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Neuropeptide regulators of the juvenile hormone biosynthesis (in vitro) in the beetle Tenebrio molitor (Coleoptera Tenebrionidae).

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A r t i c l e
NEUROPEPTIDE REGULATORS OF
THE JUVENILE HORMONE
BIOSYNTHESIS (IN VITRO) IN THE
BEETLE, Tenebrio molitor
(COLEOPTERA, TENEBRIONIDAE)
Mohatmed Abdel-latief
Institut für Biologie, Freie Universita¨t Berlin, Berlin, Germany
Klaus H. Hoffmann
Lehrstuhl für Tiero¨kologie I, Universita¨t Bayreuth, Bayreuth, Germany
The genome of Tribolium castaneum encodes two allatostatin [AS type B;
W(X)6Wamide and AS type C; PISCF-OH] and one allatotropin (AT)
precursor, but no AS type A (FGLamide) (Tribolium Genome Sequencing
Consortium, 2008: Nature 452:949–955). Here we studied the activity
(in vitro) of peptides derived from these precursors on the synthesis/release
of juvenile hormone (JH) III. The corpora cardiaca-corpora allata (CCCA) complexes of adult females of another tenebrionid beetle, the
mealworm Tenebrio molitor, were used. Incubating the gland complexes
in a medium containing Trica-AS B3 peptide, we showed that the peptide
has allatostatic function in T. molitor. The activity of the type C AS
depended on the age of the test animals and their intrinsic rate of JH III
biosynthesis. The Trica-AS C peptide inhibited the JH release from CA of
3-day-old females with a high intrinsic rate of JH synthesis, but activated
JH release from the CA of 7-day-old females with a lower intrinsic rate
of JH production. The allatotropin peptide (Trica-AT) also activated the
JH release from the CA of 7-day-old females in a dose-dependent and
reversible manner. Unexpectedly, a type A AS derived from the precursor
of the American cockroach Periplaneta americana (Peram-AS A2b)
inhibited the JH release from the CA of younger and older females in the
concentration range of 10 8 to 10 4 M, and the effects were fully
reversible in the absence of peptide. These data suggest a complex role of
Correspondence to: Klaus H. Hoffmann, Animal Ecology I, University of Bayreuth, 95440 Bayreuth,
Germany. E-mail: klaus.hoffmann@uni-bayreuth.de
ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY, Vol. 74, No. 3, 135–146 (2010)
Published online in Wiley InterScience (www.interscience.wiley.com).
& 2010 Wiley Periodicals, Inc. DOI: 10.1002/arch.20359
136
Archives of Insect Biochemistry and Physiology, July 2010
allatoactive neuropeptides in the regulation of JH III biosynthesis in
beetles. r 2010 Wiley Periodicals, Inc.
Keywords: Coleoptera; Tenebrio molitor; Tribolium castaneum; juvenile
hormone biosynthesis; corpora allata; allatostatin; allatotropin
INTRODUCTION
Juvenile hormones (JH) are unique hormones, sesquiterpenoids, which play a crucial
role in many aspects of insect development, reproduction, and behaviour. The
synthesis of JH in the corpora allata (CA) can be either stimulated or inhibited by
neuropeptides termed allatotropin (AT) and allatostatin (AS) (for review see Gäde
et al., 1997; Hoffmann et al., 1999; Stay, 2000; Gäde, 2002). In insects, three families of
structurally unrelated AS have been isolated and characterized: the type A AS
(FGLamides), type B AS [W(X6)Wamides], and type C AS (Manduca sexta-type). The
type A AS has been described for many insect orders, but seems to act as true AS only
in cockroaches, crickets, and termites (Stay, 2000; Yagi et al., 2008). However, Clark
et al. (2008) have demonstrated that a type A AS also alters the production and release
of JH in the locust, Locusta migratoria. The myoinhibitory peptide (MIP)/type B AS
peptides were first identified by their myoinhibitory action on locust oviduct muscle
(Schoofs et al., 1991). Later, four related peptides with allatostatic activity were isolated
from the Mediterranean field cricket, Gryllus bimaculatus (Lorenz et al., 1995). During
the last decade, many of the MIP/AS type B peptides were isolated from various insect
orders, but their allatostatic activity is restricted to the order Ensifera (for review see
Weaver and Audsley, 2009). The third family consists of a small number of nonamidated peptides with a C-terminal PISCF-OH sequence, first isolated from the moth
M. sexta (Kramer et al., 1991). These peptides inhibit JH biosynthesis in Lepidoptera
and Diptera (Li et al., 2006). Allatostatins are pleiotropic in function and in addition to
their allatostatic activity (in vivo and in vitro) all peptides show myoinhibitory
properties on visceral muscles in insects and other invertebrates (Hoffmann et al.,
1999; Stay, 2000).
