Molecular characterization of pheromone biosynthesis activating neuropeptide from the diamondback moth, Plutella xylostella (L.)

Pheromone biosynthesis activating neuropeptide (PBAN) produced in the subesophageal ganglion stimulates pheromone production in the pheromone gland. A cDNA isolated from female adult heads of the diamondback moth (Plutella xylostella (L.)) encodes 193 amino acids including PBAN, designated as Plx-PBAN, and four other neuropeptides (NPs): diapause hormone (DH) homologue, -NP, β-NP and γ-NP. All of the peptides are amidated in their C-termini and shared a conserved motif, FXPR(or K)L structure, as reported from other PBAN cDNAs. Plx-PBAN consists of 30 amino acids, the shortest PBAN so far reported. Plx-PBAN exhibited below 50% homology, compared with other known PBANs. The Plx-DH homologue is structurally different from DH of Bombyx mori. The length of Plx-β-NP (16 amino acids) was the shortest and showed relatively low similarity, whereas γ-NP (10 amino acids in length) was the longest among examined γ-NPs. When female adults were injected with synthetic Plx-PBAN, pheromone production showed a maximal increase 1 h post-injection. RT-PCR screening revealed that Plx-PBAN cDNA was expressed in all examined body parts, with the highest expression level in the head of female adults. Analysis of RT-PCR products indicated the Plx-PBAN sequence was identical in all examined body parts of both sexes. Phylogenetic analysis revealed that the Plx-PBAN gene is distantly related to other PBANs, demonstrated by the relatively low similarity.     

Structure and activity of Bombyx PBAN

Two structurally related molecular species of pheromone biosynthesis activating neuropeptides (PBANs), PBAN-I and -II, were isolated from adult heads of the silkworm, Bombyx mori, and characterized. PBAN-I is a carboxyl-terminally amidated 33-residue peptide. Structure-activity relationship studies revealed that 1) its carboxyl-terminal pentapeptide is the smallest size showing activity, 2) the carboxyl-terminal amide is indispensable for activity, and 3) oxidation of three Met residues in PBAN-I to Met(O) (methionine sulfoxide) caused marked enhancement of activity, and the three Met(O) residues contribute equally to the enhancement of activity. Molecular design of PBAN analogs using a carboxyl-terminal hexapeptide showed that modification of the amino-terminal amino group brought about a dramatic increase in activity. This increase was presumed to be mainly due to the increased stability in hemolymph. PBANs share the common carboxyl-terminal sequence, -Phe-Xaa-Pro-Arg-Leu-NH₂, with myotropic peptides isolated from locust and cockroach. Examination of cross-activity of these two groups of peptides revealed that PBAN and its analogs exhibited myotropic activity comparable to myotropic peptides, while myotropic peptides showed extremely high pheromonotropic activity. In B. mori, PBAN activates sex pheromone (bombykol) production presumably by promoting the reduction reaction from acyl to alcohol, which is the last step in the biosynthesis of bombykol. © 1994 Wiley-Liss, Inc.     

cDNA CLONING AND SEQUENCE DETERMINATION OF THE PHEROMONE BIOSYNTHESIS ACTIVATING NEUROPEPTIDE FROM THE SEABUCKTHORN CARPENTERWORM,Holcocerus hippophaecolus (LEPIDOPTERA: COSSIDAE)

The PBAN (pheromone biosynthesis activating neuropeptide)/pyrokinin peptides comprise a major neuropeptide family characterized by a common FXPRL amide at the C‐terminus. These peptides are actively involved in many essential endocrine functions. For the first time, we reported the cDNA cloning and sequence determination of the PBAN from the seabuckthorn carpenterworm, Holcocerus hippophaecolus, by using rapid amplification of cDNA ends. The full‐length cDNA of Hh‐DH‐PBAN contained five peptides: diapause hormone (DH) homolog, α‐neuropeptide (NP), β‐NP, PBAN, and γ‐NP. All of the peptides were amidated at their C‐terminus and shared a conserved motif, FXPR (or K) L. Moreover, Hh‐DH‐PBAN had high homology to the other members of the PBAN peptide family: 56% with Manduca sexta, 66% with Bombyx mori, 77% with Helicoverpa zea, and 47% with Plutella xylostella. Phylogenetic analysis revealed that Hh‐DH‐PBAN was closely related to PBANs from Noctuidae, demonstrated by the relatively higher similarity compared with H. zea. In addition, real‐time quantitative PCR (qRT‐PCR) analysis showed that Hh‐DH‐PBAN mRNA expression peaked in the brain–subesophageal ganglion (Br–SOG) complex, and was also detected at high levels during larval and adult stages. The expression decreased significantly after pupation. These results provided information concerning molecular structure characteristics of Hh‐DH‐PBAN, whose expression profile suggested that the Hh‐DH‐PBAN gene might be correlated with larval development and sex pheromone biosynthesis in females of the H. hippophaecolus.     

