Archives of Insect Biochemistry and Physiology 22:153-168 (1 993) Neuroendocrine Control of Sex Pheromone Biosynthesis in Heliothis pelfigera Miriam Altstein, Yoav Gazit, and Ezra Dunkelblum lnstitute of Plant Protection, Volcani Center, Agricultural Research Organization, Bet Dagan, Israel Sex pheromone production in female moths i s controlled by a cerebral factor termed pheromone biosynthesis activating neuropeptide (PBAN). Our understanding of the sequence of events common to the neuroendocrine process of PBAN, namely, its biosynthesis, secretion, transport, biological activity, and degradation, is very limited, Moreover, some of the studies are controversial, mainly those related to: (1) the route of transport of PBAN to its target organ, (2) the nature of the target organ of PBAN, (3) the possible involvement of other stimulatory and inhibitory neural and nonneural factors in the regulation of sex pheromone biosynthesis, (4) the structure-function relationship of the neuropeptide, and (5) the effect of PBAN on the enzymatic steps involved in pheromone biosynthesis in the gland. In this work, we summarize the results obtained in our laboratory on the regulation of sex pheromone biosynthesis by PBAN in Heliothis pdtigera and shed light on some of these controversial issues. G 19Y3 w ~ l r y - ~ t s Inc. s, Key words: pheromone biosynthesis activating neuropeptide, insect neuropeptide, Lepidoptera, antiserum, terminal abdominal ganglion INTRODUCTION The sexual communication between male and female moths is regulated mainly by female-produced sex pheromones [1,2]. Sex pheromones in moths are synthesized and secreted from specialized glandular cells, which are located in the intersegmental membrane between the eighth and the ninth abdominal segments . Because of their crucial role in sexual communication and mating, sex pheromones have been studied intensively. Several hundred Acknowledgments: We thank Mrs. Marina Benichus and Mrs. Orna Ben-Aziz for their part in rearing insects and for excellent technical assistance. This manuscript is part of the Ph.D. thesis of Yoav Gazit, a graduate student at the Hebrew University of Jerusalem. This rexarch was supported by the Fund for Basic Research administered by the Israel Academy of Sciences and Humanities. This is article 3507-E, 1992 series, a contribution from the Agricultural Research Organization (ARO), Bet Dagan, Israel. Received April 6, 1992; accepted June 29, 1992 Address reprint requests to Miriam Altstein, Department of Entomology, Instituteof Plant Protection, The Volcani Center, Bet Dagan, 50250, Israel. 6 1993 Wiley-Liss, Inc. 154 Altstein et al. moth sex pheromones isolated from pheromone gland extracts or as volatiles have been identified in the last two decades [4,5], and their mode of action has been studied . Sex pheromones in moths are generally C10-C18 aliphatic compounds, most of which are unsaturated. Their structural diversity is acquired by the chain length, by the position and configuration of the olefinic bonds, which may vary between one and four bonds, and be located at different positions along the carbon chain having either Z or E configurations, and by the functional group that is located at the first carbon of the chain and is usually an aldehyde, alcohol, or acetate [7-91. In recent years, a series of unsaturated C17-C21 hydrocarbon and epoxide pheromone components has been identified in several Lepidoptera families, particularly in Geometridae [lo]. The possible involvement of an endocrine factor in the biosynthesis of sex pheromones was suggested some years ago by Riddiford and Williams [ll] and by Hollander and Yin 1121. Direct evidence of the involvement of a neuroendocrine factor in the regulation of sex pheromone biosynthesis had been demonstrated for the first time in 1984 by Raina and Klun , who showed that ligation of virgin Helicoverpa (Helidhis)zed females inhibited the activation of the biosynthetic process in the pheromone gland. Injection of head ganglia extracts into the abdomen of the ligated females reactivated the mechanism, producing amounts of pheromone