Insect Biochemistry and Molecular Biology 30 2000 691–702 www.elsevier.comlocateibmb
Molecular characterization of a cDNA from the true armyworm Pseudaletia unipuncta
encoding Manduca sexta allatotropin peptide
1
Peter F. Truesdell
a
, Peter M. Koladich
b
, Hiroshi Kataoka
c
, Kuniaki Kojima
c
, Akinori Suzuki
c
, Jeremy N. McNeil
d
, Akira Mizoguchi
e
, Stephen S. Tobe
b
, William G. Bendena
a,
a
Department of Biology, Queen’s University, Kingston, ON, Canada
b
Department of Zoology, University of Toronto, Toronto, ON, Canada
c
Graduate School of Agriculture and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan
d
Departement de biologie, Universite Laval, Ste. Foy, P.Q., Canada
e
Biological Sciences, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya-shi, Aichi 464, Japan Received 31 October 1999; received in revised form 31 December 1999; accepted 25 January 2000
Abstract
Allatotropin AT is an insect neuropeptide isolated from the tobacco hornworm, Manduca sexta, stimulates juvenile hormone JH biosynthesis by the corpora allata. A cDNA isolated from the true armyworm, Pseudaletia unipuncta, encodes a 135 amino
acid AT precursor peptide which contains the AT peptide, with processing sites necessary for its endoproteolytic cleavage and amidation, plus two additional peptides of unknown function. The encoded AT peptide is identical to that isolated from M. sexta
and Agrius convolvuli.
Southern blot analysis indicated that AT is a single copy gene per haploid genome and is present in two allelic forms. A single transcript of approximately 1.5 kilobases was detected by northern blot analysis. The expression of the AT gene was analyzed
during development from sixth instar larvae to five day-old moths. Initial expression was observed in late pupae and this expression was maintained throughout the adult stages in both sexes. In one day-old moths, expression was at its lowest level of the stages
that express AT mRNA but levels increased in day 3 and day 5 adults. This pattern of AT expression in adult P. unipuncta moths mirrors that of JH biosynthesis and supports the notion that AT may act in the adult stages.
Immunohistochemistry and in situ hybridization revealed that AT expression was localized to numerous structures of the nervous system, suggesting that AT may have functions distinct from regulation of JH biosynthesis.
2000 Elsevier Science Ltd. All
rights reserved.
Keywords: Juvenile hormone; Lepidoptera; Neuropeptide; Insect
1. Introduction
Juvenile hormone JH exerts a central role in meta- morphosis, adult sexual maturation and reproduction in
most insect species Tobe and Stay, 1985. The
Abbreviations: AT, allatotropin; AST, allatostatin; CA, corpora allata;
NCC, nervi corporis cardiaci; SOG, suboesophageal ganglia; JH, juv- enile hormone.
Corresponding author. Tel.: +
1-613-533-6121; fax: +
1-613-533- 6617.
E-mail address: bendenawbiology.queensu.ca W.G. Bendena.
0965-174800 - see front matter
2000 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 5 - 1 7 4 8 0 0 0 0 0 4 0 - 0
biosynthesis of JH by the corpora allata CA of insects can either be stimulated or inhibited by neuropeptides
termed allatotropins ATs or allatostatins ASTs,
1
Peptide designations have been assigned using the first three let- ters of the Genus name followed by the first two letters of the species
name as proposed by the insect neuropeptide community attending the 19th Winter Neuropeptide Symposium, Breckenridge, CO, 1998. This
change was proposed to ensure a unique organism abbreviation as the number of organisms from which peptides are purified increases and
to conform with Swiss PRO. Thus Manse-AT is equivalent to the Man- duca sexta
allatotropin formerly abbreviated Mas-AT.
692 P.F. Truesdell et al. Insect Biochemistry and Molecular Biology 30 2000 691–702
respectively. Several different AST peptide sequences have been determined both within and between Orders
Bendena et al., 1997. Much less is known about AT although several allatotropic factors have been identified
in a variety of different insects. Lorenz and Hoffmann 1995 partially purified an allatotropic substance from
suboesophageal ganglion SOG extracts of crickets, Gryllus bimaculatus
and Acheta domesticus, which stimulated JH biosynthesis in CA. In adult Locusta
migratoria , allatotropic activity was detected in the
SOG, brain and corpora cardiaca CC Gadot et al., 1987. Gradient separation of extracts of each of these
tissues suggested the presence of more than one allato- tropic factor with molecular mass estimated to be 0.7 to
2.0 kDa Rembold et al., 1986; Ferenz and Diehl, 1983. In contrast, a much larger 20 kDa AT stimulated JH
biosynthesis in the wax moth, Galleria mellonella Bogus and Scheller, 1994. Immunocytochemical analy-
sis localized this protein to two medial neurosecretory cells in the pars intercerebralis PI and to the CC Bogus
and Scheller, 1994. Cells of the PI were implicated as a source of AT in G. mellonella, as they elicited super-
numerary larval moults when implanted into fifth instar larvae and stimulated JH biosynthesis in vitro by larval
CA Muszynska-Pytel, 1987. Removal of these medial cells from the brain destroyed the allatotropic activity
Bogus and Scheller, 1994.