Endogenous control of circadian rhythms of pheromone production in the turnip moth, Agrotis segetum

The circadian variation of pheromone production in the turnip moth, Agrotis segetum, was characterized by quantifying (Z)-7-dodecenyl acetate (Z7-12:OAc), the most abundant pheromone component produced by female turnip moth, at different times of day. Under 17:7 h light-dark cycle (LD), the peak of Z7-12:OAc production occurred around 4 h into the scotophase, while there was very little pheromone production during the photophase. When females were maintained under constant darkness (DD), the periodicity of pheromone production was sustained for 3 consecutive days. Furthermore, the rhythm in pheromone production could be entrained to a shifted LD. These results demonstrate that the pheromone production in the turnip moth is regulated endogenously by a circadian clock. To understand how the circadian rhythm of pheromone production is generated, circadian variation of pheromone- biosynthesis-activating neuropeptide (PBAN)-like activity in the brain-suboesophageal ganglion complexes (Br-SOG), hemolymph, and ventral nerve cord (VNC) was also examined. Under both LD and DD, only the VNC displayed a circadian variation in the PBAN-like activity, which was significantly higher during the late-photophase than that in the scotophase. In addition, the present study showed that removal of VNC in isolated abdomen did not affect PBAN stimulation of pheromone production, while severing the VNC impaired normal pheromone production. The role of Br-SOG, VNC, and hemolymph in the regulation of the periodicity of pheromone production is discussed.     

cDNA cloning and sequence determination of the pheromone biosynthesis activating neuropeptide of the silkworm, Bombyx mori.

We have identified the cDNAs encoding pheromone biosynthesis activating neuropeptide (PBAN) using PCR technique. The nucleotide sequence showed that the PBAN gene encodes, besides PBAN, diapause hormone and three putative amidated peptides. These four peptides share with PBAN the C-terminal pentapeptide amide which is corresponding to the shortest fragment with pheromonotropic activity. The organization of the PBAN gene is characteristic of several short neuropeptides and has some degree of similarity to that of the gene for the insect neuropeptide FMRFamide. Thus, the PBAN gene products construct a family of structurally related peptides and have various biological functions.     

Identification of a new member of PBAN family and immunoreactivity in the central nervous system from Adoxophyes sp. (Lepidoptera: Tortricidae)

Production of sex pheromones, Z9-14:OAc and Z11-14:OAc, of the smaller tea tortrix, Adoxophyes sp. was stimulated by injection of the female or male head extracts as well as synthetic pheromone biosynthesis activating neuropeptide (PBAN) into decapitated females. The amount of pheromone produced reached a maximum level 3 h after injection of synthetic PBAN into females. A cDNA isolated from brain-suboesophageal ganglion complex (Br-SEG) of A. sp. females contained an ORF of 576 nucleotides encoding 192 amino acids. Based on endoproteolytic sites, it can be predicted to be cleaved into five putative peptide domains including PBAN and four other neuropeptides. Ado-PBAN consisting of 31-amino acids is the shortest PBAN so far reported. Four other putative PBAN-encoding gene neuropeptides (PGN) are predicted with PGN-24, PGN-7, PGN-20, and PGN-8 amino acids. All of the peptides are amidated in their C-termini with a FXPR(or I, K)L structure, except for PGN-8 (TVKLTPRLamide). PBAN-like immunoreactive material was observed in Br, SEG and ventral nerve cord (VNC) of the female adult. In the brain, 5-7 pairs of neurons containing PBAN-like immunoreactivity were found in each protocerebral hemisphere. Three groups of cell clusters found in the SEG corresponded to the mandibular, maxillary and labial neurons as in other moths. PBAN-like immunoreactive neurons in the VNC were found in thoracic (three pairs) and abdominal ganglia (two pairs). As compared to other moths, a relatively low similarity of peptide sequences deduced from Ado-PBAN gene and a different expression pattern of PBAN-like immunoreactivity could indicate phylogenetical distance from the other species.     