similar to those found in untreated females. Since 1984, the presence of a neuroendocrine factor with pheromonotropic activity, termed PBAN," has been demonstrated in a variety of moth species [14-241 (Table 1)as well as in non-Lepidopteran insects such as locusta , two cockroach species, and a cricket [15,24]. However, sex pheromone biosynthesis in moths is not always regulated by PBAN and studies performed on the cabbage looper moth, Trichoplusia ni 1241, indicated that sex pheromone biosynthesis in this moth is not regulated by PBAN. Recently, three different PBAN peptides have been isolated from two moth species. Hez-PBAN was isolated from the brain SOG complex of male and female H . zea  and Bom-PBAN-I and Bom-PBAN-I1 were isolated from heads of male Bombyx mori [27,28]. The entire primary structures of all three peptides have been determined. Hez-PBAN and Bom-PBAN-I are amidated neuropeptides consisting of 33 amino acids. Bom-PBAN-I1has the same amino acid sequence as Bom-PBAN-I except for an extra arginine at the amino-terminus. Hez-PBAN and Bom-PBAN-I share -80% homology. The PBAN gene has also been recently isolated and contains in addition to the PBAN sequence, sequences of 7- and $-residue amidated peptides. These peptides share with PBAN the core C-terminal pentapeptide Phe-Ser-(or Thr)-Pro-Arg-Leu-NH2  and bear a high degree of homology to the pyrokinin family of insect peptides with myotropic activity [30,31]. 'Abbreviations used: ANOVA = analysis of variance; Bom-PBAN = Bornbyx rnori PBAN; CC = corpora cardiaca; ELISA = enzyme linked irnmunosorbent assay; Hez-PBAN = Helicoverpa zea PBAN; IR = immunoreactivity; PBAN = pheromone biosynthesis activating neuropeptide; PMSF = phenylrnethylsulfonyl fluoride; SOG = suboesophageal ganglion; TAG = terminal abdominal ganglion; TFA = trifluoroacetic acid; TLCK = N,a-p-tosyl-L-lysine chloromethyl ketone; Z8-13:Ac = (Z)-&tridecenyl acetate; Z l l - 1 6 : A l d = ( Z ) - l l-hexadecenal. Regulation of Sex Pheromone Biosynthesis 155 TABLE 1. Summary of Moths Reported to Contain and Use Compounds With PBAN Activity Tested moth BOMBYCIDAE Bombyx mori LYMANTRIIDAE Lymantria dispar NOCTUIDAE Chrysodeixis chalcites Cornutiplusia circurrtflexa Helicoverpa arvnigera Helicoverpa zea Heliotlzis peltigera Heliothis virescens Mamcstra brassicae Pseudaletia unipuncta Spodoptera lit toralis PYRALIDAE Chilo suppresalis Ostrin ia n ubilalis Referencea [W  [I61 ~ 7 1 [I81 [I31 [I91 ~ 5 1 [201  l’ P I ~231 ~ 5 1 TORTRICIDAE Argyrotaenia velutinana ~ 4 1 aIndicates only the first published study for each moth. bAnalysis of PBAN content in homologous recipient moths. ‘Analysis of PBAN content in heterologous recipient moths. Studies from several laboratories reveal that both natural and synthetic PBAN activate sex pheromone biosynthesis during photophase and scotophase [22,32,33], that the neuropeptide is active among heterologous moth species [15,18,26,34], that a PBAN-like factor is found in male and female moths [13,14,18,22], that its activity is mediated by the cyclic nucleotide adenosine 3‘5’-monophosphate (c-AMP) and is dependent on Ca’ [33,35], and that it originates from the neurosecretory cells of the SOG [15,36]. Despite the intensive studies on the regulation of sex pheromone biosynthesis by PBAN, our understanding of the sequence of events comprising the neuroendocrine process of this neuropeptide, namely, its biosynthesis, secretion, transport, biological activity, and degradation, is very limited. Moreover, some of the studies are controversial, mainly those related to: (1)the route of transport of PBAN to its target organ (hemolymph [13,34] vs. ventral nerve cord ), (2) the target organ of PBAN (pheromone gland [33,34,37-391, TAG , or corpus bursae ), (3) the possible involvement of other stimulatory factors (octopamine  and female bursa copula trix factor ) and inhibitory factors (accessory gland male receptivity terminating factor  and female inhibitory factor ) in the regulation of sex pheromone biosynthesis in relation to the activity of PBAN, (4) the structure-function relationship of the