Central processing of sex pheromone stimuli is differentially regulated by juvenile hormone in a male moth

In the male moth, Agrotis ipsilon, the neuronal basis for juvenile hormone (JH)-linked modulation of sex pheromone responsiveness was investigated following stimulation of the antenna with i) an extract of female pheromone gland, ii) the synthetic pheromone blends from A. ipsilon and a closely related species, A. segetum, and iii) single components of the A. ipsilon blend. Response characteristics of olfactory interneurons were studied in the antennal lobe (AL) at different ages and with manipulated JH levels using intracellular recording techniques. Blend-specific, generalist and component-specific neurons were identified and described according to their response pattern. The proportion of low threshold AL interneurons increased significantly with age for all stimuli tested. Changes were, however, less pronounced for the minor single components. The proportion of low threshold AL interneurons in allatectomized (JH-deprived) mature males was significantly lower for all stimuli than in intact mature males. A large proportion of low threshold AL interneurons responding to the pheromone blends, but not as pronounced for single pheromone components, could be restored/induced by injecting JH either into JH-deprived mature males or into young immature males. The specificity for the species-specific blend compared to the A. segetum blend increased with age and JH injections.     

Is the rapid post-mating inhibition of pheromone response triggered by ecdysteroids or other factors from the sex accessory glands in the male moth Agrotis ipsilon?

In many animals, male copulation is dependent on the detection and processing of female-produced sex pheromones, which is generally followed by a sexual refractory post-ejaculatory interval (PEI). In the male moth, Agrotis ipsilon, this PEI is characterized by a transient post-mating inhibition of behavioral and central nervous responses to sex pheromone, which prevents males from re-mating until they have refilled their reproductive tracts for a potential new ejaculate. However, the timing and possible factors inducing this rapid olfactory switch-off are still unknown. Here, we determined the initial time delay and duration of the PEI. Moreover, we tested the hypothesis that the brain, the testis and/or the sex accessory glands (SAGs) could produce a factor inducing the PEI. Lastly, we investigated the possible involvement of ecdysteroids, hormones essential for development and reproduction in insects, in this olfactory plasticity. Using brain and SAG cross-injections in virgin and newly-mated males, surgical treatments, wind tunnel behavioral experiments and EIA quantifications of ecdysteroids, we show that the PEI starts very shortly after the onset of copulation, and that SAGs contain a factor, which is produced/accumulated after copulation to induce the PEI. Moreover, SAGs were found to be the main source of ecdysteroids, whose concentration decreased after mating, whereas it increased in the haemolymph. 20-Hydroxyecdysone (20E) was identified as the major ecdysteroid in SAGs of A. ipsilon males. Finally, 20E injections did not reduce the behavioral pheromone response of virgin males. Altogether our data indicate that 20E is probably not involved in the PEI.     

Identification of Differentially Expressed Genes in the Pheromone Glands of Mated and Virgin Bombyx mori by Digital Gene Expression Profiling

Background

Mating decreases female receptivity and terminates sex pheromone production in moths. Although significant progress has been made in elucidating the mating-regulated inactivation of pheromone biosynthesis-activating neuropeptide (PBAN) secretion, little is known about the mating induced gene expression profiles in pheromone glands (PGs). In this study, the associated genes involved in Bombyx mori mating were identified through digital gene expression (DGE) profiling and subsequent RNA interference (RNAi) to elucidate the molecular mechanisms underlying the mating-regulated gene expression in PGs.

Results

Eight DGE libraries were constructed from the PGs of mated and virgin females: 1 h mating (M1)/virgin (V1) PGs, 3 h mating (M3)/virgin (V3) PGs, 24 h mating (M24)/virgin (V24) PGs and 48 h mating (M48)/virgin (V48) PGs (M48 and V48). These libraries were used to investigate the gene expression profiles affected by mating. DGE profiling revealed a series of genes showing differential expression in each set of mated and virgin female samples, including immune-associated genes, sex pheromone synthesis-associated genes, juvenile hormone (JH) signal-associated genes, etc. Most interestingly, JH signal was found to be activated by mating. Application of the JH mimics, methoprene to the newly-emerged virgin females leaded to the significant reduction of sex pheromone production. RNAi-mediated knockdown of putative JH receptor gene, Methoprene tolerant 1 (Met1), in female pupa resulted in a significant decrease in sex pheromone production in mature females, suggesting the importance of JH in sex pheromone synthesis.

