Insect Biochemistry and Molecular Biology 30 2000 681–689 www.elsevier.comlocateibmb
Juvenile hormone biosynthesis by corpora allata of larval tomato moth, Lacanobia oleracea, and regulation by Manduca sexta
allatostatin and allatotropin
Neil Audsley , Robert J. Weaver, John P. Edwards
Central Science Laboratory, Sand Hutton, York YO41 1LZ, UK Received 31 October 1999; received in revised form 31 December 1999; accepted 25 January 2000
Abstract
Juvenile hormone JH biosynthesis and the effects of synthetic Manduca sexta allatostatin Mas-AS and M. sexta allatotropin Mas-AT were investigated in isolated corpora allata CA of Vth stadium larvae of the tomato moth, Lacanobia oleracea. Reversed-
phase high-performance liquid chromatography RP-HPLC of JH extracted from CA shows that larvae produce predominantly JH II and its corresponding acid. It appears that the acid homologue is a result of JH esterase activity in the CA and other tissues
rather than the lack of JH acid methyltransferase. Mean rates of synthesis 100–200 fmolprh were inhibited ca. 70 by Mas-AS and stimulated in a dose-dependent manner up to three times by Mas-AT. However, Mas-AS had no significant effect on Mas-AT-
stimulated rates of JH biosynthesis. Using RP-HPLC and an enzyme-linked immunosorbent assay ELISA to Mas-AT, a peak of Mas-AT-like immunoreactivity was detected in larval L. oleracea brain homogenates. Co-elution of this immunoreactive peak with
synthetic Mas-AT suggests that this neuropeptide is also present in L. oleracea. Crown Copyright
2000 Published by Elsevier
Science Ltd. All rights reserved.
Keywords: Allatostatin; Allatotropin; Corpora allata; Juvenile hormone; Juvenile hormone acid; Insect; Lacanobia; Manduca; Neuropeptide
1. Introduction
Very little is known about the nature of the biosynth- esis of juvenile hormone JH in the corpora allata CA
of larval Lepidoptera, research in this area having been restricted to the tobacco hornworm, Manduca sexta
Schooley et al., 1973; Kramer and Law, 1980; Bhaska- ran, 1981; Sparagana et al., 1984; Bhaskaran et al. 1986,
1987; Janzen et al., 1991, the gypsy moth, Lymantria dispar Jones and Yin, 1989, the Egyptian cotton leaf-
worm, Spodoptera littoralis Pfister-Wilhelm and Lanzr- ein, 1996, the wax moth, Galleria mellonella Bogus
and Scheller 1991, 1996, and more recently the tomato moth, Lacanobia oleracea Audsley et al., 1999a,b.
Consequently, the hormonal control of JH biosynthesis
Abbreviations: Manduca sexta
allatostatin Mas-AS
pEVRFRQCYFNPISCF–OH; Manduca sexta allatotropin Mas-AT GFKNVEMMTARGF–NH
2
. Corresponding author. Tel.:
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462111. E-mail address: n.audsleycsl.gov.uk N. Audsley.
0965-174800 - see front matter. Crown Copyright
2000 Published by Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 5 - 1 7 4 8 0 0 0 0 0 3 9 - 4
by CA in larval Lepidoptera has received even less atten- tion and has only been investigated in M. sexta Kataoka
et al., 1989; Kramer et al., 1991; Janzen et al., 1991, Pseudaletia unipuncta Jansons et al., 1996, G. mel-
lonella Bogus and Scheller 1991, 1996 and L. oleracea Audsley et al., 1999b. From the pharate adult of M.
sexta two peptides were characterised which regulate JH biosynthesis by CA in vitro: a stimulatory allatotropin
Mas-AT; Kataoka et al., 1989 and an inhibitory allatos- tatin Mas-AS; Kramer et al., 1991. However, although
Mas-AT stimulates JH biosynthesis in isolated adult female M. sexta and Heliothis virescens CA, it was
reported to have no activity in larval M. sexta Kataoka et al., 1989, leading to the suggestion that Mas-AT was
adult-specific. In contrast, Mas-AS inhibits JH biosynth- esis in vitro by CA from both larval and adult female
M. sexta and adult female H. virescens Kramer et al., 1991. An allatotropic factor was partially purified from
larval G. mellonella brains Bogus and Scheller, 1996 which promotes in vitro and in vivo synthesis of JH.
By use of immunochemical and molecular techniques, Mas-AS has been shown to be present in other larval
682 N. Audsley et al. Insect Biochemistry and Molecular Biology 30 2000 681–689
Lepidoptera, namely P. unipuncta Jansons et al., 1996, L. oleracea Audsley et al., 1998 and S. littoralis
Audsley et al., 1999a, but to date no inhibitory activity of Mas-AS has been demonstrated in any of these larvae.
Synthetic Mas-AS does, however, cause up to 60 inhi- bition of JH biosynthesis in CA of adult female P.
unipuncta Jansons et al., 1996 and 50–60 inhibition of CA output in adult male and female L. oleracea
Audsley et al., 1999a,b. Recently, Oeh et al. 1999 reported that Mas-AS is active on CA of adult female
Spodoptera frugiperda, but only on Mas-AT-stimulated levels of JH production.
Bhatt and Horodyski 1999 have demonstrated Mas- AT mRNA and immunoreactivity in M. sexta larval ner-
vous system, but this peptide does not stimulate JH biosynthesis in larval CA of M. sexta Kataoka et al.,
1989 or L. oleracea Audsley et al., 1999b. M. sexta allatotropin does, however, stimulate JH synthesis in CA
of adult female M. sexta Kataoka et al., 1989, L. olera- cea Audsley et al., 1999b and S. frugiperda Oeh et
al., 1999. Audsley et al. 1999a,b suggest that the low rates of JH biosynthesis observed in larval L. oleracea
and the apparent inactivity of Mas-AS and Mas-AT may be due to the assay procedures used to measure JH lev-
els, rather than to an actual lack of bioactivity.
This study investigates these possibilities and deter- mines that JHs produced by CA of larval L. oleracea
are converted to acid homologues by JH esterases, and reports for the first time the regulation of JH biosynthesis
in the CA of a larval lepidopteran by both an allatostatin and allatotropin first characterised from M. sexta
Kataoka et al., 1989; Kramer et al., 1991. In addition to the identification of Mas-AS in L. oleracea Audsley
et al., 1998, evidence is presented that a Mas-AT immu- noreactive peptide is present in this noctuid.
2. Materials and methods
