volatile components (3,4-dehydro-exo-brevicomin; 2-sec-butyl-4,5-dihydrothiazole) but also a component of low volatility (the sex steroid testosterone), and (ii) that the sex steroids progesterone and estradiol are sex pheromone components of female house mice. Here we tested the hypothesis that the sex attractant pheromone blend of female mice, analogous to that of male mice, also comprises volatile pheromone components. Analyzing by GC-MS the head space volatiles of bedding soiled with urine and feces of laboratory-kept females and males revealed three candidate pheromone components (CPCs) that were adult female-specific: butyric acid, 2-methyl butyric acid and 4-heptanone. In a two-choice laboratory experiment, adult males spent significantly more time in the treatment chamber baited with both the synthetic steroids (progesterone, estradiol) and the synthetic CPCs than in the paired control chamber baited only with the synthetic steroids. In field experiments, trap boxes baited with both the CPCs and the steroids captured 6.7-times more adult males and 4.7-times more juvenile males than trap boxes baited with the steroids alone. Conversely, trap boxes baited with both the CPCs and the steroids captured 4.3-times more adult males and 2.7-fold fewer adult females than trap boxes baited with the CPCs alone. In combination, these data support the conclusion that butyric acid, 2-methyl butyric acid and 4-heptanone are part of the sex attractant pheromone of female house mice. With progesterone and estradiol being pheromone components of both female brown rats, Rattus norvegicus, and female house mice, these three volatile components could impart specificity to the sexual communication system of house mice, brown rats and possibly other rodent species.

Feminization of Male Brown Treesnake Methyl Ketone Expression via Steroid Hormone Manipulation.

Pheromones are useful tools for the management of invasive invertebrates, but have proven less successful in field applications for invasive vertebrates. The brown treesnake, Boiga irregularis, is an invasive predator that has fundamentally altered the ecology of Guam. The development of control tools to manage Boiga remains ongoing. Skin-based, lipophilic pheromone components facilitate mating in brown treesnakes, with females producing the same long-chain, saturated and monounsaturated (ketomonoene) methyl ketones known to function as pheromones in garter snakes, Thamnophis sirtalis. Boiga also express novel, diunsaturated methyl ketones (ketodienes) with a purported function as a sex pheromone. In our study, we implanted 17 ß-estradiol in adult male brown treesnakes in order to manipulate methyl ketone expression as sex attractants, an effect that would mirror findings with garter snakes. Specifically, estrogen promoted production of two ketomonoenes, pentatriaconten-2-one and hexatriaconten-2-one, and suppressed production of one ketodiene, heptatriacontadien-2-one. In bioassays, estrogen-implanted males elicited tongue-flicking and chin rubbing behavior from unmanipulated males, though the responses were weaker than those elicited by females. On Guam, wild males exhibited greatest responses to whole female skin lipid extracts and only weak responses to the methyl ketone fractions from females and implanted males. Our results suggest that sex identity in brown treesnakes may be conferred by the ratio of ketomonoenes (female) to ketodienes (male) from skin lipids and may be augmented by a sex-specific endocrine signal (estradiol). However, a blend of long-chain methyl ketones alone is not sufficient to elicit maximal reproductive behaviors in male Boiga.

PSA-NCAM in the posterodorsal medial amygdala is necessary for the pubertal emergence of attraction to female odors in male hamsters.

During puberty, attention turns away from same-sex socialization to focus on the opposite sex. How the brain mediates this change in perception and motivation is unknown. Polysialylated neural cell adhesion molecule (PSA-NCAM) virtually disappears from most of the central nervous system after embryogenesis, but it remains elevated in discrete regions of the adult brain. One such brain area is the posterodorsal subnucleus of the medial amygdala (MePD). The MePD has been implicated in male sexual attraction, measured here as the preference to investigate female odors. We hypothesize that PSA-NCAM gates hormone-dependent plasticity necessary for the emergence of males' attraction to females. To evaluate this idea, we first measured PSA-NCAM levels across puberty in several brain regions, and identified when female odor preference normally emerges in male Syrian hamsters. We found that MePD PSA-NCAM staining peaks shortly before the surge of pubertal androgen and the emergence of preference. To test the necessity of PSA-NCAM for female odor preference, we infused endo-neuraminidase-N into the MePD to deplete it of PSAs before female odor preference normally appears. This blocked female odor preference, which suggests that PSA-NCAM facilitates behaviorally relevant, hormone-driven plasticity.

Evidence that progestins play an important role in spermiation and pheromone production in male sea lamprey (Petromyzon marinus).

