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Effect of ace inhibitors and TMOF on growth development and trypsin activity of larval Spodoptera littoralis.

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Archives of Insect Biochemistry and Physiology 69:199–208 (2008)
Effect of Ace Inhibitors and TMOF on Growth,
Development, and Trypsin Activity of Larval
Spodoptera littoralis
Els Lemeire,1,3 Dov Borovsky,2 John Van Camp,3 and Guy Smagghe1
Angiotensin converting enzyme (ACE) is a zinc metallopeptidase capable of cleaving dipeptide or dipeptideamide moieties at the
C-terminal end of peptides. ACE is present in the hemolymph and reproductive tissues of insects. The presence of ACE in the
hemolymph and its broad substrate specificity suggests an important role in processing of bioactive peptides. This study reports
the effects of ACE inhibitors on larval growth in the cotton leafworm Spodoptera littoralis. Feeding ACE inhibitors ad lib decreased
the growth rate, inhibited ACE activity in the larval hemolymph, and down-regulated trypsin activity in the larval gut. These
results indicate that S. littoralis ACE may influence trypsin biosynthesis in the larval gut by interacting with a trypsin-modulating
oostatic factor (TMOF). Injecting third instar larvae with a combination of Aea-TMOF and the ACE inhibitor captopril, downregulated trypsin biosynthesis in the larval gut indicating that an Aea-TMOF gut receptor analogue could be present. Injecting
captopril and enalapril into newly molted fifth instar larvae stopped larval feeding and decreased weight gain. Together, these
results indicate that ACE inhibitors are efficacious in stunting larval growth and ACE plays an important role in larval growth and
development. Arch Insect Biochem Physiol. 69:199–208, 2008.
& 2008 Wiley-Liss, Inc.
KEYWORDS: angiotensin converting enzyme; captopril; enalapril; larval development; Lepidoptera; ACE activity; AeaTMOF; L-BApNA; trypsin activity
INTRODUCTION
Little is known about the in vivo role of the
invertebrate angiotensin converting enzyme (ACE).
The vertebrate counterpart, mammalian ACE
(peptidyl dipeptidase A, EC 3.4.15.1) plays an
important role in blood pressure regulation.
It activates angiotensin I by converting it into the
vasoconstrictive peptide angiotensin II and deactivates the vasodilator, bradykinin I, resulting in an
increase in blood pressure (Erdös and Skidgel,
1987). The prevalence of insect ACE in hemo-
lymph (Macours et al., 2003; Ekbote et al., 2003,
Lemeire et al., 2008), together with its broad in
vitro substrate specificity suggests a role in the
processing of neuropeptides and peptide hormones (Isaac et al., 2000). The first described insect ACE from the housefly Musca domestica
hydrolyzes several C-terminally amidated neuropeptides like leucokinin I and II, locustatachykinin
I and II, allatostatin I, SchistoFLRFamide, and
Culex depolarizing peptides I and II (Lamango
et al., 1997). These results show that M. domestica
ACE can hydrolyse peptides from the small kinin
1
Laboratory of Agrozoology, Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium
2
University of Florida, Institute of Food and Agricultural Sciences, Florida Medical Entomology Laboratory, Vero Beach, Florida
3
Research Group of Food Chemistry and Human Nutrition, Department of Food Safety and Food Quality, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium
Contract grant sponsor: Special Research Foundation of Ghent University; Contract grant number: BOF01102703; Contract grant sponsor: Fund for Scientific Research (FWO-Vlaanderen, Brussels,
Belgium).
*Correspondence to: Guy Smagghe, Laboratory of Agrozoology, Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium
E-mail: guy.smagghe@ugent.be
Received 12 December 2007; Accepted 1 March 2008
& 2008 Wiley-Liss, Inc.
DOI: 10.1002/arch.20270
Published online in Wiley InterScience (www.interscience.wiley.com)
200
Lemeire et al.
_____________________________________________________________
family. Insect neuropeptides of the kinin class
are characterized by their C-terminally amidated
pentapeptide sequence Phe-X1-X2-Trp-Gly-NH2
(X1 5 His, Asn, Phe, Ser, or Tyr; X2 5 Pro, Ser, or
Ala), which represents their active core. They have
been isolated from a number of insects and stimulate contractions in the isolated cockroach
hindgut and are also involved in ion and water
balance through their diuretic activity (Holman et
al., 1987; Coast et al., 1990; Zubrzak et al., 2007).