neuropeptide (role of central  vs. terminal regions of the molecule ), and (5) the effect of PBAN on the enzymatic steps involved in pheromone biosynthesis in the gland (prior to fatty acid biosynthesis [24,39], desaturation [16,20], or reduction of fatty acyl moieties [45,46]). + 156 Altstein e t al. In this study, we summarize the results obtained in our laboratory on the regulation of sex pheromone biosynthesis by PBAN in Heliothis pdtigertl and shed light on some of these controversial issues. RESULTS AND DISCUSSION Biological Characterization of PBAN Characterization of PBAN was performed using either ligated females at scotophase, or nonligated females at photophase . Pheromone content in the pheromone glands of treated and control females was monitored with the major component, Z11-16:Ald , by capillary gas chromatography. An internal standard (Z8-13:Ac)was used for the quantification of the pheromone content . Studies of the regulation of sex pheromone biosynthesis in the moth H . peltigem revealed that: (1)this process is dependent, as in many other moths, on the presence of an endocrine factor , (2) the activity of PBAN is timeand dose-dependent , (3) both natural and synthetic PBAN are active among heterologous moth species [431, and (4)PBAN-like factor is present in both male and female head extracts . These data correlated well with studies on other Heliothinae and non-Heliothinae moths [13-15,18,22,34]. The presence of a PBAN-like factor in male head extracts suggested that males may use a similar mechanism to regulate their sex pheromone production (which have recently been reported to play a role in the attraction of female moths, ), or that the PBAN-like factor regulates other functions in the male. The heterologous activity of PBAN indicated that the same or a closely related neuropeptide is responsible for the regulation of pheromone biosynthesis in a variety of moth species. The high homology between Hez-PBAN and Bom-PBAN [26,27] supported this suggestion. Sex pheromone biosynthesis is affected by a variety of physical, environmental and physiological factors (for review see  and [50,51]). Among these factors photoperiodicity has the most profound effect. In H . peltigem, sex pheromone production begins at the onset of scotophase and ends toward the onset of photophase, suggesting that light may have an inhibitory effect or darkness may have a stimulatory effect on this process. The mechanism by which photoperiodicity regulates sex pheromone biosynthesis is not clear, however, since PBAN controls sex pheromone production it is possible that it may affect PBAN release, transport, or action at the target organ. Studies of the biological activity of PBAN obtained from donors at photophase and scotophase revealed that the two head extracts show comparable activities upon injection into ligated females at scotophase and to both ligated and nonligated females at photophase . Comparison of the kinetic pattern of sex pheromone biosynthesis, activated by PBAN, at the two photoperiods (Fig. 1) revealed that the biosynthesis in ligated females at photophase and scotophase does not differ statistically, whereas the pheromone content in nonligated females at photophase is significantly higher at 15 and 60 min postinjection and significantly lower at 4 h postinjection. The explanation of this phenomenon is not clear. The differences result, most likely, from the use 1 Time post injection (h) Fig. 1 . Time curve of sex pheromone production in H. peltigera at photophase (0-0, nonligated; (A-A, ligated) and scotophase (@-a, ligated) in response to injection of H. peltigera head extracts obtained during scotophase. Injection and pheromonotropic activity of ligated and nonligated females were determined as described by Cazit et al. [431. Statistical analysis was performed for each curve separate1 (indicated by letters), and among the various treatments at each time point byANOVA,aftera X+l transformationofthedata. Eachpointrepresentsthemeanf S.E.ofnine glands. Differences between means were tested for significance by Duncan’s multiple range test at P < 0.05. Values with the same letter do not differ significantly. Asterisk ( * ) indicates a significant difference in pheromone content of the nonligated females at 15 min and 1 and 4 h postinjection. & of nonligated females, which may respond quicker to the neuropeptide (quicker onset) and eliminate sex pheromone from the gland at a faster rate (quicker decline). The ability of PBAN to induce sex pheromone biosynthesis during photophase and the fact that the activities of head extracts from the two photoperiods were similar suggested that photoperiodicity affects the release of PBAN from the neurosecretory cells. Examination of sex pheromone biosynthesis as a function of time revealed that it declines 3 4 h postinjection (Fig. l),regardless of the photoperiod or the state of the moth (ligated or nonligated). This decline may result either from degradation of the injected PBAN by proteolytic enzymes, present in the hemolymph or at the target organ, which are known to inactivate neuropeptide hormones , or, alternatively from an accelerated rate of pheromone elimination (degradation or release) from the gland. Analysis of sex pheromone production using a proteolytically stable analog of PBAN (PBAN 9-33, which acquired its stability from the presence of a proline at the N-terminus and an amide at its C-terminus) revealed that sex pheromone levels remained high for over 10 h postinjection . Moreover, examination of sex pheromone production stimulated by H . peltigem head extracts injected together with protease inhibitors (cocktail of aprotinin, bacitracin, TLCK, and PMSF, 1 pg each) revealed a three times higher content as compared to that obtained in the absence of the inhibitor [159.4 & 31 ng/gland; ( 2S.E.; n = 6) and 52.1 & 24.8 ng/gland ( ? S.E.; n = 4), respectively]. Based on these results, we assume that degradation of PBAN, rather than acceleration of pheromone 158 Altstein et al. elimination from the gland, causes the decline in pheromone content 3-4 h postinjection. Analysis of the pheromonotropic activity as a function of age in H . peZtigeru male and female moth donors revealed that head extracts of 3- and 7-day-old moths exhibit similar activities . Studies on the activity of the neuropeptide at earlier developmental stages indicated the presence of PBAN in larval and pupal (male and female) head extracts and an increase in activity as a function of development . The presence of pheromonotropic activity at early developmental stages suggested that PBAN is involved in functions other than sex pheromone biosynthesis. Indeed, in 1990 Matsumoto et al.  reported that PBAN is homologous to the melanization and reddish coloration hormone and that endogenous as well as synthetic PBAN induces melanization upon injection into larvae. Since larval development is accompanied by color changes, it is very likely that PBAN participates in processes related to larval melanization. Studies of the Target Organ of PBAN A study on H . zea suggested that the target site of PBAN is not the pheromone gland but the TAG that innervates the gland . This study raised the possibility that an additional neuroendocrine or neural factor (present in the TAG) mediates sex pheromone production. We tested this assumption by analyzing the ability of H . peltigeru head extracts to stimulate sex pheromone biosynthesis in the presence or absence of the TAG and by studying the ability of PBAN to stimulate sex pheromone biosynthesis after topically applying the neuropeptide on the TAG or the pheromone gland. Our data revealed that sex pheromone biosynthesis is stimulated by PBAN in the presence or absence of the TAG (Table 2) and that the pheromone glands TABLE 2. Sex Pheromone Biosynthesis in Presence and Absence of the TAG* Z11-16:Ald contcnt (ngigland r S.E.) Treatment Without protease inhibitors TAG intact (sham) TAG disconnected TAG removed TAG intact + + + + head extract head extract head extract buffer With protease inhibitors TAG intact (sham) + head extract head extract TAG removed + 21.5 2 5.9a 21.5 i 7.7a 38.0 2 10.2a <0.3 b 86.8 2 58.8 k 33.1 a 12.9a +TAGS-Wereexposed