An allatotropin was first structurally characterized from M. sexta AT (Kataoka et al.,
1989). The initiate assumption that Manse-AT is a lepidopteran-specific AT has been
questioned (Elekonich and Horodyski, 2003). A structurally closely related AT is a true
allatotropin in the dipterans Aedes aegypti and Anopheles gambiae (Li et al., 2003).
Although, the naming of the peptides (type A, B, and C allatostatins, allatotropin)
might be inadequate, particularly for species where the peptides have multiple
functions (Weaver and Audsley, 2009), we decided to use this popular terminology in
the present study.
The Coleoptera are one of the insect orders in which little is known concerning the
regulator of JH biosynthesis and the peptides involved. Recent proteomic and
genomic studies on the red flour beetle, Tribolium castaneum, failed to reveal A-type
allatostatin peptides and their preprohormone (Li et al., 2008; Weaver and Audsley,
2008), or the receptor (Hauser et al., 2008). However, one allatotropin-like peptide,
one allatostatin type C peptide, and six type B peptides and their precursors were
identified (Amare and Sweedler, 2007; Li et al., 2008; Tribolium Genome Sequencing
Consortium, 2008). Using MALDI-TOF mass spectrometry to identify neuropeptides
of the mealworm, Tenebrio molitor, also no evidence was found for the presence of type A
Archives of Insect Biochemistry and Physiology
JH Biosynthesis in Beetles
137
AS (Weaver and Audsley, 2008). In earlier experiments, T. molitor CA were not sensitive
to these allatostatins (Weaver and Edwards, 1998). Also no ion masses matching the
putative type B and type C AS, and the assumed AT, could be detected in either the
brain or the corpora cardiaca (CC)-CA complexes of T. molitor using mass spectrometry
(Weaver and Audsley, 2008).
Recently, Wasielewski et al. (2009) extracted a type A allatostatin-like brain factor
from T. molitor females, which cross-reacts with Grybi-AS A1 antibodies (antibodies
against a type A AS of G. bimaculatus) and regulated the reproductive cycle of T. molitor.
Extract of brains from the beetles inhibited JH biosynthesis by cockroach (Diploptera
punctata) CA in a dose-dependent manner and reversibly, an indication for an A-typelike AS in T. molitor (Elliott et al., 2009). However, the T. molitor CA are not innervated
by type A AS immunoreactive axons.
Basically, clear evidence is lacking on neuropeptide regulation of JH biosynthesis
in beetles. In this study, we describe the activity of putative allatoregulating peptides
in beetles using synthetic peptides whose structures were derived from the
T. castaneum genome sequence and employing the radiochemical CA assay in vitro
for measuring JH biosynthesis/release from the CA of T. molitor (Weaver et al., 1980;
Feyereisen and Tobe, 1981). T. castaneum is simply too small for dissection of the CA. In
the case of type A AS, the Peram-AS A2b peptide from the cockroach Periplaneta
americana was used.
MATERIALS AND METHODS
Insect Rearing
Tenebrio molitor larvae were reared under constant laboratory conditions at 25711C in
the dark, and fed with wheat bran and moist paper. Freshly emerged adult females
were collected daily and used at appropriate age in the radiochemical JH assay.