Kinins and kinin analogues also inhibit weight
gain in larval Heliothis virescens and Helicoverpa zea
(Nachman et al., 2002; Seinsche et al., 2000). Kinin peptide analogues that are resistant to housefly
ACE and Drosophila Ance, respectively, were synthesized by incorporating the sterically hindered
aminoisobutyric acid (Aib) or by replacing a-amino acids by b-amino acids (Nachman et al., 2002;
Zubrzak et al., 2007). These reports indicate that
ACE can function as a peptide-metabolizing enzyme controlling a key metabolic pathway regulated by peptide hormones.
Neobellieria bullata trypsin-modulating oostatic
factor (Neb-TMOF, NPTNLH), which inhibits
both trypsin and ecdysone biosynthesis, needs
ACE for activation. When ACE was inhibited by
captopril and lisinopril, Neb-TMOF’s hydrolysis
was also inhibited. It was suggested that a cleavage
product by itself is the most active trypsin and
ecdysone inhibitor (Zhu et al., 2001). Vandingenen et al. (2001) reported that Neb-TMOF is the
first known in vivo ACE substrate. A complete
knock down of ACE in the hemolymph caused
an up-regulation of trypsin activity in the gut
after a liver meal and elevated levels of yolk
polypeptides in adult fleshflies. A. aegypti TMOF
(Aea-TMOF), on the other hand, down-regulates
trypsin biosynthesis in the female mosquito gut.
It is secreted from the ovary after a blood meal
and binds to a gut receptor on the hemolymph
side of the gut (Borovsky et al., 1994). Aea-TMOF
and Neb-TMOF have different amino acids
and are different, although both are resistant to
degradation by gut proteolytic enzymes. AeaTMOF is a decapeptide with six proline residues
at the C-terminal tail forming a very stable three-
dimensional structure (Curto et al., 1993). It inhibits trypsin biosynthesis in the gut of A. aegypti
and Culex quinquefasciatus larvae, stunts larval
growth and development, and causes anorexia,
starvation, and death (Borovsky and Meola,
2004). Injecting Aea-TMOF into second and fourth
instar larvae of H. virescens also stopped trypsin
biosynthesis (Nauen et al., 2001), suggesting
that trypsin biosynthesis in this lepidopteran is
regulated by a TMOF-like factor, closely related to
Aea-TMOF.
Recently, we described the characterization
of SlACE, an angiotensin-converting enzyme of the
cotton leafworm Spodoptera littoralis (ABW34729)
(Lemeire et al., 2008). S. littoralis is an economically important pest insect, causing high damage
in cotton, maize, and many vegetables around
the world; besides many populations have developed high levels of resistance to conventional
and new insecticide groups (Smagghe and
Degheele, 1997). Since the role of ACE in insect
larval growth and development is not known, we
applied ACE inhibitors to S. littoralis larvae. This
report describes the effect of ACE inhibitors
and Aea-TMOF on trypsin activity and larval
development.
MATERIALS AND METHODS
Insects
An established colony of the cotton leafworm
S. littoralis was reared as described (Smagghe et al.,
2002). Larvae were fed a regular wheat germ–based
artificial diet (Poitout et al., 1972). Under these
conditions, the duration of the 1st (L1) until the 5th
(L5) larval stage is about 3 days each, the sixth and
last larval stage (L6) takes approximately 6 days;
larval stages were determined by measuring
the head capsule width (Degheele, 1987). ACE
inhibitors were fed to the larvae using Premix food
(Stonefly Industries, Inc., Bryan, TX) (Marchetti
et al., 1998; Krishnan and Kodrı́k, 2006). This is a
wheat-germ artificial diet that can be prepared by
adding cold water; ACE inhibitors were mixed
during the preparation of the diet when needed.