Progestins (progestogens, C21 steroids) have been shown to regulate key physiological activities for reproduction in both sexes in all classes of vertebrates except for Agnathans. Progesterone (P) and 15α-hydroxyprogesterone (15α-P) have been detected in sea lamprey (Petromyzon marinus) plasma, but the expression patterns and functions of putative progestin receptor genes have not yet been investigated. The first objective of this study was to determine the differences in mRNA expression levels of nuclear progestin receptor (nPR) and the membrane receptor adaptor protein 'progesterone receptor membrane component 1' (pgrmc1) in putative target tissues in males at different life stages, with and without lamprey GnRH-I and -III treatment. The second objective was to demonstrate the function of progestins by implanting prespermiating males (PSM) with time-release pellets of P and measuring the latency to the onset of spermiation and plasma concentrations of sex pheromones and steroids. The third objective was to measure the binding affinity of P in the nuclear and membrane fractions of the target tissues. Expression levels of nPR and pgrmc1 differed between life stages and tissues, and in some cases were differentially responsive to lamprey GnRH-I and -III. Increases in nPR and pgrmc1 gene expressions were correlated to the late stages of sexual maturation in males. The highest expression levels of these genes were found in the liver and gill of spermiating males. These organs are, respectively, the site of production and release of the sex pheromone 3 keto-petromyzonol sulfate (3kPZS). The hypothesis that pheromone production may be under hormonal control was tested in vivo by implanting PSM with time-release pellets of P. Concentrations of 3kPZS in plasma after 1week were 50-fold higher than in controls or in males that had been implanted with androstenedione, supporting the hypothesis that P is responsible for regulating the production of the sex pheromone. P treatment also accelerated the onset of spermiation. Saturation and Scatchard analyses of the target tissues showed that both nuclear and membrane fractions bound P with high affinity and low capacity (KD 0.53pmol/g testis and 0.22 pmol/g testis, and Bmax 1.8 and 5.7 nM, respectively), similar to the characteristics of nPR and mPR in other fish. The fact that a high proportion of P was also converted in vivo to 15α-P means that it is not yet possible to determine which of these two steroids is the natural ligand in the sea lamprey.

Chemosensory receptor genes in the Oriental tobacco budworm Helicoverpa assulta.

The Oriental tobacco budworm (Helicoverpa assulta) is a specialist herbivore moth and its larvae feed on Solanaceous plants. (Z)-9-hexadecenal (Z9-16: Ald) is the major sex pheromone component in H. assulta but the specific pheromone receptor (PR) against Z9-16: Ald has not yet been identified. In the present study, we integrated transcriptomic, bioinformatic and functional characterization approaches to investigate the chemosensory receptor genes of H. assulta. We identified seven potential PRs with 44 olfactory receptors, 18 gustatory receptors and 24 ionotropic receptors, which were further studied by in silico gene expression profile, phylogenetic analysis, reverse transcription PCR and calcium imaging assays. The candidate PR, HassOR13, showed a strong response to the minor sex pheromone component, (Z)-11-hexadecenal, but not the major component, Z9-16: Ald, in calcium imaging assays. This study provides the molecular basis for comparative studies of chemosensory receptors between H. assulta and other Helicoverpa species and will advance our understanding of the evolution and function of Lepidoptera insect chemosensation.

Acp70A regulates Drosophila pheromones through juvenile hormone induction.

Mated Drosophila melanogaster females show a decrease in mating receptivity, enhanced ovogenesis, egg-laying and activation of juvenile hormone (JH) production. Components in the male seminal fluid, especially the sex peptide ACP70A stimulate these responses in females. Here we demonstrate that ACP70A is involved in the down-regulation of female sex pheromones and hydrocarbon (CHC) production. Drosophila G10 females which express Acp70A under the control of the vitellogenin gene yp1, produced fewer pheromones and CHCs. There was a dose-dependent relationship between the number of yp1-Acp70A alleles and the reduction of these compounds. Similarly, a decrease in CHCs and diene pheromones was observed in da > Acp70A flies that ubiquitously overexpress Acp70A. Quantitative-PCR experiments showed that the expression of Acp70A in G10 females was the same as in control males and 5 times lower than in da > Acp70A females. Three to four days after injection with 4.8 pmol ACP70A, females from two different strains, exhibited a significant decrease in CHC and pheromone levels. Similar phenotypes were observed in ACP70A injected flies whose ACP70A receptor expression was knocked-down by RNAi and in flies which overexpress ACP70A N-terminal domain. These results suggest that the action of ACP70A on CHCs could be a consequence of JH activation. Female flies exposed to a JH analog had reduced amounts of pheromones, whereas genetic ablation of the corpora allata or knock-down of the JH receptor Met, resulted in higher amounts of both CHCs and pheromonal dienes. Mating had negligible effects on CHC levels, however pheromone amounts were slightly reduced 3 and 4 days post copulation. The physiological significance of ACP70A on female pheromone synthesis is discussed.