Regulation of sex pheromone biosynthesis in the oriental tobacco budworm,Helicoverpa assulta (Lepidoptera: Noctuidae)

The five components, Z9-16:Ald, 16:Ald, Z11-16:Ald, Z9-16:Ac and Z11-16:Ac, of the sex pheromone in Helicoverpa assulta were mostly detected during the scotophase, with their titer peaking at the 4th hour during the scotophase under a 15L/9D regime. They were not detected during the photophase, but were produced during the photophase when decapitated females were injected with extracts of virgin female (FHE), male heads (MHE), homogenates of the brain-suboesophageal ganglion complex (Br-SOG), or synthetic Hez-PBAN. Production of Z9-16:Ald increased during the first 45min after FHE injection and then declined to a very low level after 2h during the photophase. Synthetic Hez-PBAN stimulated the sex pheromone glands for at least 2h and the effect was more or less proportional to the concentration of the peptide. From the present results, we suggest the following: PBAN is released continuously into the haemolymph to stimulate pheromone biosynthesis at least during the first half of the scotophase, PBAN is synthesized and accumulated independent of photoperiod or sex, and the release starts just prior (about 1h) to the beginning of the scotophase.     

Identification and Determination of the Partial cDNA Encoding the Pheromone Biosynthesis Activating Neuropeptide in Helicoverpa armigera

Sex pheromone biosynthesis is induced in many moths by a neuropeptide, PBAN, consisting of 33-amino acid amidated at the C-terminus. The present study is concerned with cloning and characterizing the partial sequence of Hea-PBAN cDNA which is isolated from the brain and suboesophageal ganglion complex (Br-Sg) of Helicoverpa armigera adults. From the cDNA sequence, it can be predicted that the cDNA has a PBAN domain with 33 amino acids, LSDDMPATPADQEMYRQDPEQIDSRTKYFSPRL, with FSPRL amidated at the C-terminus. The amino acid sequence of predicted peptides including the PBAN, is identical to that of H. zea and H. assulta.     

Identification of a unique pheromonotropic neuropeptide including double FXPRL motifs from a geometrid species, Ascotis selenaria cretacea, which produces an epoxyalkenyl sex pheromone

Virgin females of the Japanese giant looper (Ascotis selenaria cretacea, Assc) in the family of Geometridae secrete an epoxyalkenyl sex pheromone to attract males. To regulate its biosynthesis in the pheromone gland, Assc females produce a pheromone biosynthesis-activating neuropeptide (PBAN) in the suboesophageal ganglion (SG), as do females in many lepidopteran species. We have isolated Assc-PBAN cDNA, which encodes 181 amino acids, including a PBAN homologue and four other putative peptides: a diapause hormone (DH) homologue, alpha-SG neuropeptide (SGNP), beta-SGNP, and gamma-SGNP, all of which shared an FXPR(K)L motif on their C-termini. Although PBANs with 30-35 amino acids have been characterized from 15 other species, the Assc-PBAN homologue consisted of 28 amino acids and showed low homology (<46%) compared with the others. Assc-beta-SGNP with eight amino acids was also shorter than the other beta-SGNPs (16-22 amino acids). Furthermore, all of the known PBAN cDNAs have a GRR sequence between beta-SGNP and PBAN as a cleavage site, but the Assc-PBAN cDNA showed an unusual GR sequence at the corresponding position, indicating the possibility of non-cleavage between the beta-SGNP and PBAN. When the GR sequence was a cleavage site, the question arose of whether or not the glutamine residue at the N-terminus of the Assc-PBAN homologue was cyclized. To identify the sequence of the Assc-PBAN, the brain-SG extract was fractionated by HPLC referring to three synthetic peptides with the predicted sequences. The chromatographic behavior of the natural pheromonotropic peptide revealed the unique structure of Assc-PBAN including beta-SGNP, i.e., SVDFTPRLGRQLVDDVPQRQQIEEDRLGSRTRFFSPRL-NH(2), as the first determination of PBAN from the insects producing an epoxyalkenyl sex pheromone.     

Molecular cloning of pheromone biosynthesis activating neuropeptide in silkworm, Bombyx mori

Pheromone biosynthesis activating neuropeptide (PBAN) is a suboesophageal ganglion secretory polypeptide of insect, which activates the pheromone gland to produce sex pheromone biosynthesis in female silkworm, Bombyx mori. A Bombyx genomic library was screened by the method of plaque hybridization using the 32P-labeled BomDH cDNA as a probe. The genomic sequence encoding PBAN has been cloned and its structure is analyzed. The PBAN gene comprises two exons interspersed by a single intron 697 bp in length. Preceding the PBAN amino acid sequence is a 32-amino acid sequence containing two FXPRL amide peptides, which are α-SGNP (Ile-Ile-Phe-Thr-Pro-Lys-Leu) and β-SGNP (Ser-Val-Ala-Asn-Pro-Arg-Thr-His-Glu-Ser-Leu-Glu-Phe-Ile-Pro-Arg-Leu), which is followed by a Gly-Arg processing site. Immediately, after the PBAN amino acid sequence is a Gly-Arg processing site and a FXPRL amide peptide γ-SGNP (Thr-Met-Ser-Phe-Ser-Pro-Arg-Leu). It is suggested that besides PBAN, 7-, 8-, and 17-residue amidated peptides wer     

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.     