Archives of Insect Biochemistry and Physiology December 2008
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Effect of Ace Inhibitors and TMOF on S. littoralis
Chemicals
Captopril (D-3-mercapto-2-methyl-propionylL-proline), enalapril-maleate salt (Na-[(S)-1-ethoxycarbonyl-3-phenylpropyl]-L-analyl-L-proline maleate salt) and the trypsin substrate N-benzoylL-arginine p-nitroanilide (L-BApNA) were purchased from Sigma (Bornem, Belgium). Lisinopril
(Na-[(S)-1-carboxy-3-phenylpropyl]-L-lysyl-L-proline)
was provided by Merck & Co., Inc. (Whitehouse
Station, NJ). Aedes aegypti trypsin modulating
oostatic factor was available in house. The
fluorogenic ACE substrate ortho-aminobenzoic
acid-Phe-Arg-Lys-2,4-dinitrophenyl-Pro (Abz-FRK(Dnp)P-OH) was a kind gift of A. K. Carmona
(Universidade Federal de Sao Paulo, Escola Paulista de Medicina, Departamento de Biofisica, Sao
Paulo, Brazil). All other chemicals were purchased
from Sigma (Bornem, Belgium) unless stated
otherwise.
Effects of ACE Inhibitor Feeding on Larval Growth
Newly hatched larvae (L1, day 1) were transferred to Premix diet. The diet was treated by adding
5 mg captopril, enalapril, or lisinopril per mg diet.
Control diet did not contain any inhibitor. Premix
control and treated diets were refreshed when needed. Larval bioassays were repeated twice each, with
at least 10 larvae per treatment, placed individually
in 25-well plates. Larval age and weight were evaluated regularly until the larval-pupal molt. The
duration of the larval period was determined for
each treatment. Larval period started at L1 day 1,
until the pre-pupal stage. During treatment, hemolymph was collected to assess the effect on ACE
activity. Therefore, larvae of stage L5, 24712 h old
and larvae of stage L6, 72712 h old were selected.
After collecting hemolymph, guts were isolated
from these larvae to determine trypsin activity.
Injection of ACE Inhibitors and/or Aea-TMOF Into
Larvae
Third instar larvae (24 h old) and newly molted
fifth instar larvae and fifth instar larvae 24 h old
Archives of Insect Biochemistry and Physiology December 2008
201
were used in experiments. Larvae were fed on artificial diet. ACE inhibitors and TMOF were prepared in insect Ringer (86 mM NaCl, 5.4 mM KCl,
3 mM CaCl2.2H20) (Nauen et al., 2001). Stock
solutions of ACE inhibitor captopril (40 mg/ml) and
enalapril (20 mg/ml) were prepared in Ringer. L3
larvae were injected into the dorsal abdomen with
0.5 ml Ringer containing 20 mg of captopril or 10 mg
of enalapril using finely drawn glass capillary
tubes. Prior to injections, larvae were anaesthetized
with diethyl ether. L3 larvae were also injected with
20 mg of Aea-TMOF, a combination of 20 mg captopril and 20 mg Aea-TMOF and a combination of
10 mg enalapril and 10 mg Aea-TMOF. L5 larvae
were injected with 80 mg of captopril or 40 mg of
enalapril in a volume of 2 ml. In the control groups,
larvae were injected with the corresponding volume of Ringer. After treatment, individual larvae
were placed into a 25-well plate containing artificial diet. Larval weight was measured before
the injections and at 48 h (captopril) or 24 h
(enalapril) later. At these intervals, guts of L3 larvae were dissected out and separately homogenized and assayed for trypsin activity. The
inhibition of ACE activity in the hemolymph was
analyzed 24 h after treating L5 larvae.
Hemolymph ACE Activity and Inhibition Assays
Larvae were fed or injected with ACE inhibitors.
To measure the activity of ACE in the hemolymph
after feeding or injecting ACE inhibitors, control
and treated larvae were cold anaesthetized and a
leg was cut off. Hemolymph (30–150 ml/larva) was
collected in an Eppendorf tube. The protein content was measured by a Coomassie Bradford assay
and diluted with Tris buffer (10 mM Tris, pH 7.4)
to 500 mg/ml. Protein concentration of the diluted
samples was determined in the same assay.
ACE activity measurements were performed as
described previously (Lemeire et al., 2007) with the
fluorescent substrate Abz-FRK-(Dnp)P-OH, which
has an lem 5 420 nm and an lex 5 320 nm (Alves
et al., 2005). Briefly, a 10-ml sample was added to
170 ml of MOPS buffer [50 mM 3-(N-morpholino)propanesulfonic acid, 50 mM NaCl, pH 8]
202
Lemeire et al.
_____________________________________________________________
followed by 20 ml of a 50-mM Abz-FRK-(Dnp)P-OH
stock solution in dimethyl sulfoxide to a final
volume of 200 ml. Hydrolysis of the substrate
by ACE was kinetically measured at 241C with a
SPECTRAmaxs Gemini XS spectrofluorometer
(Sopachem, Wageningen, The Netherlands).
