Neural activation of the sex-pheromone gland in the moth Manduca sexta: real-time measurement of pheromone release

Abstract. Investigations of various species of moths have suggested that the biosynthesis of sex pheromone in the abdominal pheromone glands of females may be at least partly regulated by neuroendocrine mechanisms. Few studies, however, have explored the mechanisms underlying the release of sex pheromone. In experiments on the sphinx moth Manduca sexta (L.) (Lepidoptera: Sphingidae), we have monitored the time course of sex-pheromone release in scotophase females with the aid of an electroantennogram bioassay based on the highly sensitive and selective sex-pheromone receptor neurones of the male antenna. Pheromone release was evoked by orthodromic stimulation of the ventral nerve cord. Neurally stimulated release occurred with a subsecond latency and did not depend on bioactive factors in the haemolymph or on movement of the abdomen or the ovipositor. Severing the most medial pair of nerves posterior to the terminal abdominal ganglion (the terminal nerves) eliminated pheromone release, but not abdominal contractions. Release was also inhibited reversibly if the descending Ca²⁺-dependent synaptic input to the terminal ganglion was blocked by exposure to elevated concentrations of Mg²⁺. These findings indicate that the release of sex pheromone from the pheromone gland in female M. sexta is a true neuroeffector response and that the gland appears to be controlled by neurones that project to it from the terminal abdominal ganglion.     

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。     

Regulation of sex pheromone biosynthesis in two noctuid species,S. littoralis and M. brassicae,may involve both PBAN and the ventral nerve cord

In order to understand better the mechanism of regulation of pheromone production in moth species, we performed ELISA analyses to detect and follow pheromone biosynthesis activating neuropeptide-like immunoreactivity (PBAN-IR) in different tissues of the two noctuidae species, Spodoptera littoralis and Mamestra brassicae. Male S. littoralis and both male and female M. brassicae brain-subesophageal ganglion (Br-SEG), corpora cardiaca-corpora allata complex, and terminal abdominal ganglion extracts showed the presence of PBAN-IR during both the photophase and the scotophase. However, PBAN-IR was found only in scotophase in female hemolymph. Analysis of extracts of Br-SEG, terminal abdominal ganglion, and hemolymph after HPLC fractionation showed that the most immunoreactive fraction in all the extracts exhibited the same retention time as Hez-PBAN, suggesting that similar PBAN-like material is present in all these tissues. In vivo studies demonstrated that severing the ventral nerve cord in M. brassicae anterior to the terminal abdominal ganglion impaired normal sex pheromone production by third-scotophase females, as was previously shown in S. littoralis. Additionally, PBAN-IR levels were lower in hemolymph samples obtained at the peak of pheromone production in both S. littoralis and M. brassicae females that had the ventral nerve cord severed compared with sham operated animals. These results, along with earlier reported data, indicate that control of pheromone production in both species may involve both PBAN (or PBAN-like peptides) and the ventral nerve cord and support the hypothesis that a neural input from the ventral nerve cord triggers the release of the pheromonotropic peptide(s) into the hemolymph, which then acts directly on the pheromone gland to stimulate pheromone biosynthesis. Arch. Insect Biochem. Physiol. 37:295–304, 1998. © 1998 Wiley-Liss, Inc.

1 We thank Germán Lázaro for insect rearing.

     

Evidence for both humoral and neural regulation of sex pheromone biosynthesis in Spodoptera littoralis

Selected tissues presumably involved in the control of sex pheromone production were analyzed by ELISA for the presence of PBAN-like immunoreactivity (PBAN-IR) in Spodoptera littoralis. The temporal distribution pattern of PBAN-IR in the hemolymph is similar to that of pheromone production in the gland. On the other hand, analysis of the retrocerebral complex, brain-subesophageal ganglion complex, and terminal abdominal ganglion (TAG) revealed similar PBAN-IR levels in both photophase and scotophase periods. Pheromonotropic activity exhibited by both hemolymph and TAG, as determined by a modified in vitro bioassay, agrees with the results of the immunochemical analyses. Severing the ventral nerve cord anterior to the TAG impaired normal sex pheromone production by second-scotophase females. These results are discussed in the context of how sex pheromone biosynthesis is regulated by PBAN in S. littoralis. © 1996 Wiley-Liss, Inc.     