Neurones in the preoptic area of the male goldfish are activated by a sex pheromone 17α,20ß-dihydroxy-4-pregnen-3-one.

Pheromones are interesting molecules given their ability to evoke changes in the endocrine state and behaviours of animals. In goldfish, a sex pheromone, 17α,20ß-dihydroxy-4-pregnen-3-one (17,20ß-P), which is released by preovulatory females, is known to trigger the elevation of luteinising hormone (LH) levels, as well as reproductive behaviour in males. Interestingly, when 11-ketotestosterone (11-KT) is implanted into adult female fish, LH levels increase in response to the pheromone at any time of the day, which is normally a male-specific response. However, the neural mechanisms underlying the male-specific information processing of 17,20ß-P and its androgen dependence are yet unknown. In the present study, we focused on the preoptic area (POA), which plays important roles in the regulation of reproduction and reproductive behaviours. We mapped activity in the POA evoked by 17,20ß-P exposure using the immediate-early gene c-fos. We found that a population of ventral POA neurones close to kisspeptin2 (kiss2) neurones that appear to have important roles in reproduction was activated by 17,20ß-P exposure, suggesting that these activated neurones are important for the 17,20ß-P response. Next, we investigated the distribution of androgen receptor (ar) in the POA and its relationship with 17,20ß-P-responsive and kiss2 neurones. We found that ar is widely expressed in the ventral POA, whereas it is only expressed in approximately 10% of 17,20ß-P-activated neurones. On the other hand, it is expressed in almost 90% of the kiss2 neurones. Taken together, it is possible that ar expressing neurones in the ventral POA, most of which were not labelled by c-fos in the present study, may at least partly account for androgen effects on responses to primer pheromones; the ar-positive kiss2 neurones in the ventral POA may be a candidate. These results offer a novel insight into the mechanisms underlying male-specific information processing of 17,20ß-P in goldfish.

A novel mechanism regulating a sexual signal: the testosterone-based inhibition of female sex pheromone expression in garter snakes.

Vertebrates communicate their sex to conspecifics through the use of sexually dimorphic signals, such as ornaments, behaviors and scents. Furthermore, the physiological connection between hormones and secondary sexual signal expression is key to understanding their dimorphism, seasonality and evolution. The red-sided garter snake (Thamnophis sirtalis parietalis) is the only reptile for which a described pheromone currently exists, and because garter snakes rely completely on the sexual attractiveness pheromone for species identification and mate choice, they constitute a unique model species for exploring the relationship between pheromones and the endocrine system. We recently demonstrated that estrogen can activate female pheromone production in male garter snakes. The purpose of this study was to determine the mechanism(s) acting to prevent female pheromone production in males. We found that castrated males (GX) are courted by wild males in the field and produce appreciable amounts of female sex pheromone. Furthermore, pheromone production is inhibited in castrates given testosterone implants (GX+T), suggesting that pheromone production is actively inhibited by the presence of testosterone. Lastly, testosterone supplementation alone (T) increased the production of several saturated methyl ketones in the pheromone but not the unsaturated ketones; this may indicate that saturated ketones are testosterone-activated components of the garter snake's skin lipid milieu. Collectively, our research has shown that pheromone expression in snakes results from two processes: activation by the feminizing steroid estradiol and inhibition by testosterone. We suggest that basal birds and garter snakes share common pathways of activation that modulate crucial intraspecific signals that originate from skin.

Sex-specific trail pheromone mediates complex mate finding behavior in Anoplophora glabripennis.

Anoplophora glabripennis (Motsch.) is a polyphagous member of the Cerambycidae, and is considered, worldwide, to be one of the most serious quarantine pests of deciduous trees. We isolated four chemicals from the trail of A. glabripennis virgin and mated females that were not present in trails of mature males. These compounds were identified as 2-methyldocosane and (Z)-9-tricosene (major components), as well as (Z)-9-pentacosene and (Z)-7-pentacosene (minor components); every trail wash sample contained all four chemical components, although the amounts and ratios changed with age of the female. Males responded to the full pheromone blend, regardless of mating status, but virgin females chose the control over the pheromone, suggesting that they may use it as a spacing pheromone to avoid intraspecific competition and maximize resources. Virgin, but not mated, males also chose the major pheromone components in the absence of the minor components, over the control. Taken together, these results indicate that all four chemicals are components of the trail pheromone. The timing of production of the ratios of the pheromone blend components that produced positive responses from males coincided with the timing of sexual maturation of the female.