The inhibition of ACE activity was calculated by
expressing the specific ACE activity (relative fluorescence units/time unit/mg protein) (Lemeire et al.,
2007) of treated larvae relative to the specific ACE
activity of control larvae. Each value is a mean of at
least three independent samples of each treatment.
Trypsin Activity in the Gut
Whole guts of treated and control larvae were
isolated in Ringer solution, rinsed in Tris buffer
(10 mM Tris-HCl, pH 7.4), and transferred to an
Eppendorf tube. The guts were homogenized in
Tris buffer with a hand homogenizer and centrifuged (21,460 g, 15’, 41C), and the supernatant
was transferred to a clean tube and stored at 201C
until used. Protein concentration was measured by
the Coomassie Bradford assay. Trypsin activity was
determined with L-BApNA as substrate. A stock
solution was prepared by dissolving 40 mg of
L-BApNA in 0.5 ml of dimethyl sulfoxide; to the
solution, 9.5 mL of glycine buffer (100 mM glycine, pH 10.5) was added. A known amount of gut
equivalent in 10 ml was incubated at 371C, for 30
(L5, L6) or 60 min (L3) in 90 ml of glycine buffer
and with 50 ml of substrate (final concentration
3 mM). The absorbance was monitored at 405 nm.
Control reactions were incubated without gut extract. To calculate trypsin activity, a standard curve
of p-nitroaniline was constructed (0 to 1 mM) and
a regression curve was plotted. The amount of
p-nitroaniline was calculated from the calibration
graph. Trypsin activity of the guts is expressed as
nmol p-nitroaniline/min/gut.
Statistical Analysis
Data were analyzed by the Kruskal-Wallis nonparametric test for ‘‘k independent samples’’ in
SPSS 12.0 and S-plus. The weight gain results were
analyzed on a significance level of a 5 0.05 after 7
and 15 days in treatment. After 10 days in treatment, a equaled 0.10 for captopril and 0.05 for
enalapril. The data of larval period duration were
analyzed on a significance level of a 5 0.05 and
successful adult formation for enalapril-treated
larvae was compared to the control on a level of
a 5 0.10. In the feeding experiments, ACE activity
results were analyzed on a significance level of
a 5 0.05; trypsin activity results on a level of
a 5 0.05 for L5 larvae and a 5 0.10 for L6 larvae.
The ACE activity results for injected L5 larvae were
analyzed on a significance level of a 5 0.05; weight
data of newly molted L5 larvae were analyzed on a
level of a 5 0.05 and of L5 larvae 24 h old on a
level of a 5 0.10. The results of the injection
experiments with L3 larvae were analyzed on a
significance level of a 5 0.05.
RESULTS
Effect of Feeding ACE Inhibitors on Larval Growth and
Development
ACE inhibitors (captopril, lisinopril, and enalapril; 5 mg/mg Premix diet) were fed to first instar
larvae ad lib. Captopril and enalapril were the
most potent ACE inhibitors reducing larval weight
significantly during treatment. No reduction in
weight gain was observed with lisinopril (Fig. 1).
In contrast, when hemolymph of last instar
S. littoralis larvae was incubated with these ACE inhibitors, lisinopril was as potent as captopril and
inhibited SlACE in the nM range, while enalapril was
effective in mM concentrations (results not shown).
Larvae fed enalapril were slow to develop but molted
normally and the larval stage from L1 to pre-pupa
took 2370.5 days. The critical weight needed for the
larvae to start metamorphosis was 515.3759.6 mg.
The duration of the larval period after feeding captopril and lisinopril was 17.870.4 and 17.170.2
days, respectively, and did not differ from the control group (Table 1). At day 2 during the treatment, a
significant difference in larval development was observed in larvae treated with 5 mg enalapril/mg Premix (stage 1.2770.12 compared to 1.9470.06 for
the control). Further experiments showed that 5 mg
Archives of Insect Biochemistry and Physiology December 2008
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Effect of Ace Inhibitors and TMOF on S. littoralis
203
TABLE 2. The Influence of ACE Inhibitors on Hemolymph ACE Activity and
on Trypsin Activity Was Determined for L5 Larvae (24712 h Old) and L6
Larvae (72712 h Old)
ACE Inhibition (%) 7sem
Fig. 1. Weight gain of S. littoralis larvae when fed Premix
diet containing 5 mg/mg captopril, enalapril, or lisinopril
during the larval development. Larvae in the control
treatment were fed Premix diet without ACE inhibitor.