Conclusion

A series of differentially expressed genes in PGs in response to mating was identified. This study improves our understanding of the role of JH signaling on the mating-elicited termination of sex pheromone production.     

Early manipulation of juvenile hormone has sexually dimorphic effects on mature adult behavior in Drosophila melanogaster

Hormones are critical for the development, maturation, and maintenance of physiological systems; therefore, understanding their involvement during maturation of the brain is important for the elucidation of mechanisms by which adults become behaviorally competent. Changes in exogenous and endogenous factors encountered during sexual maturation can have long lasting effects in mature adults. In this study, we investigated the role of the gonadotropic hormone, juvenile hormone (JH), in the modulation of adult behaviors in Drosophila. Here we utilized methoprene (a synthetic JH analog) and precocene (a JH synthesis inhibitor) to manipulate levels of JH in sexually immature male and female Drosophila with or without decreased synthesis of neuronal dopamine (DA). Locomotion and courtship behavior were assayed once the animals had grown to sexual maturity. The results demonstrate a sexually dimorphic role for JH in the modulation of these centrally controlled behaviors in mature animals that is dependent on the age of the animals assayed, and present DA as a candidate neuronal factor that differentially interacts with JH depending on the sex of the animal. The data also suggest that JH modulates these behaviors through an indirect mechanism. Since gonadotropic hormones and DA interact in mammals to affect brain development and later function, our results suggest that this mechanism for the development of adult behavioral competence may be evolutionarily conserved.     

Pheromonal effects on sexual maturation,yellowing, and the vibration reaction in immature male desert locusts (Schistocerca gregaria)

Both yellow (crowded) and non-yellow (isolated) mature males of Schistocerca gregaria are equally capable of accelerating the sexual maturation and inducing integumental yellowing in immature adult males. The bioassay of a mature male extract shows conclusively the presence of the maturation-accelerating pheromone in this species. The pheromone does not appear to be effective in inducing yellowing, or in initiating the vibration reaction, in immature males.     

Low doses of a neonicotinoid insecticide modify pheromone response thresholds of central but not peripheral olfactory neurons in a pest insect

Insect pest management relies mainly on neurotoxic insecticides, including neonicotinoids, leaving residues in the environment. There is now evidence that low doses of insecticides can have positive effects on pest insects by enhancing various life traits. Because pest insects often rely on sex pheromones for reproduction, and olfactory synaptic transmission is cholinergic, neonicotinoid residues could modify chemical communication. We recently showed that treatments with different sublethal doses of clothianidin could either enhance or decrease behavioural sex pheromone responses in the male moth, Agrotis ipsilon. We investigated now effects of the behaviourally active clothianidin doses on the sensitivity of the peripheral and central olfactory system. We show with extracellular recordings that both tested clothianidin doses do not influence pheromone responses in olfactory receptor neurons. Similarly, in vivo optical imaging does not reveal any changes in glomerular response intensities to the sex pheromone after clothianidin treatments. The sensitivity of intracellularly recorded antennal lobe output neurons, however, is upregulated by a lethal dose 20 times and downregulated by a dose 10 times lower than the lethal dose 0. This correlates with the changes of behavioural responses after clothianidin treatment and suggests the antennal lobe as neural substrate involved in clothianidin-induced behavioural changes.     

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.     

