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
2.1. Synthesis of allatostatin analogues The AST pseudopeptide mimetic analogues 396-1
Ala–Arg–Pro–Tyr–Asn–Aic–Gly–Leu–NH
2
, Aic
= 2-
amino-indane-2-carboxyl-, 397-2 Ala–Arg–Pro–Tyr– Asn–Phe–Cpa–Leu–NH
2
, Cpa
= cyclopropylAla-,
ASTb φ
2 Hca–Asn–Phe–Cpa–Leu–NH
2
, Hca =
hydroc- innamyl- and 419 Ala–Arg–Pro–Tyr–Asn–Aic–Cpa–
Leu–NH
2
were synthesized as previously described Nachman et al. 1998, 1999.
2.2. Animals Newly emerged mated females were isolated from the
stock culture and kept at 27 ±
1 °
C until dissection and experimental manipulations. The relative humidity was
approximately 50 ±
5 with a 12 h light12 h dark cycle. Insects were reared on Purina Lab Chow and water ad
libitum. Mating was confirmed by the presence of a sper- matophore. Day 4 mated females were the source of
corpora allata for analysis of JH biosynthesis and for measurement of basal oocyte length.
2.3. Degradation assay Haemolymph collection and assay were as previously
described Garside et al., 1997a. Tissue collection, membrane preparations and assay
were as previously described Garside et al., 1997b. 2.4. Injections
All injections were administered to non-anaesthetized mated female D. punctata. Water was chosen as the sol-
vent for injected compounds because Woodhead et al. 1993 found a greater effect on rates of JH biosynthesis
and length of basal oocytes following injection of cock- roach
saline than
of water
B. Stay,
personal
705 C.S. Garside et al. Insect Biochemistry and Molecular Biology 30 2000 703–710
communication. Assuming a haemolymph volume of 50
µ l for adult female D. punctata Mundall et al., 1981,
the initial concentration of the injected peptide or ana- logue in the haemolymph was approximately 100
µ M.
Injections of 6 nmol 1 nmol µ
l were administered once daily, approximately 24 h apart on days 0–3 inclusive,
using a 26 gauge 10 µ
l Hamilton syringe. The needle was inserted into the membranous joint between the coxa
and femur on the metathoracic leg. Control insects were similarly injected, but with 6
µ l of autoclaved, double
distilled water. On day 4, approximately 18 h after day 3 injection, animals were sacrificed; JH biosynthesis by
CA was measured in vitro by the radiochemical assay Tobe and Clarke, 1985 and the length of basal oocytes
was measured using an ocular micrometer. Each group of peptide-injected animals 4
× 6 nmol was compared
with a group of water-injected animals treated concur- rently.
2.5. Animal weight, feeding, excretion and soluble protein content of fat body
On days 1 to 4 d
1–4
, animals were measured for food consumption, production of frass, and weight. Food pel-
lets and animals were weighed prior to segregation in jars. Food consumption and production of frass were
each approximated by weighing of food pellets and frass approximately 18 h following the previous day’s injec-
tion on each of d
1–4
. Food consumption was determined by the subtraction of weight of food pellet on d
X
from weight on d
X21
for each day. Animal weight was meas- ured approximately 18 h following the previous days
injection on each of days 1 to 4. Percentage weight gain
= [Weight
d4
2Weight
d0
Weight
d0
] ×
100. Soluble
protein content of fat body from day 4 mated females was also determined in these same animals by transfer
of abdominal fat body from each animal to a pre- weighed vessel. Excess fluid was removed by blotting
with filter paper and the wet weight measured. The fat body was homogenized in 1 N NaOH and centrifuged at
4000g for 10 min. The quantity of soluble protein was determined by the Bradford 1976 method using bovine
serum albumin as standard. Data are presented as
µ g
proteinmg fat body. 2.6. Radiochemical assay
Rates of JH release were determined by the in vitro radiochemical method of Feyereisen and Tobe 1981
and Tobe and Clarke 1985. The incorporation of l-[
14
C-S-methyl]-methionine 50 µ
M, specific radioactivity 1.48–2.03 GBqmmol; Amersham into JH III by pairs
of CA incubated in 100 µ
l TC 199 GIBCO; 1.3 mM Ca
2 +
, 2 Ficoll, methionine-free was used to quantify JH release. Animals were anaesthetized on ice prior to
dissection. Corpora allata were dissected directly into non-radioactive medium. Individual pairs of CA were
transferred from non-radioactive medium to radioactive medium and were incubated for 3 h. The amount of inhi-
bition was expressed as the percentage reduction from the
untreated rate:
[12treated rateuntreated
rate] ×
100. 2.7. Statistics
Data were analysed using a one-way analysis of vari- ance ANOVA with a Dunnett’s multiple comparison
test as the post hoc determination of significance and a Student’s t-test using Prism Graph Pad. Variability is
reported as ±
standard error of the mean SEM.
3. Results