Synthetic Peptides
The peptides used in this study (Table 1) were custom-synthesized by Biosyntan
(Berlin). All peptides were assayed as 95% pure by analytical reversed-phase HPLC.
Assay for JH Synthesis
Assays of JH synthesis/release by T. molitor CC-CA complexes were used to test the
allatostatic or allatotropic effects of the synthesized peptides. CC-CA complexes from
3- and 7-day-old adult females were dissected, and the 3-day-old CC-CA (n 5 20 per
experiment; glands with a generally high intrinsic rate of JH biosynthesis during
vitellogenesis; Z120 pmol h 1 CA 1) were used in the JH assay when putative
allatostatic peptides were tested. The day-7 glands [n 5 20 for each experiment; glands
with a generally lower intrinsic rate of JH release (r90 pmol hr 1 CA 1) at the
beginning of oviposition] were used in assays measuring putative allatotropic activity.
In a first screening experiment (see Fig. 1), CC-CA complexes from 2-day-old females
(high intrinsic rate of JH biosynthesis) were used. The modified rapid radiochemical
partition assay (Feyereisen and Tobe, 1981) was used as previously described (Abdellatief et al., 2004). Gland complexes were assayed in modified TC 199 medium
(Weaver et al., 1980), to which 3H-acetate (Moravek; initial specific activity
Archives of Insect Biochemistry and Physiology
138
Archives of Insect Biochemistry and Physiology, July 2010
Table 1. Primary Sequences of Allatoregulating Peptides Deduced from the T. castaneum Genome
(Weaver and Audsley, 2008)
Peptide name
Trica-AT
Trica-AS C
Trica-AS B1
Trica-AS B2
Trica-AS B3
Trica-AS B4
Trica-AS B5
Trica-AS B6
Peram-AS A2b
Species
Peptide sequence
Tribolium castaneum
T. castaneum
T. castaneum
T. castaneum
T. castaneum
T. castaneum
T. castaneum
T. castaneum
Periplaneta americana
Molecular mass
GIEALKYHNMDLGTARGY-NH2
QSRYRQCYFNPISCF-OH
DWNKDKHIW-NH2
GWNHLHEGW-NH2
AWQSLQSGW-NH2
NWGQFHGGW-NH2
SKWDNFRGSW-NH2
EPAWSNLKGIW-NH2
LPVYNFGL-NH2
2,010.7
1,894.5
1,062.5
921.7
Rates of JH release (pmol x h-1 x CA-1)
Custom-synthesized peptides used in this study are shown in bold type and their molecular masses are given.
The N-terminally non-blocked peptide was used in this study.
200
ns
*
160
120
80
40
0
Peram-AS A2b
C(2-4)
Trica-AS B3
Trica-AS C
10(-7)
Figure 1. Screening of the corpora allata (CA) activity (in vitro) of the synthetic putative allatostatins. CA
activity is presented in pmol JH release per hour and pair CA. CC/CA complexes of 2-day-old females of
T. molitor were incubated for 2 h in the presence of the respective peptide. Control: Control incubation for 2 h
without peptide. Mean values 7SE of 40 determinations. Statistical analysis was done using the MannWhitney U-test. Ns, not significant. Po0.05, Po0.001 (different from the control).
740 GBq mmol 1) was added to reach a final specific activity of 0.59 GBq mmol 1 (final
acetate concentration ca. 0.6 mM). Gland complexes were at first preincubated in nonradioactive medium for 2 h before the rate of JH III (Weaver et al., 1980) was
determined for 2 h in untreated labelled medium [control rate; C(2–4)]. Other glands
were transferred to peptide supplemented medium for 2 h. Finally, in some cases
glands were retransferred to untreated medium for another 2 h to measure the
reversibility of peptide effects. Control glands stayed in the untreated medium also for
another 2 h [C(4–6)].
Statistical Analysis
All values are means7SE of measurements indicated in the figure legends. The significant
differences between the various groups were evaluated by LSD Mann-Whitney U-tests.