by removal of the dorsal cuticle of the 6th abdominal segment and disconnection was performed by cutting the ventral nerve cord anteriorly and posteriorly to the TAG. Injcction of H . peltiyfvu head extracts was performed without and with protease inhibitors (cocktail of aprotinin, bacitracin, PMSF, and TLCK, 1 pg each) and analysis of pheromone content was peformrd on nonligated females at photophase, as described by Gazit et al. . Statistical analysis was performed (separately for the two treatment groups) by ANOVA after a transformation of the data. Each value represents the mean 2 S.E. of 7 glands. Differences between means were tested for significance by Duncan’s multiple range test at ’I < 0.05. Values with the same letter d o not differ significantly. a Regulation of Sex Pheromone Biosynthesis 159 TABLE 3. Response of Pheromone Glands to Topically Applied PBAN* Treatment Application of PBAN o n gland Application of PBAN on gland Application of buffer on gland Time postapplication (min) Z11-16:Ald content (nglgland 2 S.E.) 30 60 60 15.9 i 5.9 b 32.7 2 5.9 a 1.2 2 1.2c *Application of PBAN 9-33 o n the gland was performed in 0.25 pl of 0.1 M sodium phosphate buffer, p H 7.4, as described by Dunkelblum et al. [5Y]. Analysis of pheromone content was performed using nonligated females at photophase, as described by Gazit et al. . Statistical analysis was performed by ANOVA after 6 1 transformation of the data. Each value represents the mean f S.E. of four glands. Differences between means were tested for significance by Duncan’s multiple range test at P < 0.05. Values with different letters differ significantly. respond to the topically applied neuropeptide in a time-dependent manner (Table 3). Topical application of H . pcltigera head extracts on the TAG (enclosed with an inert grease) did not evoke sex pheromone production (data not shown). Our data indicate that the TAG does not play a direct role in sex pheromone biosynthesis and that the pheromone gland is probably the target organ for the neuropeptide. It is possible that the inability of the H. zed (in the study of Teal et al. ) to respond to the injected brain-SOG extracts in the absence of the TAG resulted from proteolytic degradation of the neuropeptide, caused by proteases released from the dissected tissue. Our results, which show a significant increase in pheromone production in the presence of protease inhibitors (Table 2) support this hypothesis. In vivo studies on the role of the TAG in Ostvinia fuvnacalis  and H . zed , and in vitro studies (performed with isolated pheromone glands where the TAG was removed) in Argyrotaeniu velutinana , H . zea [33,39] and H . avrnigcra [18,37], support our conclusion that the TAG is not essential for pheromone biosynthesis and that the gland is the target organ for PBAN. Immunochemical Characterization of PBAN In order to widen the characterization of PBAN and our understanding of its role in the regulation of sex pheromone biosynthesis in vivo, polyclonal antisera were raised in rabbits (using Hez-PBAN 1-33 as an antigen) and used, in combination with HPLC and ELISA , to identify and quantify the PBAN-like factor in H.p e l t i p a head extracts. Analysis of the antisera collections from the immunized rabbits by means of ELISA revealed that the sixth collection of rabbit YG-16 [marked YG-16(6)]and the third collection of rabbit YG-17 [marked YG-17(3)]exhibited high affinities toward Hez-PBAN . Cross-reactivity analysis of the two antisera, using Hez-PBAN and C-terminally amidated and free acid peptides derived from its sequence, revealed that YG-16(6) recognizes only the complete PBAN sequence, either amidated or as the free acid, and displays no cross reactivity with any of the other N-terminally truncated fragments (Table 4). YG-17(3), in contrast, cross reacted with all the C-terminally amidated peptides, regardless of their length, and did not recognize the free acid fragments (Table 4). The ability of YG-16(6) to recognize only the complete peptide indicated that this antiserum is Altstein et al. 160 TABLE 4. Cross-Reactivityof YG-16(6) and YG-17(3) Antisera With Fragments Derived From the N- and C-terminal Region of Synthetic Hez-PBAN) Cross-Reactivity (%) YG-l6(h) YC-17(3) Peptide PBAN 1-33 PBAN Y-33 PBAN 1.3-33 PBAN 17-33 PBAN 19-33 PBAN 26-33 PBAN 28-33 PBAN PBAN PBAN PBAN 1-33 9-33 19-33 9-18 NH2 NHz NH2 NH2 NHz NH2 NH2 OH OH OH OH q0.3 <0.3 <0.3 100 205 63 68 <0.3 95 <0.3 95 c0.3 95 100 0.2 0.2 36 ~ 0 . 