Juvenile hormones: Their role in the regulation of the pheromonal communication system of the armyworm moth,Pseudaletia unipuncta

Recently, much effort has been devoted to the elucidation of the neuro-endocrine mechanisms regulating the biosynthesis and emission of sex pheromones in the Lepidoptera. The available data indicate that the hormonal mechanisms involved vary considerably among species. For example, compelling evidence that juvenile hormones (JH) play a role in the control of sex pheromone production has been presented only for the armyworm moth, Pseudaletia unipuncta. In this species, females that are allatectomized at emergence neither produce nor release pheromone, but both activities are restored following replacement therapy with synthetic JH. However, injection of synthetic JH into neck-ligated females does not induce pheromone biosynthesis, whereas treatment with either a brain homogenate or synthetic PBAN results in a rise in the pheromone titer. These results indicate that the role played by JH is an indirect one and that the tropic factor is a PBAN-like substance. Studies on in vitro JH biosynthesis by isolated corpora allata of P. unipuncta have shown that the low JH output observed early in the life of adult females coincides with the absence of both calling behavior and pheromone production. The subsequent increase in the rates of JH biosynthesis correlates with the onset of pheromone production and release. We have therefore proposed that JH titers must pass a threshold level before the circadian release of PBAN and calling behavior can begin. Furthermore, recent experiments suggest that the continuous presence of JH is necessary for calling behavior to be maintained once initiated. Lastly, we present data suggesting a role for JH or JH acids in the receptivity of P. unipuncta males to the female sex pheromone. © 1994 Wiley-Liss, Inc.     

Juvenile hormone induction of pheromone gland PBAN-responsiveness in Helicoverpa armigera females

The maturation of corpora allata (CA) and the competence of pheromone glands in the adult moth Helicoverpa armigera, are both age-related and appear to be correlated. Sex pheromone glands of pharate adults do not produce sex pheromone independently, nor do they respond to exogenous PBAN. Newly emerged moths produce significantly less pheromone than day one moths. JH (juvenile hormone) II was found to be the main JH form produced by CA in vitro. JH II primed pheromone glands of pharate adults to respond to PBAN. In addition, injection or topical application of JH II to newly-emerged females induced pheromone production in the presence of PBAN. Our findings suggest that JH is involved in the initiation of pheromone production of Helicoverpa armigera.     

铃夜蛾属昆虫性信息素生物合成及内分泌调控

综述了铃夜蛾属Helicoverpa昆虫性信息素生物合成途径及内分泌因子的调控作用 ,包括信息素生物合成激活神经肽 (PBAN)和信息素生物合成抑制肽 (PSP)等的来源、结构和作用机制及一些种中保幼激素 (JH)和章鱼胺 (OA)对性信息素生物合成的作用 ,并展望了未来的研究方向。     

Unexpected Effects of Low Doses of a Neonicotinoid Insecticide on Behavioral Responses to Sex Pheromone in a Pest Insect

In moths, which include many agricultural pest species, males are attracted by female-emitted sex pheromones. Although integrated pest management strategies are increasingly developed, most insect pest treatments rely on widespread use of neurotoxic chemicals, including neonicotinoid insecticides. Residual accumulation of low concentrations of these insecticides in the environment is known to be harmful to beneficial insects such as honey bees. This environmental stress probably acts as an “info-disruptor” by modifying the chemical communication system, and therefore decreases chances of reproduction in target insects that largely rely on olfactory communication. However, low doses of pollutants could on the contrary induce adaptive processes in the olfactory pathway, thus enhancing reproduction. Here we tested the effects of acute oral treatments with different low doses of the neonicotinoid clothianidin on the behavioral responses to sex pheromone in the moth Agrotis ipsilon using wind tunnel experiments. We show that low doses of clothianidin induce a biphasic effect on pheromone-guided behavior. Surprisingly, we found a hormetic-like effect, improving orientation behavior at the LD20 dose corresponding to 10 ng clothianidin. On the contrary, a negative effect, disturbing orientation behavior, was elicited by a treatment with a dose below the LD0 dose corresponding to 0.25 ng clothianidin. No clothianidin effect was observed on behavioral responses to plant odor. Our results indicate that risk assessment has to include unexpected effects of residues on the life history traits of pest insects, which could then lead to their adaptation to environmental stress.     