Archives of Insect Biochemistry and Physiology
JH Biosynthesis in Beetles
139
RESULTS
In order to investigate the allatoregulatory activity of the peptides, whose sequences
were deduced from the Tribolium castaneum genome, the Trica-AT, Trica-AS C, and
Trica- AS B3 peptides were commercially synthesized (Table 1). Since no allatostatin
type A structure is known for a coleopteran species, the Periplaneta americana type A
allatostatin-2b (Peram-AS A2b) was also synthesized and included in our experiments.
A first screening of putative allatostatin functions (in vitro) using the CC/CA
complexes of 2-day-old adult females of Tenebrio molitor (high intrinsic rate of JH
release) resulted in a moderate but significant reduction in JH release during a 2-h
incubation period with either Trica-AS B3 or Trica-AS C at concentrations of 10 7 M.
Surprisingly, a similar reduction of JH release was observed when CC/CA complexes
were incubated for 2 h in the presence of 10 7 M Peram-AS A2b (Fig. 1).
A more detailed study on the inhibition of JH release by putative allatostatins is
presented in Figure 2. Treatment of individual pairs of CC/CA from 3-day-old females
of T. molitor with 10 8 to 10 4 M Trica-AS C resulted in a dose-dependent reduction of
JH release with an ED50 value of about 5 10 6 M (Fig. 2A). Incubation of the CC/CA
in the presence of Trica-AS B3 also reduced the JH release in a dose-dependent
manner, but with a slightly higher ED50 dose (Fig. 2B). Almost the same results were
obtained when the CC/CA complexes from 3-day-old females were incubated with the
type A allatostatin from P. americana. At 10 6 M Peram-AS A2b, JH release was reduced
from 186 pmol h 1 pair CA 1 in the control to 122 pmol h 1 pair CA 1, a reduction of
about 35% (Fig. 2C). A repeat of this experiment with another cohort of CC-CA from
200
c
160
b
120
a
80
40
Rates of JH release
(pmol x h-1 x CA-1)
Rates of JH release
(pmol x h-1 x CA-1)
200
0
C(2-4)
10(-6)
120
a
80
40
10(-4)
Trica-AS C
10(-8)
C(2-4)
B
10(-6)
10(-4)
Trica-AS B3
200
c
160
b
120
a
80
40
Rates of JH release
(pmol x h-1 x CA-1)
200
Rates of JH release
(pmol x h-1 x CA-1)
b
0
10(-8)
A
c
160
160
120
80
40
0
10(-8)
C
C(2-4)
10(-6)
Peram-AS A2b
10(-4)
0
C(4-6)
D
Peram-AS Trica-AS Trica-AS C
B3
A2b
Figure 2. Rates of JH release (pmol h 1 pair CA 1) from the CC/CA of 3-day-old females of T. molitor
treated for 2 h with (A) 10 8 to 10 4 M T. castaneum allatostatin type C (Trica-AS C), (B) allatostatin type B
(Trica-AS B3), or (C) P. americana allatostatin type A (Peram-AS A2b). C(2–4), control incubation for 2 h
without peptide. (D) Reversibility of the inhibition of JH release. Another 2-h incubation was carried out in
a peptide-free medium. C(4–6), control incubation for the second 2-h incubation without peptide. The data
represent means7SE of 20 determinations. Significant differences are indicated by different letters
(Po0.05).
Archives of Insect Biochemistry and Physiology
140
Archives of Insect Biochemistry and Physiology, July 2010
a
C
Rates of JH release (pmol x h-1 x CA-1)
B
Rates of JH release (pmol x h-1 x CA-1)
A
Rates of JH release (pmol x h-1 x CA-1)
200
b
160
c
120
80
40
0
10(-8)
C(2-4)
10(-6)
10(-4)
Trica-AT
200
a
b
160
c
120
80
40
0
10(-8)
C(2-4)
10(-6)
10(-4)
Trica-AS C
120
c
80
b
40
a
0
10(-8)
C(2-4)
10(-6)
10(-4)
Trica-AS B3
Figure 3. Rates of JH release (pmol h 1 pair CA 1) from the CC/CA of 7-day-old females of T. molitor treated
for 2 h with (A) 10 8 to 10 4 M T. castaneum allatotropin (Trica-AT), (B) Trica-AS C, and (C) Trica-AS B3. The data
represent means7SE of 20 determinations. Significant differences are indicated by different letters (Po0.05).