Temporal distribution of PBAN‐like immunoreactivity in the hemolymph of Mamestra brassicae females in relation to sex pheromone production and calling behavior

A direct enzyme‐linked immunosorbent assay has been developed and applied to the analysis of PBAN immunoreactivity in female hemolymph of the cabbage armyworm, Mamestra brassicae. PBAN‐IR determinations have been carried out with third scotophase insects at different times of the photoperiod. The rhythm of calling and the pattern of pheromone production by third scotophase females at different times of the photoperiod have also been determined. PBAN‐IR and calling are well correlated. However, whereas pheromone titers decrease, both PBAN‐IR levels and percentage of calling females remain high in the last hours of the scotophase. These results are discussed in the context of the regulation of sex pheromone biosynthesis in M. brassicae. Arch. Insect Biochem. Physiol. 40:80–87, 1999. © 1999 Wiley‐Liss, Inc.     

Immunochemical and biological analysis of pheromone biosynthesis activating neuropeptide in Heliothis peltigera.

This study describes the preparation and characterization of a highly specific antiserum to Helicoverpa zea pheromone biosynthesis activating neuropeptide (Hez-PBAN), and the use of this antiserum, in an enzyme linked immunosorbent assay (ELISA), to determine: a) the content of endogenous PBAN in head extracts of male and female Heliothis peltigera; b) the level of PBAN at different developmental stages; and c) the content of PBAN in four different moth species. Cross-reactivity studies revealed that the antiserum is directed mainly toward the N-terminal region of the neuropeptide, and that it exhibits similar binding affinities toward the oxidized and reduced forms of PBAN. Analysis of PBAN content in head extracts of male and female H. peltigera, at scotophase, revealed the presence of 4.97 and 4.58 pmol, respectively, in 3-day-old moths, and 5.33 and 4.78 pmol, respectively, in 7-day-old moths. The similarity in the content of PBAN at both ages and sexes was in accordance with the amount of pheromonotropic activity in these extracts which stimulated pheromone biosynthesis to a similar level. Analysis of PBAN-like immunoreactivity (IR) in head extracts of H. peltigera larvae and pupae demonstrated the existence of the neuropeptide in the 4th larval instar and continued to increase as a function of development. No IR could be detected in the first three larval instars. The larval and pupal extracts also exerted pheromonotropic activity which followed a similar pattern. The activity in these extracts, however, was considerably lower than that found in adult male and female heads. IR was also detected in head extracts of three other Noctuidae moths: Helicoverpa armigera, Cornutiplusia circumflexa and Spodoptera littoralis, indicating a high degree of chemical and structural similarity of PBAN in these moths.     

Female sex pheromone suppression and the fate of sex-peptide-like peptides in mated moths of Helicoverpa armigera

Insect males produce accessory gland (MAG) factors that are transferred in the seminal fluid to females during copulation, and elicit changes in the mated female's behavior and physiology. Our previous studies showed that the injection of synthetic Drosophila melanogaster sex-peptide (DrmSP) into virgin females of the moth Helicoverpa armigera causes a significant inhibition of pheromone production. In this and other moth species, pheromone production, correlated with female receptivity, is under neuroendocrine control due to the circadian release of the neuropeptide PBAN. In this study, we show that PBAN, present in the hemolymph during the scotophase in females, is drastically reduced after mating. We also identify 4 DrmSP-like HPLC peaks (Peaks A, S1, S2, and B) in MAGs, with increasing levels of DrmSP immunoreactivity during the scotophase, when compared to their levels observed during the photophase. In H. armigera MAGs, a significant reduction in the pheromonostatic peak (Peak B) was already evident after 15 min of copulation, and depletion of an additional peak (Peak S2) was evident after complete mating. Peak A is also detected in female brains, increasing significantly 1 h after mating, at which time inhibition of pheromone biosynthesis also occurs. However, changes corresponding to the other MAG peaks were not detected in mated female tissues.     