Weight (mg) is shown for 7, 10, and 15 days after the
treatment was started (L1, d1). Data are based on at least
10 larvae per treatment. Significant differences compared
to the control are indicated by ().
TABLE 1. Duration of the Larval Stage (Days) From L1, Day 1 Until the
Beginning of Pre-Pupal Stage for Larvae Fed Premix Diet Containing 5 mg/
mg ACE Inhibitor During Larval Development
Treatment
Duration of larval stage (days) 7 sem
Control
Captopril
Lisinopril
Enalapril
17.170.2a
17.870.4a
17.170.2a
2370.5b (515.3759.6)
The significant difference of enalapril treatment compared to the other treatments is
indicated by lowercase letters (a, b). The weight (mg) is indicated at the onset of the
pre-pupal stage for larvae treated with enalapril.
enalapril/mg Premix was effective. Lower doses of
0.2, 0.5, or 1 mg enalapril/mg Premix did not reduce
weight, nor did the treatments retard the larval
growth. In addition to the retarded larval development, successful adult formation was significantly
lower for larvae treated with 5 mg enalapril/mg Premix. Only 46717% of the larvae became adults
compared to 80713% of the control treated larvae.
Effect on ACE Activity After Feeding or Injecting ACE
Inhibitors
Feeding first instar larvae, ad lib, captopril, or
enalapril caused a drop in ACE activity in the
Archives of Insect Biochemistry and Physiology December 2008
Trypsin activity (nmol p-nitroaniline/min/
gut) 7sem
Feeding L5 (24712 h) L6 (72712 h)
L5 (24712 h)
L6 (72712 h)
Control 0.0714.3 aA 0.076.5 aA
Captopril 24.874.9 aA 80.571.1 bB
Enalapril 87.173.9 bB 74.972.9 bC
309729 a
506799 a
2637123 b
30407546 a
15287493 b
16677568 b
Larvae (L1,d1) were fed ad lib Premix diet containing 5 mg/mg captopril or enalapril
or on untreated diet. ACE activity was measured in a fluorescence assay and
statistically analyzed. Significant differences between means in columns are indicated
with lowercase letters (a,b), significant differences between means in rows with
capital letters (A–C). For trypsin activity, the guts were analyzed individually in an
L-BapNA assay.
hemolymph of L5 and L6 larvae (Table 2). Captopril and enalapril traversed the gut epithelium
and exerted their inhibitory effect on SlACE in the
hemolymph. Enalapril was more potent than
captopril on fifth instar larvae (24712 h old),
knocking down the ACE activity by 8774%. ACE
activity
in captopril-treated insects decreased only by
2575%. During the sixth and last instar (72712 h
old), both enalapril and captopril inhibited ACE
activity by 7573 and 8171%, respectively.
In another experimental series, captopril (80 mg)
and enalapril (40 mg) were injected into larval hemolymph. After newly molted (0–7 h old) fifth
instar larvae were injected with 40 mg of enalapril
(0.9870.09 mg/mg weight), the ACE activity in the
hemolymph was inhibited by 9471.2%. In contrast, injecting 80 mg of captopril (1.7870.08 mg/
mg weight) inhibited 52.5712.9% of the ACE
activity in the hemolymph after 24 h. Newly molted larvae injected with enalapril did not gain
weight after 24 h, while the larvae that were injected with insect Ringer gained 34.875.3 mg.
After injecting captopril, there was no significant
effect on weight gain, indicating that enalapril is a
more potent inhibitor when injected.
ACE activity in hemolymph of L5 larvae,
24 h old and injected with 40 mg of enalapril
(0.3970.02 mg/mg weight) or 80 mg of captopril
(0.7470.02 mg/mg weight), was inhibited by
3774 and 4678%, respectively (Table 3). Inject-
204
Lemeire et al.