3-day-old females (n 5 20) with a slightly lower intrinsic rate of JH release resulted in a
reduction of the JH release of 36% (157.7717.6 pmol h 1 pair CA 1 in the untreated
control versus 101.3713.5 pmol h 1 pair CA 1 at 10 6 M Peram-AS A2b) (data not
shown in figures). The effects for the Peram-AS A2b peptide were dose-dependent,
but less pronounced than for the T. castaneum B- and C-type allatostatin, respectively.
In all three cases, however, the inhibitory effects were fully reversible after another 2-h
incubation in the absence of an allatostatin (Fig. 2D).
Incubation of the CC/CA complexes from 7-day-old females in the presence of the
allatotropin peptide (Trica-AT) resulted in a dose-dependent and strong activation of
JH biosynthesis/release (Fig. 3A). At a concentration of 10 6 M Trica-AT, JH release in
Archives of Insect Biochemistry and Physiology
Rates of JH release (pmol x h-1 x CA-1)
JH Biosynthesis in Beetles
141
160
120
c
80
b
40
a
0
C(2-4)
10(-6)
10(-6)
10(-6)
Peram-AS A2b
Trica-AS B3
Peram-AS A2b
+Trica-AS B3
Figure 4. Rates of JH release (pmol h 1 pair CA 1) from the CC/CA of 3-day-old females of T. molitor
treated for 2 h with either 10 6 M Peram-AS 2b, 10 6 M Trica-AS B3, or a mixture of both (10 6 M each).
The data represent means7SE of 20 determinations. Significant differences are indicated by different letters
(Po0.05).
vitro was almost doubled (123.0710.9 pmol h 1 pair CA 1) compared to the untreated
control (69.677.1 pmol h 1 pair CA 1). Treatment of glands with a lower intrinsic rate
of JH release with 10 8 to 10 4 M T. castaneum type C allatostatin (Trica-AS C) did not
inhibit the JH synthesis (see Fig. 2A), but led to a significant and dose-dependent
increase in JH release (Fig. 3B). In contrast, treatment of such glands with 10 8 to
10 4 M Trica-AS B3 showed the most effective inhibition of JH release with an ED50
value of 8 10 7 M (Fig. 3C).
Double incubations were performed to confirm the allatostatic effect of the PeramAS A2b peptide on the JH release from CA of T. molitor. Incubating the gland
complexes from 3-day-old females with Trica-AS B3 and Peram-AS A2b (10 6 M
each) resulted in an additive effect (Fig. 4). In this case, JH III release was suppressed
to about 15% of the control (17.6710.0 pmol h 1 pair CA 1 compared to
122.9714.0 pmol h 1 pair CA 1). An additive effect was also observed when the gland
complexes from 7-day-old females were incubated in the presence of 10 6 M Trica-AT
and 10 6 M Trica-AS C (Fig. 5). The rate of JH III release then increased more than
3-fold compared to the control incubation (317.3732.9 pmol h 1 pair CA 1 vs.
89.7711.6 pmol h 1 pair CA 1).
DISCUSSION
Juvenile hormone (JH) III seems to be the only JH homolog synthesized in the CA
and present in the haemolymph of the Coleoptera (Scott et al., 2001). As in other insect
orders, the functions of JH in beetles are highly diverse. For example, in the yellowspotted longicorn beetle, Psacothea hilaris, metamorphosis and diapause induction are
strongly correlated with the haemolymph JH III titers (Munyiri and Ishakawa, 2004).