There appears to be a large discrepancy in molecular mass between allatotropic factors found in different
stages of development andor species of insects. In con- trast to the 20 kDa AT in larval brains reported by Bogus
and Scheller 1994, an a-amidated AT peptide Manse-
AT thirteen residues in length, has been purified from head extracts of pharate adult Manduca sexta Kataoka
et al., 1989. To date, this is the only known AT whose primary structure has been determined. This Manse-AT
stimulated JH biosynthesis by CA of adult M. sexta but had no effect on the CA of larvae or pupae of this spec-
ies. It also strongly stimulated CA activity of the adult female moth, Heliothis virescens, but was unable to do
so in the beetle, Tenebrio molitor, the grasshopper, Schi- stocerca nitens
, or the cockroach, Periplaneta americana Kataoka et al., 1989. This evidence suggests that the
activity of Manse-AT may be restricted to the adult stage of Lepidoptera.
Taylor et al. 1996 isolated and sequenced a Manse- AT genomic clone from M. sexta and provided evidence
to suggest that the Manse-AT gene is expressed as three alternatively spliced mRNAs which encode distinct pre-
cursor proteins which upon processing would release the AT peptide. Manse-AT gene is expressed in the brain
and nerve cord of larvae, pupae and adults of M. sexta Taylor et al., 1996. Manse-AT has no effect on CA of
M. sexta
larvae, and pupal CA do not produce appreci- able JH, so the expression of Manse-AT in these stages
of development may be related to activities independent of the stimulation of JH biosynthesis in the adult CA.
Functional assays and distribution studies support additional roles for Manse-AT. Manse-AT has been
shown to function as a cardioaccelerator in adult M. sexta
Veenstra et al., 1994 and inhibit midgut ion trans- port in day 2 fifth instar M. sexta larvae Lee et al.,
1998. Multiple functional activities is also suggested by the distribution of Manse-AT immunoreactive cells
throughout M. sexta development that encompasses numerous structures in the nervous system Veenstra and
Hagedorn, 1993; Veenstra et al., 1994; Zitnan et al. 1993, 1995. Immunological evidence suggests that
Manse-AT-like peptides may be present in the nervous system of other insects, e.g. Drosophila melanogaster
Zitnan et al., 1993, however, the function of these pep- tides in these species remain unknown. It is possible that
distantly related insects contain very different ATs. The inability of Manse-AT to stimulate JH biosynthesis in
species outside the Order Lepidoptera suggests that if similar peptides do exist, they are not involved in reg-
ulating CA activity.
Barth 1965 proposed that in long lived insects, where mating may be suppressed for varying lengths of
time, a complex neuroendocrine system would have evolved to regulate pheromone production and mating
behaviour. This has been examined in the true army- worm, Pseudaletia unipuncta, a migrant species, and
several aspects of reproduction in both sexes involve JH. JH is involved, either indirectly or directly, in the regu-
lation of the female pheromone communication system and oocyte maturation, including vitellogenin synthesis
Cusson and McNeil, 1989a,b; Cusson et al., 1990, 1994b. In males, JH plays a role in the regulation of
the responsiveness to the female sex pheromone and, probably in the development of accessory reproductive
glands Cusson et al., 1994a. In some species of insects, there is evidence to suggest that migration is also under
the control of JH Rankin et al., 1986, with different JH titer thresholds for either migration or reproduction
Rankin and Riddiford, 1978. In response to cues of an impending habitat deterioration the titer of JH would
exceed a minimal level to initiate migration but not the higher threshold required for the initiation of sexual
maturation and reproduction. In contrast, under favor- able conditions the titer of JH would rise rapidly to
exceed the upper threshold to promote events associated with reproduction.
Because of the differences in their life history stra- tegies, it was of interest to know whether M. sexta AT
peptide and an AT gene of similar organization would be present in P. unipuncta. As Manse-AT stimulated JH
biosynthesis in M. sexta and H. virescens, the expression of the AT gene in P. unipuncta was examined through-
out development to determine if abundant AT mRNA levels coincided with high release rates of JH, and to
693 P.F. Truesdell et al. Insect Biochemistry and Molecular Biology 30 2000 691–702
determine if there was any sex- or stage-specific expression indicative of additional functions.
Specific regions of the brain are known to synthesize AT peptides, which are transported to the CA and influ-
ence its activity. The cellular localization of AT expression was determined, to provide evidence for a
role in JH regulation and to provide insight into additional functions for this peptide in P. unipuncta.
2. Materials and methods