Neurological influences on pheromone release and calling behaviour in the gypsy moth, Lymantria dispar

ABSTRACT. Surgical removal of the brain or disconnection of the last abdominal ganglion from the ventral nerve cord prevented sex pheromone release in female Lymantria dispar (L.) (Lymantriidae), as assayed by the male wing-fanning response. The calling behaviour continued to occur in individuals whose terminal abdominal ganglion had been thus isolated, however, indicating that the neural mechanisms controlling calling function independently in the last abdominal ganglion.     

Spatial and temporal distribution of pheromone biosynthesis-activating neuropeptide in Helicoverpa (Heliothis) armigera using RIA and in vitro bioassay.

A [3H]-PBAN (pheromone biosynthesis-activating neuropeptide) analog was synthesized, and binding of the radioligand to a specific PBAN-antiserum was achieved. The inhibition of binding of the radioligand by unlabeled PBAN, several PBAN analogs, and other competitors was studied and a specific radio-immunoassay was developed. Using this radioimmunoassay we found PBAN-like immunoreactivity in methanol extracts of hemolymph and neural tissues from females. Higher levels of PBAN-like immunoreactivity in extracts of brain-suboesophageal ganglion complexes, corpora cardiaca, thoracic ganglia, and abdominal ganglia were observed during the 4-5th h scotophase when compared to the PBAN-like immunoactivity levels during the 6-11th h photophase. On the other hand, the concentrations of PBAN-like immunoreactivity, in the terminal abdominal ganglion were higher during the photophase relative to minimal levels observed during the scotophase, indicating an accumulation before the onset of pheromone production. These differences in concentrations of PBAN were also reflected in the stimulation of in vitro pheromone glands, whereby significant stimulations were obtained by scotophase and photophase brain extracts, scotophase thoracic ganglia extracts, and photophase terminal abdominal ganglia extracts. No detectable levels of PBAN were found in hemolymph extracts during the sampling periods.     

Release mechanism of sex pheromone in the female gypsy moth <Emphasis Type="Italic">Lymantria dispar</Emphasis>: a morpho-functional approach

A morpho-functional investigation of the sex pheromone-producing area was correlated with the pheromone release mechanism in the female gypsy moth Lymantria dispar. As assessed by male electroantennograms (EAG) and morphological observations, the pheromone gland consists of a single-layered epithelium both in the dorsal and ventral halves of the intersegmental membrane between the 8th and 9th abdominal segments. By using the male EAG as a biosensor of real-time release of sex pheromone from whole calling females, we found this process time coupled with extension movements of the ovipositor. Nevertheless, in females in which normal calling behavior was prevented, pheromone release was detected neither in absence nor in presence of electrical stimulation of the ventral nerve cord/terminal abdominal ganglion (TAG) complex. Tetramethylrhodamine-conjugated dextran amine stainings also confirm the lack of any innervation of the gland from nerves IV to VI emerging from the TAG. These findings indicate that the release of sex pheromone from the glands in female gypsy moths is independent of any neural control exerted by the TAG on the glands, at least by way of its three most caudally located pairs of nerves, and appears as a consequence of a squeezing mechanism in the pheromone-producing area.     

Diel changes in the presence and physiological actions of octopamine in the female sex-pheromone glands of heliothine moths

Recent evidence suggests that the biogenic monoamine octopamine (OA) may be involved in the regulation of female sex-pheromone production in Lepidoptera. A radioenzymatic assay coupled with high performance liquid chromatography revealed the presence of OA in the innervated sex-pheromone gland of the corn earworm moth Helicoverpa (Heliothis) zea. Significantly more OA was found in glands just before the onset of scotophase (ca 320 fmol/gland), compared to levels at mid-photophase or just after the onset of scotophase (ca 160 fmol/gland).