_____________________________________________________________
TABLE 3. Newly Molted L5 Larvae and L5 Larvae 24 h Old Were Injected
With 80 mg of Captopril or 40 mg Enalapril
ACE inhibition (%) 7 sem
Injection (2 ml)
Ringer
Captopril (80 mg)
Enalapril (40 mg)
Weight (mg) 7 sem
L5 (0–7 h)
L5 (24 h)
L5 (0–7 h)
L5 (24 h)
0.0078.4 aA
52.5712.9 bB
94.571.2 cC
0.0076.5 aA
46.078.2 bB
37.073.9 bB
80.678.6 a
57.373.8 a
42.277.4 b
138715 a
93.8711.2 a
118715 a
Control insects were injected with 2 ml of insect Ringer. The hemolymph ACE
activity was determined in a fluorescence assay, 24 h after treatment. Significant
differences between means in columns are indicated with lowercase letters (a–c) and
between means in rows by capital letters (A–C). The measured weight 24 h after
treatment is indicated.
ing captopril into L5 larvae, aged 24 h equally
inhibited ACE as compared with newly molted
larvae. On the other hand, enalapril inhibition of
ACE was significantly lower for L5 larvae treated at
24 h than immediately after emergence, and their
weight gain was not affected.
Trypsin Activity After Treatment With ACE Inhibitors
and Aea-TMOF
Trypsin activity was measured in the gut after
feeding or injecting larvae with captopril or enalapril. Gut preparations from fifth and sixth instar
larvae, fed a Premix diet containing ACE inhibitors,
were incubated with L-BApNA. In fifth instar larvae
(24 h old) treated with enalapril, the trypsin activity was significantly lower than in the control or
captopril-fed larvae (Table 2). Enalapril-fed larvae
(L5, 24 h old) had a lower weight than controls.
In L6 larvae, aged 72 h, the trypsin activity was
lower after feeding enalapril as well as captopril
(Table 2). The captopril-fed larvae, though, did not
show a significant weight difference.
Trypsin activity was also determined in L3 larvae. Newly molted L3 larvae or 24-h-old L3 larvae
were injected with Ringer or ACE inhibitors
(0.5 ml). Newly molted larvae (0–5 h old) treated
with 20 mg of captopril (5.4470.35 mg/mg weight)
were all killed by the treatment (results not
shown). Injecting the same amount of captopril
(2.2070.20 mg/mg weight) or 10 mg of enalapril
(1.8570.13 mg/mg weight) into L3 larvae (24 h
old) did not affect weight gain or trypsin activity in
the insect gut as compared with Ringer injections
(Table 4).
To determine whether an Aea-TMOF-like factor
is present in S. littoralis caterpillars, L3 larvae (24 h
old) were treated with 10 or 20 mg of Aea-TMOF.
Individual guts were analyzed 24 or 48 h later. No
significant difference was found in the trypsin
activity after treating with TMOF alone (Table 4).
An assay for the interaction of the fluorescent
substrate Abz-FRK-(Dnp)P-OH and Aea-TMOF was
undertaken. A Dixon and Cornish-Bowden plot
indicated a competitive inhibition of the fluorescent substrate with Aea-TMOF when incubated
with ACE (results not shown). The in vitro assay
indicated that Aea-TMOF can bind to the active site
of SlACE. Thus, a combination of Aea-TMOF and
ACE inhibitor was injected into L3 larvae (24 h
old). When trypsin activity was analyzed 24 h later,
10 mg Aea-TMOF combined with 10 mg of enalapril
did not affect the trypsin activity in the gut and no
difference in weight was found. However, analysis
of guts 48 h after injection of larvae with a combination of 20 mg Aea-TMOF and 20 mg of captopril
significantly inhibited trypsin activity in the gut
as compared to treatments with captopril and AeaTMOF alone (Table 4). Insects that were treated
also showed a decrease in weight as compared with
controls.
DISCUSSION
This study evaluated the effect of ACE inhibitors
and TMOF on growth and development of
S. littoralis larvae. In addition, the inhibition of
ACE activity in the hemolymph was followed and
correlated with trypsin activity in the gut.
