Insect Biochemistry and Molecular Biology 30 2000 703–710 www.elsevier.comlocateibmb
Injection of Dip-allatostatin or Dip-allatostatin pseudopeptides into mated female Diploptera punctata inhibits endogenous rates of JH
biosynthesis and basal oocyte growth
Chris S. Garside
a
, Ronald J. Nachman
b
, Stephen S. Tobe
a,
a
Department of Zoology, University of Toronto, Toronto, On, M5S 3G5, Canada
b
Veterinary Entomology Research Unit, Southern Plains Area Research Center, ARS, US Department of Agriculture, 2881 FB Road, College Station, TX 77845, USA
Received 31 October 1999; received in revised form 31 December 1999; accepted 25 January 2000
Abstract
Studies on the catabolism of allatostatins ASTs provided the rationale for the design of a series of Dip-allatostatin-derived pseudopeptide mimetic analogues. In vitro, the Dip-ASTs and pseudopeptides show varying degrees of resistance to catabolism
and all show significant inhibition of juvenile hormone JH biosynthesis. This study was undertaken to determine whether potent Dip-ASTs andor their pseudopeptide mimetic counterparts caused ‘allatostatic’ effects in vivo following injection into mated female
Diploptera punctata. Animals injected with aqueous solvent or Dip-AST 71–7 N-terminal fragment, which excludes the active core region of the ASTs, were used as controls. An in vitro radiochemical assay revealed that injection of Dip-AST 5, 7 or
pseudopeptide analogues 397-2 or ASTb
φ 2 significantly inhibited the biosynthesis of JH P,0.05. The results also indicate that
basal oocyte growth was significantly inhibited by injection of these same compounds, with the exception of Dip-AST 7 P,0.05. Analogues 396-1 and 419 did not significantly inhibit rates of JH biosynthesis but did significantly inhibit the growth of basal
oocytes. Analyses of feeding, excretion and food absorptionutilization patterns of these same animals suggested that these com- pounds are not toxic to the insect; rather they directly inhibit the biosynthesis of JH by the corpora allata, and reduce the rate of
growth of basal oocytes. Disruption of critical reproductive andor developmental processes by pseudopeptide analogues of the ASTs could provide novel and selective strategies for future insect pest management.
2000 Elsevier Science Ltd. All rights reserved.
Keywords: Allatostatin; Juvenile hormone biosynthesis; Diploptera punctata; Pseudopeptide mimetic analogues
1. Introduction
Cockroach allatostatins ASTs are a family of pep- tides, six to 18 amino acids in length, sharing the com-
mon C-terminus YFXFGLamide. This pentapeptide sequence is the minimal sequence required for inhibition
of juvenile hormone JH biosynthesis in vitro and is generally regarded as the core sequence responsible for
direct receptor interaction. In many insect species, a reduction in endogenous titres of JH is critical for meta-
morphosis from the nymph to the adult stage, whereas in adult females, oocyte growth and maturation show an
Corresponding author. Tel.: +
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1-416-978- 3522.
E-mail address: stephen.tobeutoronto.ca S.S. Tobe.
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 1 - 2
absolute dependence on the presence of juvenile hor- mones. ASTs were first isolated from the Pacific beetle
cockroach, Diploptera punctata Woodhead et al. 1989, 1994; Pratt et al. 1989, 1991. Additional members of
this family that inhibit the biosynthesis of JH in the hom- ologous insect have since been isolated from the cock-
roaches Periplaneta americana Weaver et al., 1994; Predel et al., 1999 and Blattella germanica Belle´s et
al., 1994, and the cricket Gryllus bimaculatus Lorenz et al., 1995. Following their designation as ‘allatostat-
ins’, these peptides have been shown to be modulators of muscle contraction in vitro Lange et al. 1993, 1995,
inhibitors of vitellogenin production by fat body Martı´n et al., 1996, inhibitors of ovarian ecdysteroid biosynth-
esis Lorenz et al., 1998 and, most recently, stimulators of the activity of carbohydrate-metabolizing enzymes in
midgut Fuse´ et al., 1999.
704 C.S. Garside et al. Insect Biochemistry and Molecular Biology 30 2000 703–710
Dip-ASTs are released directly at the corpora allata CA andor indirectly by release from the corpora car-
diaca Stay et al., 1992, midgut endocrine cells Yu et al., 1995 or potentially from haemocytes Skinner et al.,
1997, travelling by way of the haemolymph to the CA or other targets. In vivo, ASTs in the haemolymph may
inhibit JH biosynthesis by the CA. However, AST bioac- tivity can be reduced and ultimately terminated through
catabolism by soluble haemolymph enzymes and by membrane-bound proteolytic enzymes Garside et al.,
1997a,b. The observed rates of catabolism suggest that haemolymph ASTs may not be effective humoral inhibi-
tors of JH biosynthesis.
The results of these metabolic studies led to the design of a series of Dip-AST-derived pseudopeptide mimetic
analogues. Our approach to the design of Dip-AST ana- logues with reduced susceptibility to metabolic inacti-
vation has been both to eliminate those amino acids that are not required for biological activity by replacement
with non-peptide moieties and to increase the rigidity of the molecule Nachman et al., 1998. Radiochemical
assay for JH biosynthesis indicates that the activities of these analogues approach those of the native neuropep-
tides Nachman et al. 1997, 1999. Studies on the catab- olism of Dip-AST analogues showed that selected ana-
logues had increased resistance to catabolism by enzymes in the haemolymph or on the surface of tissues
Garside et al., 1997c; Nachman et al., 1999. It is likely that the significant biological activity of these peptidomi-
metics is attributable in part to their increased resistance to catabolism.
The effects of ASTs in vivo have been comparatively understudied. Woodhead et al. 1993 observed a sig-
nificant reduction in both rates of JH biosynthesis and in length of basal oocytes with Dip-AST 7 but only a
significant reduction in rates of JH biosynthesis, not in length of basal oocytes, following injection of Dip-AST
2. In virgin P. americana, Weaver et al. 1995 demon- strated that both Dip-AST 5 and 7 were effective in low-
ering total body JH III levels 12 h post injection, but not after 2 or 24 h. Injections of Dip-AST 7 into mid-cycle
mated females produced no apparent effect but injection of Dip-AST 5 did result in a substantial reduction of
endogenous total body JH III levels. Lorenz et al. 1998 injected Grb-AST A1 or B1 into G. bimaculatus and
observed reductions in a number of physiological para- meters, including ovarian ecdysteroid biosynthesis and
haemolymph vitellogenin titres compared with Ringer- injected controls.
Piulachs et al. 1997 synthesized a methyleneamino and a ketomethylene AST analogue with the aim of
increasing resistance to degradation of ASTs by haemo- lymph peptidases. They showed that both analogues
were similarly active to the model peptides with respect to the inhibition of JH biosynthesis in vitro from CA of
virgin B. germanica. The methyleneamino analogue was less active as an inhibitor of vitellogenin production in
vitro by the fat body of B. germanica, but was more active in vivo in terms of both inhibition of JH biosynth-
esis and as an inhibitor of vitellogenin production by the fat body.
In this study, we investigated the effects of injection of Dip-AST or Dip-AST analogue on rates of JH
biosynthesis by CA in vitro and on basal oocyte growth of mated female D. punctata. Disruption in the cycle of
JH biosynthesis could inhibit production or release of vitellogenin by fat body or uptake of vitellogenin by
oocytes. Alternatively, reduction in the growth of basal oocytes may inhibit production of JH, either neurally or
by release of an unknown humoral factors.
2. Materials and methods