Exogenous OA had several actions on pheromone production. H. zea virgin females normally do not produce pheromone during the photophase, but highly significant levels of pheromone were induced by injection of OA into intact, day-2 photophase females. Importantly, this effect was absent in older females that showed increased levels of flight and oviposition activity. A second action of OA was revealed in isolated abdomen preparations from day-2 H. virescens females. Exogenous OA stimulated highly significant increases in pheromone production if abdomens were treated at the onset of scotophase, but not if they were treated in photophase. This critical period for OA action in these reduced preparations coincided with the time when peak levels of OA were present in the pheromone gland tissue. OA is therefore sufficient to induce pheromone production, but its actions in these short-lived insects depend on factor such as age and photoperiod. Diel fluctuations in OA levels in the pheromone gland, together with the observed phermonotropic actions of this amine, support the hypothesis that OA is involved in the regulation of pheromone production in these insects.     

Physiological control of pheromone production in Choristoneura fumiferana and C. rosaceana

The diel periodicity of calling behavior and pheromone production are synchronous in virgin females of both Choristoneura fumiferana and C. rosaceana (Lepidoptera: Tortricidae). Newly emerged females decapitated prior to scotophase produced no or very little pheromone 24 h later. However, injection of PBAN or Br-SEG homogenates, obtained from donors of the same or the other species, stimulated pheromone production to normal levels. Transection of the ventral nerve cord (VNC) or extirpation of the terminal abdominal ganglion (TAG) did not affect pheromone production in control females. Similarly, injections of PBAN or Br-SEG homogenates into decapitated females reactivated pheromone production to normal levels, whether or not the VNC was intact or the TAG present. Furthermore, octopamine was not effective in stimulating pheromone production in decapitated females. Taken together, these results indicate that the regulation of pheromone production is not neurally mediated in either Choristoneura species. However, there was no evidence that hemolymph collected from pheromone-producing females contained pheromonotropic activity. Similarly, isolated glands incubated with PBAN did not produce pheromone. The presence of the bursa copulatrix was required to produce pheromone in both tortricids as production was not restored in decapitated bursa-less females injected with PBAN or a Br-SEG homogenate. However, an extract of the bursa copulatrix did not elicit pheromonotropic activity in decapitated females or incubated glands of either species. The bursa copulatrix is only involved in pheromone production of some species of tortricids but our results do not support the current explanation for such interspecific differences. We postulate that the relative importance of a bursa factor may be related to the evolution of different desaturation systems used for pheromone biosynthesis in the Tortricidae. Arch.     

Sex pheromone biosynthesis,storage and release in a female moth: making a little go a long way

Moth pheromone research has pioneered much of our understanding of long-distance chemical communication. Two important characteristics of this communication have, however, remained largely unaddressed: the release of small quantities of pheromone by most moth species, despite potential advantages of releasing greater amounts, and the intermittency of release in some species, limiting the time of mate attraction. We addressed the proximate mechanisms underlying these characteristics by manipulating biosynthesis, storage and release of pheromone in females of the noctuid moth Chloridea virescens. We found that (i) mass release is determined by pheromone mass on the gland surface; (ii) amounts synthesized are limited by pheromone biosynthesis activating neuropeptide concentration, not precursor availability; (iii) some gland structural feature limits mass release rate; (iv) intermittent calling enables release at a mass rate greater than biosynthetic rate; and (v) at typical mass release rates, the periodicity of pheromone availability on the gland surface roughly matches the periodicity (intermittency) of calling. We conclude that mass release in C. virescens and possibly many other species is low because of constraints on biosynthesis, storage and gland structure. Further, it appears the behaviour of intermittent calling in C. virescens may have evolved as a co-adaptation with pheromone availability, allowing females to release pheromone intermittently at higher mass rates than the biosynthesis rate.     