Feeding newly hatched larvae (L1, day 1) captopril, or enalapril inhibited larval growth rate. Similarly, Isaac et al. (2007) found that ACE
inhibitors (2 mmol) injected into Manduca sexta
larvae (4th and 5th instars) slowed down larval
growth. Lisinopril and captopril reduced growth
during 3 days post-injection. The effect of fosinopril, however, was irreversible for 8 days following
treatment. Lisinopril was administered through
feeding in this report and did not affect larval
Archives of Insect Biochemistry and Physiology December 2008
_______________________________
Effect of Ace Inhibitors and TMOF on S. littoralis
205
TABLE 4. Trypsin Activity in the Gut and Weight Was Determined 24 and 48 h After Injection of 0.5 ml of Enalapril (10 mg), Captopril (20 mg), Aea-TMOF
(10 or 20 mg), or a Combination of Enalapril and Aea-TMOF (10 mg Each) or Captopril and Aea-TMOF (20 mg Each) Into L3 Larvae 24 h Old
Time of analysis
24 h
Injection L3
Ringer
Enalapril (E) (10 mg)
Aea-TMOF (10 mg)
E1TMOF (10110 mg)
Trypsin activity (nmol
pnitroaniline/min/gut) 7sem
4.9771.95
6.5172.59
10.673.65
11.373.30
a
a
a
a
48 h
Weight (mg)7sem
9.6970.7
8.0671.1
13.972.4
12.671.4
a
a
a
a
Injection L3
Ringer
Captopril (C) (20 mg)
Aea-TMOF (20 mg)
C1TMOF (20120 mg)
Trypsin activity (nmol
pnitroaniline/min/gut)7sem
Weight (mg)7sem
5.7271.23 a
4.5170.59 a
7.5472.00 a
1.9770.86 b
19.372.3 a
18.071.8 a
19.471.9 a
11.071.2 b
Significant differences between means in columns are indicated by lowercase letters (a,b).
weight gain. It is possible that it has a high turnover
rate in the gut or most of it is excreted before it can
reach the hemolymph and inhibit ACE. In M. sexta,
fosinopril was far more potent than the other inhibitors including its active analogue, fosinoprilat
(Isaac et al., 2007). In this study, feeding enalapril
to S. littoralis ad lib dramatically decreased weight
gain. Both fosinopril and enalapril are prodrugs and
need to be activated by esterases before they can
bind and inhibit ACE. The esterification of fosinoprilat renders the inhibitor more lipophilic and
facilitates its translocation across cell membranes
(Isaac et al., 2007). This could also be the case for
enalapril. The deesterification reaction that converts
enalapril into the active inhibitor enalaprilat takes
place in vivo. Its potency in vitro appeared much
less than captopril and lisinopril. It is possible that
specific esterases needed for activation are limited in
the in vitro assay, or may not function well. Vercruysse et al. (2004) reported no effect on larval
growth when newly molted L1 larvae were fed
captopril ad lib. These authors applied captopril as a
thin layer to the diet surface by distributing 75 ml of
a 10 mg/ml captopril concentration in methanol and
let the solvent evaporate. In this study, we thoroughly mixed 5 mg captopril with the Premix diet
forcing the larvae to continuously ingest both the
food and the ACE inhibitor.
Isaac et al. (2007) reported that injection of
inhibitors was most effective when larvae had just
molted into the fifth instar. In contrast to the
feeding assay, where larvae gradually increase the
amount of ingested ACE inhibitor, injection into
Archives of Insect Biochemistry and Physiology December 2008
newly molted larvae in our study led to a higher
dose/weight ratio. This was reflected by the high
level of ACE inhibition in fifth instar larvae of less
than 7 h old compared to larvae 24 h old (Table 3).
In addition, injecting newly molted larvae led to a
significantly higher decrease in weight 24 h after
treatment than injecting larvae at 24 h and analyzing them at 48 h. When larvae grow, their
weight increases during the larval stage and decreases prior to molting. At this point, they stop
feeding, start contracting, lose water, and the ACE
activity is at a maximum. Lemeire et al. (2008)
reported that 24 h after the fourth or fifth larval
molt of S. littoralis larvae, the ACE activity is low as
compared to 48 h or 62 h after the molt. Increase in
ACE activity during the second day after the fifth
larval stage is probably the cause for a lower inhibitory effect of ACE inhibitors. The enhanced
sensitivity of newly molted larvae to ACE inhibitors and especially enalapril is probably not
due to an increase in esterase activity; esterases are
very common in insects and found at high concentrations in the hemolymph. Our data suggest
that during the first day after the molt,
S. littoralis larvae have low ACE activity and this is
the most appropriate time to treat the larvae. Based
on these observations, it is now possible to speculate that this may also be true for larvae of other
Lepidopteran species.
Injecting enalapril (90 nmol) into fifth instar
larvae (0–7 h old) or feeding larvae ad lib led in
both cases to high levels of ACE inhibition and
weight reduction. Larvae injected with enalapril
206
Lemeire et al.