In the Colorado potato beetle, Leptinotarsa decemlineata, ovarian development is
positively correlated with rates of JH biosynthesis as well as JH haemolymph titers
(Kramer, 1978). In the red flour beetle, T. castaneum, JH regulates the secretion of
proteins from the male accessory reproductive glands, which are transferred to
females along with sperm during copulation and thereby affect the progeny
Archives of Insect Biochemistry and Physiology
Archives of Insect Biochemistry and Physiology, July 2010
Rates of JH release (pmol x h-1 x CA-1)
142
400
a
320
240
160
b
b
10(-6)
10(-6)
10(-6)
Trica-AT
Trica-AS C
Trica-AT +
Trica-AS C
80
0
C(2-4)
Figure 5. Rates of JH release (pmol h 1 pair CA 1) from the CC/CA of 7-day-old females of T. molitor
treated for 2 h with either 10 6 M Trica-AT, 10 6 M Trica-AS C, or a mixture of both (10 6 M each). The data
represent means7SE of 20 determinations. Significant differences are indicated by different letters
(Po0.05).
production by females mated with JH-deficient males (Parthasarathy et al., 2009). In
the mealworm beetle, T. molitor (and perhaps also in other beetles), mating reduces
immunity and this down-regulation is also mediated by JH (Rolff and Siva-Jothy, 2002;
Rantala et al., 2003). The molecular mechanisms of JH action in controlling the
metamorphosis of beetles were recently elucidated (Minakuchi et al., 2008;
Parthasarathy and Palli, 2009). Although, a JH receptor has not yet been identified,
there is strong evidence supporting the hypothesis that the methoprene-tolerant gene
product serves as a receptor for JH controlling the entry into metamorphosis in
T. castaneum (Konopova and Jindra, 2007). The CA seem to be the only site of JH
biosynthesis in beetles (Scott et al., 2001).
Little is known about the regulation of JH biosynthesis in beetles by allatoregulating neuropeptides. Yi and Adams (2001) demonstrated that M. sexta allatotropin
(Manse-AT) mimics the effects of a pyriproxifen (JH analog) treatment of the potato
beetle, L. decemlineata, with regard to their phosphatase activities in the intestine and
Malpighian tubules. Kramer et al. (1991) could not find an effect of Manse-AS on the
JH biosynthesis in the CA of T. molitor. The whole genome sequences from T. castaneum
provided the opportunity to identify the genes for allatoregulating peptides and their
receptors (Amare and Sweedler, 2007; Hauser et al., 2008; Li et al., 2008). The
deduced amino acid sequences revealed the presence of three peptide precursors
encoding a potential coleopteran allatotropin, a type C allatostatin, and six peptides of
the type B allatostatin, whereas neither type A allatostatin gene nor its receptor were
found. Using MALDI-TOF mass spectrometry to identify neuropeptides of the brain
and CC-CA of adult T. molitor, again no evidence for the existence of a type A AS was
found (Weaver and Audsley, 2008). However, in that study, other candidates for the
regulation of JH synthesis, such as the B- and C-type allatostatins, were also
undetectable. The lack of information about the allatoregulating neuropeptides in
T. molitor, together with the recent findings of type A allatostatin-like compounds in
the brain/CC of T. molitor (Elliott et al., 2009; Wasielewski et al., 2009), motivated the
current investigation on the effects of putative allatostatins and an allatotropin on
the synthesis/release of JH III from CC-CA complexes, in vitro, of adult females of the
beetle. The reason why we used T. molitor instead of T. castaneum for our experiments
Archives of Insect Biochemistry and Physiology
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was that the adult T. molitor beetles are an average of 6.5-times longer and 60-times
heavier than T. castaneum (Weaver and Audsley, 2008), making it easier to dissect the
CC-CA complexes and to get reliable values for JH biosynthesis rates with single gland
incubations. This study assumes that sequences of allatoregulating peptides are
conserved between the two closely related tenebrionid beetles (Weaver and Audsley,
2008).