3 c0.3 <o. 1 <0.3 <0.1 *Cross-reactivity was performed using the two-step competitive ELISA a s described by Gazit et al. , using a 1:1,000 dilution of each antisera. Synthetic Hez-PBAN and derived peptides were synthesized by the solid phase method as described by Gazit et al. (431. Cross-reactivity represents the ratio (in Yo) between the concentrations of PBAN 1-33 NH2 causing 50% inhibition in the binding of an antiserum to the antigen adsorbed o n the solid phase, and any of the tested peptides causing the same inhibition. directed toward the N-terminal region of the molecule. The cross reactivity of YC-17(3)with all the C-terminally amidated PBAN derived fragments, but not with the free acid peptides, indicated that this antiserum is directed mainly toward the C-terminal region of PBAN. The use of YG-16(6) to determine the content of PBAN-like IR in scotophase H . peltigera male and female head extracts at various days postemergence revealed values ranging from 4.6 to 5.3 pmol PBAN-like IWhead at 3 and 7 days in both sexes . Analysis of the content of PBAN-like IR as a function of development confirmed the biological data on the presence of PBAN in larvae and pupae and its increase as a function of development . Studies performed by Rafaeli et al.  in H . armigera, using a different PBAN antiserum in RIA, revealed that the CC and Brain-SOG complexes contain -1 pmol PBAN-like IR. Our studies on H . armigcra revealed the presence of 4 pmol PBAN-like IWhead .The reason for these differences may result from variations in the preparations used for IR determination (dissected CC and Brain-SOG complex vs. whole heads), from differences in the extraction procedures (methanolic vs. hydrochloric acid extraction) or from the presence of IR in regions other than the CC and Brain-SOG complexes (e.g., nerve terminals) that contribute to the IR, in our studies. The availability of an antiserum that recognizes fragments derived from the C-terminus of PBAN enabled us to look for the occurrence of such fragments in head extracts of H . peltigem. Analysis was performed by combining HPLC fractionation of head extracts with ELISA. Examination of the fractions with the antiserum YG-16(6), which recognizes the complete Hez-PBAN, revealed the presence of a single peak with a retention time of 4 7 4 9 min (Fig. 2A). The retention time of synthetic Hez-PBAN under the same conditions was slightly different (45 min), probably as a result of a difference in the sequence of the Regulation of Sex Pheromone Biosynthesis 1.5 n 161 Fraction 0 1.0 rr fa 0.5 0.0 Fraciion 120 lc Fraction Fig. 2. WAN-like IR (A and 6) and pheromonotropic activity (C) of HPLC fractionated H . peltigera head extracts. Head extracts (SO p.1) were subjected to J. 4 x 2.50 mm Lichrospher 100 RP C18 5 p m column (Merck, Darmstadt, Germany) and eluted using 0. I % TFA for 10 min followed by a linear gradient of 0-60% acetonitrile in 0.1% TFA for 60 rnin, at room temperature, at a flow rate of 1 ml/min on a Tracor Model 985 HPLC system (Tracor Instruments, Austin, TX), equipped with a IJV spectrometric detector Model 970. Using the two-step competitive ELISA as described by Gazit et al. 1171, 100 pl, of each fraction were analyzed for IR with YG-16(6) (1:1,000) (A) and YC-17(3) (1 : 1,000) (B) antisera. Pherornonotropic activity (C) was monitored by injecting an equivalent of 0.03 of each fraction into nonligated females at photophase, as described by Carit et al. . Arrow marks the retention time of synthetic Hez-PBAN 1-33. neuropeptides from the two Heliothinae moths. Analysis of the fractions with the antiserum YG-17(3) revealed the presence of two additional peaks, with retention times of 4 0 4 4 and 52-57 min (Fig, 2B). Evaluation