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Neuroendocrine control of sex pheromone biosynthesis in Heliothis peltigera.

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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 [3]. 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 [6]. 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 [13], 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 [25],
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 [26] 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
[29] 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
[15]
[I61
~ 7 1
[I81
[I31
[I91
~ 5 1
[201
[21] 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 [32]), (2) the target organ of PBAN (pheromone gland [33,34,37-391,
TAG [32], or corpus bursae [34]), (3) the possible involvement of other
stimulatory factors (octopamine [40] and female bursa copula trix factor
[34]) and inhibitory factors (accessory gland male receptivity terminating
factor [41] and female inhibitory factor [42]) 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 [43] vs.
terminal regions of the molecule [44]), 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 [43]. Pheromone content in
the pheromone glands of treated and control females was monitored with the
major component, Z11-16:Ald [47], by capillary gas chromatography. An
internal standard (Z8-13:Ac)was used for the quantification of the pheromone
content [43].
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 [19], (2) the activity of PBAN is timeand dose-dependent [43], (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 [19]. 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, [48]), 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 [49] 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 [19]. 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 [52], 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 [43]. 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 [17]. 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 [17]. 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. [53] 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 [32]. 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. [43]. 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. [43]. 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. [32]) 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 [54] and H . zed [55], and in vitro studies
(performed with isolated pheromone glands where the TAG was removed)
in Argyrotaeniu velutinana [34], 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 [56], 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 [17].
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. [17], 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 [17]. 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 [17]. Studies
performed by Rafaeli et al. [57] 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 [17].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. [43]. 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 [58].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 [31].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 [29]. 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 [43]. 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 [43]. Similar results were
obtained, in H. zed, by Raina and Kempe [44]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. [27], 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. [27], 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. [43]. 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. This,
however, is not likely, as neuropeptides usually have similar modes of actions
across a wide range of species, and it is unlikely that PBAN will act differently
Regulation of Sex Pheromone Biosynthesis
165
in species of the same order or even in the same family or subfamily. It seems,
therefore, more likely that different laboratory procedures such as the use of
different extraction and purification methods, variable dissection protocols,
as well as the use of homologous vs. heterologous native and synthetic PBAN
in a variety of moths, account for most of the controversial results. Clearly,
there is still much to be studied and compared on the mode of action of PBAN
among the various moth species.
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