The circadian rhythm of flight activity of Spodoptera exigua males in response to sex pheromone

In many moths, male attraction to the blend of synthetic sex pheromone releasing continuously in the field shows an apparent circadian rhythm similar to that of locomotion activity. In this study, the daily rhythms of electroantennography (EAG) and behavioral responses to sex pheromone, and the daily rhythms of locomotion activity were measured in male beet armyworm, Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae). The peaks of males trapped by light and sex pheromone were all during the latter part of the night in the field. However, there was no significant variation among time intervals in the EAG responses of male antennae to sex pheromone stimuli. The principal period of locomotion activity under L15:D9 (LD) continued to occur during the scotophase and the subjective scotophase in the day of constant darkness (DD1) and the second of two consecutive days of constant darkness (DD2). The majority of males contacted the sex pheromone source in a wind tunnel during the latter part of the scotophase under LD and the subjective scotophase for DD1 and DD2. There were significant associations between the rhythm of the behavioral response to sex pheromone and locomotion activity. These results suggested that the male's behavioral response to sex pheromone in the beet armyworm could be observed only until locomotion activity of the male occurred at the end of the dark period, despite sex pheromone being released continuously from synthetic pheromone‐baited traps in the field.     

Effects of pheromone concentration and photoperiod on the behavioral response sequence to sex pheromone in the male brown-banded cockroach,Supella longipalpa

The effects of female sex pheromone concentration and time of day on behavioral responses of male brown-banded cockroaches were monitored with a two-choice olfactometer. The duration of each behavior or behavioral event in a deterministic response sequence and the probabilities of behavioral transitions were analyzed. Pheromone concentration had a significant effect on all behaviors in the response sequence. The time of testing, relative to the entrainment photoperiodic regime, significantly affected behavioral events early in the sequence but not later-occuring behaviors. Although the probability of males choosing the pheromone rather than the solvent control was a function of the pheromone concentration, it was independent of the time of testing. By affecting the probability of behavioral transitions, the pheromone concentration and time of testing determined the number of insects exhibiting specific behaviors in the response sequence. The behavioral response sequence can be characterized by probabilities of behavioral transitions along with latencies of early behavioral acts.     

Inhibition of (3H)vitellogenin uptake by isolated amphibian oocytes injected with cytoplasm from progesterone-treated mature oocytes.

Fully grown meiotically immature (germinal vesicle stage) amphibian oocytes incorporate radioactive protein ([³H]vitellogenin) following in vitro culture. In vitro exposure of such oocytes to exogenous progesterone induces germinal vesicle breakdown and inhibits incorporation of vitellogenin. In the present studies, we have investigated the effects of cytoplasm taken from mature and immature oocytes on incorporation of vitellogenin and nuclear breakdown following microinjection of this material into immature oocytes. Vitellogenin incorporation was markedly suppressed in oocytes which underwent nuclear breakdown following injection with cytoplasm from mature oocytes. Incorporation of vitellogenin into oocytes which did not mature after injection with cytoplasm taken from mature oocytes resembled that seen in oocytes injected with immature cytoplasm. The degree of suppression of vitellogenin incorporation following cytoplasmic injections was similar to that seen in uninjected oocytes treated with progesterone. Oocytes injected with cytoplasm obtained from immature oocytes did not undergo either nuclear breakdown or changes in vitellogenin incorporation. The results suggest that cytoplasm obtained from mature oocytes contains a factor(s) which alters directly or indirectly the capacity of the oocyte cell membrane to incorporate vitellogenin. Enucleated immature oocytes also incorporated [³H]vitellogenin, and injection of such oocytes with mature, but not immature, oocyte cytoplasm suppressed vitellogenin incorporation. Suppressive effects of injected cytoplasm thus appear to be mediated through physiological changes in the recipient oocyte cytoplasm rather than the nuclear component.     