Role of neuropeptides in sex pheromone production in moths

Sex pheromone biosynthesis in many moth species is controlled by a cerebral neuropeptide, termed pheromone biosynthesis activating neuropeptide (PBAN). PBAN is a 33 amino acid C-terminally amidated neuropeptide that is produced by neuroendocrine cells of the subesophageal ganglion (SEG). Studies of the regulation of sex pheromone biosynthesis in moths have revealed that this function can be elicited by additional neuropeptides all of which share the common C-terminal pentapeptide FXPRL-amide (X = S, T, G, V). In the past two decades extensive studies were carried out on the chemical, cellular and molecular aspects of PBAN and the other peptides (termed the pyrokinin (PK)/PBAN family) aiming to understand the mode of their action on sex pheromone biosynthesis. In the present review we focus on a few of these aspects, specifically on the: (i) structure-activity relationship (SAR) of the PK/PBAN family, (ii) characterization of the PK/PBAN receptor and (iii) development of a novel strategy for the generation of PK/PBAN antagonists and their employment in studying the mode of action of the PK/PBAN peptides.     

Cloning and characterization of the pheromone biosynthesis activating neuropeptide receptor gene in Spodoptera littoralis larvae

In noctuid moths cuticular pigmentation is regulated by the pyrokinin/pheromone biosynthesis activating neuropeptide (PK/PBAN) family, which also mediates a variety of other functions in moths and other insects. Numerous studies have shown that these neuropeptides exert their functions through activation of the PBAN receptor (PBAN-R), with subsequent Ca(2+) influx, followed by either activation of cAMP or direct activation of downstream kinases. Recently, several PBAN-Rs have been identified, all of which are from the pheromone gland of adult female moths, but evidence shows that functional PK/PBAN-Rs can also be expressed in insect larvae, where they mediate melanization and possibly other functions (e.g., diapause). Here, we identified a gene encoding a G-protein-coupled receptor from the 5th instar larval tissue of the moth Spodoptera littoralis. The cDNA of this gene contains an open reading frame with a length of 1050 nucleotides, which translates to a 350-amino acid, 42-kDa protein that shares 92% amino acid identity with Helicoverpa zea and Helicoverpa armigera PBAN-R, 81% with Bombyx mori PBAN-R and 72% with Plutella xylostella PBAN-R. The S. littoralis PBAN-R gene was stably expressed in NIH3T3 cells and transiently in HEK293 cells. We show that it mediates the dose-dependent PBAN-induced intracellular Ca(2+) response and activation of the MAP kinase via a PKC-dependent but Galphai-independent signaling mechanism. Other PK/PBAN family peptides (pheromonotropin and a C-terminally PBAN-derived peptide PBAN(28-33)NH(2)) also triggered MAP kinase activation. This receptor, together with the previously cloned PBAN-R, may facilitate our understanding of the cell-specific responses and functional diversities of this diverse neuropeptide family.     

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     

Firefly luciferase, synthesized to very high levels in caterpillars infected with a recombinant baculovirus, can also be used as an efficient reporter enzyme in vivo

Trichoplusia ni and Spodoptera littoralis larvae were infected with a recombinant AcNPV, having the viral polyhedrin gene replaced with the cDNA encoding firefly luciferase. Both S. littoralis and T. ni synthesized very high levels of luciferase representing greater than or equal to 25% and greater than or equal to 15%, respectively of the total Coomassie blue stainable protein. Luciferase was apparently not secreted into the hemolymph but was contained within the body tissue. Expression in S. littoralis larvae suggests that luciferase can be an excellent reporter enzyme to study virus infection, dissemination and expression in different tissues, host range determination, insect physiology and also to monitor the release of recombinant virus in the environment when used as a biocide.     

PBAN selective antagonists: inhibition of PBAN induced cuticular melanization and sex pheromone biosynthesis in moths

A D-Phe scan (sequential D-Phe replacement) library of linear peptides, synthesized on the basis of a slightly modified active sequence of PBAN (YFSPRL-amide) was employed to detect potential inhibitors of cuticular melanization in Spodoptera littoralis larvae and to compare their stimulatory and inhibitory melanization activity with their pheromonotropic agonistic and antagonistic activities. A quantitative melanotropic assay was used to monitor the extent of cuticular melanization elicited by Hez-PBAN1-33NH2 in S. littoralis larvae in the presence and absence of the D-Phe peptides. The data revealed the presence of two partial melanotropic antagonists, and disclosed the presence of selective pure melanotropic agonists and pure pheromonotropic antagonists indicating differences in the inhibitory and stimulatory patterns of the library with respect to both activities. The differences between the pheromonotropic and melanotropic inhibitory patterns of the peptides hints at the possibility that sex pheromone biosynthesis in the pheromone gland of Heliothis peltigera females and induction of cuticular melanization in S. littoralis may be mediated by different receptors (that may result either from presence of different receptor sub-types or may reflect species differences in receptor structure and/or properties) despite the fact that they are induced by the same peptide (PBAN1-33NH2).