of the pheromonotropic activity of all fractions (Fig. 2C) revealed that only the peak that elutes at 4 7 4 9 min is biologically active. The less hydrophobic and the more hydrophobic peptides were devoid of any pheromonotropic activity. These results suggest that fragments, other than complete PBAN, which are derived from the sequence of the neuropeptide and are devoid of pheromonotropic activity, are present in head extracts of H . peltigem. Such fragments may be naturally occurring components in the head ganglia or, alternatively, result from the degradation of PBAN during tissue processing. The later alternative, however, is not very likely as our dissection procedure involves the use of 162 Altstein et al. cold 0.1 M hydrochloric acid, thus decreasing to minimum any chance of proteolytic degradation of the neuropeptide during tissue preparation. The nature of the above mentioned fragments is not known and has yet to be determined. The presence of the PBAN derived fragments raises the possibility that the complete PBAN serves as a precursor molecule from which different fragments with different activities are derived, probably by the action of prohormone processing enzymes .Alternatively, it is possible that the PBAN gene or another gene carries short sequences that share a high degree of homology with the C-terminal region of PBAN and that are cleaved by similar enzymes. The first assumption was supported by several studies, including ours, where it has been demonstrated that fragments derived from the sequence of Hez-PBAN 1-33 induce pheromonotropic [43,44] as well as myotropic activity .The second assumption was recently substantiated by evidence based on the sequence of the PBAN gene, where, besides PBAN, 7and %residue amidated sequences were found, which share with PBAN the core C-terminal pentapeptide Phe-Ser(Thr or Val)-Pro-Arg-Leu-NHz . It is thus possible that both alternatives are correct. Structure-Activity Relationship of PBAN Structure-activity studies were performed using synthetic Hez-PBAN and four shorter amidated fragments derived from its C-terminus to determine sequences which are essential for the pheromonotropic activity. Comparison of the activity of Hez-PBAN 1-33 with that of the four C-terminally amidated fragments, PBAN 9-33, PBAN 19-33, PBAN 26-33, and PBAN 28-33, revealed that removal of eight amino acids from the N-terminal region had a minor effect on the biological activity . Removal of 10 additional amino acids from the N-terminus (PBAN 19-33) resulted in a decrease, but not loss, of activity. Shorter peptides derived from the C-terminus of the molecule (PBAN 2&33 and PBAN 28-33) had a much lower biological activity, and even a 100 times higher concentration of the octa- and hexapeptides failed to stimulate pheromone biosynthesis to maximal levels (Fig. 3) and . Similar results were obtained, in H. zed, by Raina and Kempe where the C-terminally amidated fragment (PBAN 28-33) was reported to be less active, compared to the full length PBAN, even at high concentrations such as 100 and 1,000 pmol. Kitamura et al. , however, demonstrated that a 10 amino acid C-terminally amidated peptide derived from Bom-PBAN is as active a s the full length molecule at a dose of 12.5 pmol. It is possible that the use of a 10 rather than an eight amino acid Bom-PBAN derived peptide accounts for the high pheromonotropic activity in Kitamura's study. The ability of PBAN 9-33 and PBAN 19-33 to stimulate sex pheromone biosynthesis demonstrated that the N-terminal region of PBAN 1-33 is not essential for the biological activity and that the region between the 9th and the 19th amino acid is important for the stimulation of sex pheromone production. Despite the fact that the C-terminally derived peptides had very low biological activity at concentrations at which PBAN 1-33 reached maximal activity, we found that the C-terminal part of PBAN does play a major role in the stimulation of sex pheromone production. This was demonstrated with Regulation of Sex Pheromone Biosynthesis I/ O 0.1 I 10 Dose of injected peptide (pmOl) 163 I00 Fig. 