Termination of sex pheromone production in mated females of the silkworm moth

A mating duration of more than 6 h was necessary to permanently terminate the production of the sex pheromone (bombykol) in the silkworm moth, Bombyx mori L. (Lepidoptera: Bombycidae), although the female formed a bursa copulatrix including a spermatophore and laid fertilized eggs even after mating for only 0.5 h. The 6-h mated female again produced bombykol if given an injection of synthetic pheromonotropic neuropeptide (PBAN), which is known to activate pheromone biosynthesis in a virgin female. Extracts of brain-suboesophageal ganglion (SG) complexes, which were removed from 6- and 24-h mated females, showed strong pheromonotropic activities. These results indicated that the pheromone gland of the mated female maintained its ability to biosynthesize bombykol; however, it could not produce pheromone due to a suppression of PBAN secretion from the SG. Furthermore, bombykol titers did not decrease after mating in females with a transected ventral nerve cord, even after the injection of a spermatophore extract, suggesting that the suppression of PBAN secretion was mediated by a neural signal and not by a substance in the spermatophore. The mated females accumulated (10E, 12Z)-10,12-hexadecadienoic acid, a precursor of bombykol biosynthesis, in their pheromone glands as did decapitated females. © 1996 Wiley-Liss, Inc.     

Pheromonotropic and pheromonostatic activity in moths

Pheromone biosynthesis in many species of moths requires a pheromonotropic neurosecretion, the pheromone biosynthesis activating neuropeptide (PBAN), from the brain-subesophageal ganglion-corpora cardiaca complex. Some investigators suggest that PBAN is released into the hemolymph and acts directly on sex pheromone glands (SPG) via a Ca⁺⁺/calmodulin-dependent adenylate cyclase. Others suggest, however, that PBAN acts via octopamine that is released by nerves from the terminal abdominal ganglion innervating the SPG. These findings suggest that there are controversies on the mode of action of PBAN and other pheromonotropic factors, sometimes even within the same species. Mating in many insects results in temporary or permanent suppression of pheromone production and/or receptivity. Such a suppression may result from physical blockage of the gonopore or deposition of pheromonostatic factor(s) by the male during copulation that result in suppressed pheromone production and/or receptivity in females either directly or by a primer effect. In several species of insects, including moths, a pheromonostatic factor is transferred in the seminal fluid of males. Similar to the controversies associated with the pheromonotropic activity of PBAN, sometimes even within the same species, there appear to be controversies in pheromonostasis in heliothines as well. This paper reviews these conflicting findings and presents some data on pheromonostatic and pheromonotropic activity in Heliothis virescens that support and conflict with current information, raising further questions. Answers to some of the questions are partly available; however, they remain to be answered unequivocally. © 1994 Wiley-Liss, Inc.     

The neurophysiology of larval firefly luminescence: Direct activation through four bifurcating (DUM) neurons