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.     

Assortative Mating in Two Pheromone Strains of the Cabbage Looper Moth, Trichoplusia ni

The evolution of animal communication systems is an integral part of speciation. In moths, species specificity of the communication channel is largely a result of unique sex pheromone blends produced by females and corresponding specificity of male behavioral response. Insights into the process of speciation may result from studies of pheromone strains within a species in which reproductive isolation is not complete. Toward this end we investigated assortative mating based on female pheromone phenotypes and male response specificity between mutant and normal colonies of the cabbage looper moth, Trichoplusia ni. There was no evidence of assortative mating in small cages in which the density of moths was high. In larger cages with lower densities of moths, assortative mating was evident. In these larger cages, matings between normal males and normal females and mutant males and mutant females were more frequent than interstrain matings. Wind tunnel tests indicated that normal males responded preferentially to pheromone released by normal females, whereas mutant males did not discriminate between normal and mutant pheromone blends. In large field cages, pheromone traps baited with normal females caught equal numbers of mutant and normal males, while pheromone traps baited with mutant females caught primarily mutant males. The overall pattern of assortative mating could be explained primarily based on the normal males' preference for the pheromone blend released by normal females.     

Agrotis segetum雄蛾触角叶神经元对性信息素时间编码的机制分析

应用压力注射,在Agrotis segetum雄蛾触角叶（AL）中33个对性信息素有反应的MGC神经元上探计了对性信息素反应模式的形成机制,压力注射100mmol/L GABA进入AL神经网引起神经元一个慢的超极化电位,并有一个长时程的放电抑制相,与用性信息素刺激诱导的神经元分应很相似,但GABA并不影响神经元对性信息素刺激的去极化反应,低Cl^-溶液可减弱AL神经元对性信息素刺激的超极化反应,甚至使超极化相逆转为兴奋反应,抑制相消失。压力注射Bicuculline使神经元放电频率增加。压力注射Bicuculline的同时给予性信息素刺激,可使性信息素刺激所致的神经元放电增加进一步加强;Bicuculline可使性信息素刺激引起的神经元超极化幅度变小,放电抑制时间变短,甚至其抑制相完全被逆转为正常放电,无超极化反应和抑制相存在,结果表明,AL神经元对性信息系反应的超极化相与GABA受体有关。     

Endocrine manipulations and ontogenesis of male sexual behaviour in Locusta: studies on precocene-induced early adultiforms

ABSTRACT. Fourth- and third-instar permanent adultiforms of Locusta migratoria migratorioides (R.&F.) were produced by applying 7-ethoxy-precocene II (precocene III) to hoppers in earlier stages. The mating behaviour of male adultiforms and the effects of injected exogenous juvenile hormone III (JH) on this behaviour were investigated quantitatively and the results were compared to those obtained for normal adult males and for adult males chemically allatectomized by precocene III. Fourth- and third-instar adultiform males exhibited sexual behaviour and injections of JH intensified this behaviour in a dose-dependent manner, though the number of repeated injections in which a cumulative dose is administered was found to be not less important than the amount of the cumulative dose itself. Huge repeated doses (6×144=864 μg) of JH temporarily induced normal intensity of mating behaviour in fourth-instar male adultiforms. The same doses of JH intensified mating behaviour also in third-instar adultiform males, but less markedly so than in fourth-instar ones. For restoring temporarily normal intensity of mating behaviour in chemically allatectomized but morphogenetically normal adult males, much smaller doses (2×36=72, or 2×72=144μg) of JH were sufficient. In contrast, even huge doses of JH were unable to induce mating behaviour in normal (no precocene-treated) male hoppers. Thus, the ethological ontogenesis (='ethogenesis') of the male's mating behaviour, including the response of the system to JH, is clearly accelerated by precocious metamorphosis. However, this is not a simple 'all or none' effect, because adultiforms in earlier stadia exhibit less intense male mating behaviour and more limited response to JH than adultiforms in later stadia or adults.