3. Dose-responsecurve of pheromonotropic activity evoked by synthetic Hez-PBAN 1-33 (0) and three C-terminally amidated shorter fragments: PBAN 9-33 (m), PBAN 26-33 (A), and PBAN 28-33 (A). Pheromonotropic activity was determined using nonligated H. pcltigera females at photophase, as described by Gazit et al. [431. Statistical analysis was performed among the four transformation of the data. peptides, at 1,10, and 100 pmol separately, by ANOVA after a Each point represents the mean -e S.E. of nine glands. Differences between means were tested for significance by Duncan's multiple range test at P < 0.05. Values with the same letter do not differ sign i f icantl y. the aid of the two antisera that are directed toward the N- and the C-terminal parts of PBAN, YG-16(6) and YG-17(3), respectively. Preincubation of H . peltiger0 head extracts with each of these antisera prior to their injection into females revealed that the N-terminally directed antiserum was unable to inhibit the pheromonotropic activity present in the head extracts, whereas the C-terminally directed antiserum completely blocked this activity (Fig. 4A), probably due to the interference of the C-terminally directed antiserum with the binding of PBAN to its receptor. Similar results were obtained when the two antisera were injected in vivo to block endogenous PBAN activity (Fig. 4B). The importance of the C-terminal region in the stimulation of the biological activity was also indicated by Kitamura et al. , who demonstrated that Bom-PBAN lacking the C-terminally Leu-NH2 moiety is devoid of any biological activity. Conclusions Our studies on sex pheromone biosynthcsis in H. petfigera moths revealed that: (1)sex pheromone biosynthesis in this moth is dependent on a cerebral factor, (2) PBAN-like factor is present in both male and female moths, (3) PBAN is active among heterologous moth species, (4) PBAN can activate pheromone biosynthesis during photophase and scotophase, and (5) I'BAN- 164 Altstein et al. I00 r a 1 c + 3 120 00 40 YG-16 YG-17 P.1 buffer Fig. 4. Induction of sex pheromone biosynthesis by exogenously administered (head extract) (A) and endogenous PBAN (B) in the presence and absence of YG-16(6) and YG-17(3) antisera. A: Head extracts (an equivalent of 0.2 head) were incubated with a final dilution of 1:10 antiserum, preimmune serum (!?I.), or 0.1 M sodium phosphate buffer, pH 7.4, for 3 h at 3 7 T , and the mixture was injected into nonligated H. pelfigera females at photophase as described by Gazit et al. 1431. 6:Ten kl undiluted antiserum, preimmune serum, or 0.1 M sodium phosphate buffer, pH 7.4, were injected into 6-day-old H.pelfigera females at scotophase, and endogenous pheromone was monitored 24 h later as described by Gazit et al. . Statistical analysis was performed by ANOVAafter a V% transformation of the data. Each point represents the mean f S.E. of eight glands. Differences between means were tested for significance by Duncan’s multiple range test at P < 0.05. Values with the same letter do not differ significantly. like factor is present in larvae and pupae and its content increases as a function of development. In addition, our studies indicate that the target organ of PBAN is the pheromone gland and that the terminal abdominal ganglion is not essential for the stimulation of the pheromonotropic activity. Structurefunction analysis revealed that the N-terminus of PBAN is not essential for pheromonotropic activity and that the region between the 9th and the 19th amino acids together with the C-terminus of the molecule are essential parts of the active site of PBAN. Although most of the aspects related to the mode of action of PBAN in H . peltzgera are similar to those reported by other laboratories for Heliothinae and non-Heliothinae moths, some data are controversial, especially those related to the structure-function relationship of PBAN and its target organ. One possible explanation for the discrepancies among the studies on PBAN is that the neuropeptide exhibits different modes of action in different moths. 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