Summary The paired lanterns of the larval fireflyPhoturis versicolor are bilaterally innervated by four dorsal unpaired median (DUM) neurons the somata of which are found in the terminal abdominal ganglion (A8) and which stain with Neutral Red (Fig. 1A). Both intra- and extracellularly recorded activity in these neurons is always associated with a bilateral glow response, or BGR (Figs. 3 and 4). Luminescence cannot be initiated or maintained in the absence of DUM neuron excitation. Furthermore, there is a linear causative relationship between the frequency of DUM neuron activity and the amplitude of the resultant BGR (Figs. 6 and 7).Due to the intrinsic bilateral morphology, firefly DUM neurons may be antidromically activated through either lantern nerve, resulting in the initiation of luminescence in the contralateral lantern (Figs. 8 and 9). This activation is unaffected by high Mg⁺⁺ saline indicating that the DUM neurons provide a direct pathway for conduction through the ganglion (Fig. 9). The DUM neurons receive synaptic input from axons descending through both anterior connectives, however, stimulation of only one connective results in a BGR since excitation is carried to both sides of the periphery through the bilateral axons.Firefly DUM neurons exhibit physiological qualities typical of neurosecretory cells: spikes are characterized by a slow time course and a long and deep afterhyperpolarization (Fig. 10). This is consistent with the observation that spontaneous firing rates are usually below 3 Hz, but nevertheless elicit a strong BGR (Figs. 3 and 5). The physiological evidence presented in this study correlates well with the morphological, pharmacological and biochemical evidence compiled from previous studies, which indicates that the four DUM neurons represent the sole photomotor output from the central nervous system to the larval lanterns. Evidence is discussed which indicates that these effects are mediated throught the release of octopamine, long presumed to be the lantern neurotransmitter. These results, therefore, describe a novel and unexpected role for DUM neurons in regulating an unusual invertebrate effector tissue and further expands the growing list of functions for octopamine in neural control mechanisms.Abbreviations A1-A7 first through seventh abdominal ganglia - A8 terminal abdominal ganglion - DUM dorsal unpaired median - BGR bilateral glow response     

Correlation between glycerolipids and pheromone aldehydes in the sex pheromone gland of female tobacco hornworm moths,Manduca sexta (L.)

Analysis by TLC and HPLC revealed that the triacylglycerols comprise the most abundant lipid class in the sex pheromone glands of Manduca sexta females. Also, conjugated olefinic acyl analogs of the major pheromone aldehydes occur principally in the triacylglycerols. The amount of triacylglycerols with conjugated diene acyl moieties significantly decreased when the period of pheromone production was extended by 7 h beyond the normal period of pheromone production by 3 injections of pheromone biosynthesis activating neuropeptide (PBAN) at 3 h intervals. This decrease indicates that the triacylglycerols stored in the gland may serve as major sources of pheromone precursors in the biosynthesis of the sex pheromone aldehydes. Furthermore, analysis of pheromone aldehydes and triacylglycerols in the gland from moths treated with PBAN showed that the proportions of the triacylglycerols with conjugated diene moieties were closely correlated with the proportions of aldehydes found in the same gland. This correlation suggests that the proportions of fatty acids bound to certain triacylglycerols regulates the proportions of aldehydes in biosynthesis of the pheromone blend in M. sexta. © 1995 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Ant Trail Pheromone Biosynthesis Is Triggered by a Neuropeptide Hormone

Our understanding of insect chemical communication including pheromone identification, synthesis, and their role in behavior has advanced tremendously over the last half-century. However, endocrine regulation of pheromone biosynthesis has progressed slowly due to the complexity of direct and/or indirect hormonal activation of the biosynthetic cascades resulting in insect pheromones. Over 20 years ago, a neurohormone, pheromone biosynthesis activating neuropeptide (PBAN) was identified that stimulated sex pheromone biosynthesis in a lepidopteran moth. Since then, the physiological role, target site, and signal transduction of PBAN has become well understood for sex pheromone biosynthesis in moths. Despite that PBAN-like peptides (∼200) have been identified from various insect Orders, their role in pheromone regulation had not expanded to the other insect groups except for Lepidoptera. Here, we report that trail pheromone biosynthesis in the Dufour''s gland (DG) of the fire ant, Solenopsis invicta, is regulated by PBAN. RNAi knock down of PBAN gene (in subesophageal ganglia) or PBAN receptor gene (in DG) expression inhibited trail pheromone biosynthesis. Reduced trail pheromone was documented analytically and through a behavioral bioassay. Extension of PBAN''s role in pheromone biosynthesis to a new target insect, mode of action, and behavioral function will renew research efforts on the involvement of PBAN in pheromone biosynthesis in Insecta.     