The type A allatostatin A2 region was similar in the precursors of all arthropod
species examined so far, except in T. castaneum. The AS A2 peptide contains a KR
cleavage site, but the cleavage may not occur in all species. We used the cleaved short
form of AS A2, the AS A2b in the bioassay, which is a potent myoinhibitor in
cockroaches, but does not exert much allatostatic activity in most insects (Pratt et al.,
1991). The short form consistently occurs in insects, where the CA are insensitive to
any type A AS (Meyering-Vos et al., 2001; Abdel-latief et al., 2004). Incubation of
the CC-CA complexes of T. molitor females with an intrinsic high JH synthesis activity
in the presence of the synthetic Peram-AS A2b peptide led to an unexpected,
but distinct inhibition of JH release in the concentration range of 10 8 to 10 4 M,
which was fully reversed when the glands were transferred to peptide-free medium.
The ED50 dose (10 5 M) was slightly higher than those for the other allatostatins
tested in this study, and much higher than for the effective type A allatostatins in
cockroaches or crickets (10 7 to 10 11 M; Bendena et al., 1997; Lorenz, 2001).
Whether type A AS peptides show an allatostatic activity also in vivo remains unclear,
because no FGLamide immunoreactivity was found within the CA of T. molitor (Elliott
et al., 2009).
On the basis of our results, we suggest that type B AS [W(X)6Wamides] act as true
allatostatins in T. molitor. The Trica-AS B3 peptide inhibited JH synthesis/release in CCCA complexes with a high as well as low intrinsic rate of JH production, and the ED50
values were only slightly higher than in crickets (Lorenz, 2001). Thus, Coleoptera
(beetles) is the second order of insects, after the Ensifera, where type B AS seem to
inhibit JH biosynthesis. Peptide injection studies and RNA interference experiments
should be performed to prove the putative in vivo allatostatic effects of type B AS in
beetles.
Activity of the type C Trica-AS (PISCF-OH allatostatin) depends on the age of the
test animals and their intrinsic rate of JH production in the CA, respectively.
Treatments of glands from 3-day-old females with Trica-AS C resulted in a dosedependent reduction of JH release with an ED50 value of about 5 10 6 M, whereas
incubation of the CC-CA-complexes from 7-day-old females led to a significant
increase in JH release. The PISCF-OH peptide described here is not the first such
peptide with an allatotropic activity. We characterized previously a peptide with
allatotropic activity from the fall armyworm, Spodoptera frugiperda (Abdel-latief et al.,
2004), with high similarity in its primary structure to the Manse-AS, but without the
disulfide bridge, characteristic of type C allatostatins. RNA interference studies showed
that this peptide has an allatostatic action in last instar larvae of S. frugiperda, whereas
gene silencing in young adult females led to a large decrease in the JH titer of the
animals (Griebler et al., 2008), which is consistent with an allatotropic activity of the
peptide. The dependence of the JH-regulating activity of type C AS on the rate of JH
production has been found also in other insect species and for other allatoregulating
peptides (Clark et al., 2008). The high concentration necessary for 50% change in
activity (in vitro) may be due to the fact that we used an N-terminally non-blocked
peptide in our experiments.
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Archives of Insect Biochemistry and Physiology, July 2010
Whether allatotropins act on the CA by direct stimulation or through ‘‘suppression’’ of the allatostatin-mediated inhibition of JH synthesis is still questionable
(Yamanaka et al., 2008; Weaver and Audsley, 2009). When we tested the synthetic
peptide Trica-AT (10 6 M) on the CC-CA complexes from 7-day-old females, a
significant increase in JH release was found. An even higher rate of JH release was
observed after addition of 10 6 M Trica-AT together with 10 6 M Trica-AS C to those
gland complexes.
In conclusion, regulation of JH biosynthesis in the CA of beetles (in vitro) seems to
be more complex than in other holometabolous insects. However, the primary
sequences of the specific allatoregulating compounds and their modes of action in
T. molitor (in vivo) remain to be determined.
ACKNOWLEDGMENTS
We thank Prof. Dr. Joseph Woodring (Bayreuth) for language corrections.
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neuropeptide, coleoptera, regulatory, beetle, molitor, juvenile, tenebrio, tenebrionidae, hormone, vitro, biosynthesis
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