Mechanisms of sperm release from the receptaculum seminis of Schistocerca vaga Scudder (Orthoptera: Acrididae)

Release of spermatozoa from the receptaculum seminis of Schistocerca vaga was studied by means of electrical and mechanical stimulation. Electrical stimulation of the receptaculum nerve, or the ductus aperture nerve, leads to release of spermatozoa from the receptaculum seminis, provided the spermathecal innervation is intact. Mechanical stimulation of the ductus aperture in the genital chamber also leads to sperm release, provided the neural loop, ductus aperture/terminal abdominal ganglion/receptaculum seminis, has not been interrupted at any point. Ten somata in the terminal abdominal ganglion, including 6 dorsal unpaired medial (DUM) neurons, innervate the receptaculum seminis; some of these somata may be neurosecretory. Approximately 80 presumed sensory axons run from the ductus aperture to the same ganglion. On the basis of these neuroanatomical data and the results of electrical and mechanical stimulation, a schema of how the release mechanisms operate in S. vaga is proposed.     

Effect of tyramine and stress on sex-pheromone production in the pre- and post-mating silkworm moth, Bombyx mori

Tyramine (TA) increased significantly after mating, whereas there were no significant differences in octopamine (OA) and dopamine (DA) levels in the brain-suboesophageal ganglion (SOG) complexes between virgin and mated females. The effects of various biogenic amines were tested on pheromone production of virgin and mated females of the silkworm moth, Bombyx mori. After 8h a significant reduction by TA (46%) was observed. Meanwhile, when OA or DA was injected, a significant increase of pheromone titer was observed in both virgin and mated females. This study also presents evidence for an increase in levels of OA and DA in the brain-SOG complexes in response to mechanical stress in B. mori female. TA suppressed pheromone production in an in vitro pheromone gland (PG) homogenate preparation, thus suggesting that the target of TA is the PG. TA inhibited pheromone production in vitro in a dose-dependent manner and DA had a lower inhibitory activity than TA, whereas OA had no effect, suggesting that TA is a candidate for regulating pheromone production in the PG, although other factors could be responsible for the pheromonostatic function.     

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.     

The distribution of pheromone-biosynthesis-activating neuropeptide (PBAN) immunoreactivity in the central nervous system of the corn earworm moth,Helicoverpa zea

Summary Production of sex pheromone in several species of moths has been shown to be under the control of a neuropeptide termed pheromone-biosynthesis-activating neuropeptide (PBAN). We have produced an antiserum to PBAN from Helicoverpa zea (Lepidoptera: Noctuidae) and used it to investigate the distribution of immunoreactive peptide in the brain-suboesophageal ganglion complex and its associated neurohemal structures, and the segmental ganglia of the ventral nerve cord. Immunocytochemical methods reveal three clusters of cells along the ventral midline in the suboesophageal ganglion (SOG), one cluster each in the presumptive mandibular (4 cells), maxillary (12–14 cells), and labial neuromeres (4 cells). The proximal neurites of these cells are similar in their dorsal and lateral patterns of projection, indicating a serial homology among the three clusters. Members of the mandibular and maxillary clusters have axons projecting into the maxillary nerve, while two additional pairs of axons from the maxillary cluster project into the ventral nerve cord. Members of the labial cluster project to the retrocerebral complex (corpora cardiaca and cephalic aorta) via the nervus corpus cardiaci III (NCC III). The axons projecting into the ventral nerve cord appear to arborize principally in the dorsolateral region of each segmental ganglion; the terminal abdominal ganglion is distinct in containing an additional ventromedial arborization in the posterior third of the ganglion. Quantification of the extractable immunoreactive peptide in the retrocerebral complex by ELISA indicates that PBAN is gradually depleted during the scotophase, then restored to maximal levels in the photophase. Taken together, our findings provide anatomical evidence for both neurohormonal release of PBAN as well as axonal transport via the ventral nerve cord to release sites within the segmental ganglia.Abbreviations A aorta - Br-SOG brain-suboesophageal ganglion complex - CC corpus cardiacum - PBS phosphate-buffered saline - PLI PBAN-like immunoreactivity - TAG terminal abdominal ganglion - VNC ventral nerve cord