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did not feed 24 h after the treatment and captoprilinjected larvae did not feed avidly as compared to
Ringer-injected larvae. Furthermore, hemolymph
collection was difficult to obtain because the
hemolymph volume in these larvae was low.
These results indicate that perhaps a kinin-like
peptide remains active after feeding the ACE inhibitors, and caused fluid loss, reduction in hemolymph volume, and loss of appetite. Diuretic
hormones like Manse DP-II (amidated C-terminus
–FLNRVamide) and Locmi-DH (amidated C-terminus –FLQQIamide) have influence on feeding.
Manse DP II reduces food consumption and slows
growth in first instar larvae resulting in higher
mortality. Although no effect was seen when
Manse DP II was injected into M. sexta larvae, injection into H. virescens larvae elicited antifeeding
behavior. Injecting Locusta migratoria nymphs with
Locmi-DH reduced feeding and the duration of the
first meal as compared with controls (Gäde and
Goldsworthy, 2003). None of the above-mentioned peptides have been studied in vitro to see
whether they are hydrolyzed by ACE. On the other
hand, members of the diuretic peptide family of
smaller kinins are in vitro good ACE substrates.
Lamango et al. (1997) found that LK I and II and
Cus DP I and II could be hydrolyzed by purified
M. domestica ACE. Analogues of the kinin peptide
family with altered amino acids or enhanced steric
hindrance were specifically designed to prohibit
hydrolysis by housefly or fruitfly ACE (Nachman
et al., 2002; Zubrzak et al., 2007). Kinin family
peptides of insects from Orthoptera, Dictyoptera,
Lepidoptera, and Diptera are in general diuretic,
but with different levels of efficacy (Gäde and
Goldsworthy, 2003). Seinsche et al. (2000) described how a combination of helicokinin HezK I
and 1 mmol of captopril or enalapril-maleate led to
drastic weight decreases and higher mortality. It
was suggested that ACE inhibitors could prohibit
the hydrolysis of HezK I, resulting in a persistency
of the peptide causing weight loss probably by
water loss (Seinsche et al., 2000).
The treatment of ACE inhibitors did not only
retard growth but also had an effect on gut trypsin
activity. Larvae that were fed ACE inhibitors ad lib
were also analyzed for trypsin activity in the
fifth (24712 h) and sixth larval stage (72712 h)
(Table 2). ACE inhibition together with trypsin activity was determined in the gut. The majority of the
ACE activity was inhibited (87.173.9%) in L5 larvae when treated with enalapril (Table 2). Trypsin
activity was also significantly lower in treated larvae
compared to control and captopril-treated larvae
(Table 2). The ACE activity in larvae reared on ACE
inhibitors until the last instar was highly inhibited
for both enalapril and captopril and trypsin activity
in the gut was significantly lower.
An effect of ACE inhibition on TMOF is a possibility. We investigated whether an Aea-TMOF
analogue was present in S. littoralis larvae, as was
shown in H. virescens (Nauen et al., 2001). Captopril (90 nmol) injected into newly molted L3
larvae caused 100% mortality. However, when
captopril (90 nmol) was injected with Aea-TMOF
(19 nmol) into L3 larvae, 24 h old, trypsin activity
was several-fold significantly lower (2.3- to 3.8fold) as compared to insects injected with saline,
captopril, or Aea-TMOF (Table 4). In vitro, AeaTMOF can bind to the active site of ACE. TMOF is a
competitive inhibitor of the fluorescent substrate
Abz-FRK-(Dnp)P-OH, but Aea-TMOF is probably
not hydrolyzed by SlACE, although this awaits
definite proof. Lamango et al. (1997) reported that
the only substrates not hydrolyzed by the housefly
ACE are proctolin and CCAP. Apparently, the
penultimate Pro residue of proctolin prevented
hydrolysis by the housefly ACE. Mammalian ACE
poorly hydrolyzes substrates with a penultimate
Pro at their C-terminal (Ondetti et al., 1982;
Erdos, 1990) and Aea-TMOF has 6 prolines at the
C-terminus.
The interaction of Aea-TMOF with SlACE in this
study should be interpreted with caution. It is
possible that an Aea-TMOF gut receptor is present
in S. littoralis larvae. Binding of Aea-TMOF to ACE
prevents it from binding to its gut receptor and
hence prevents it from stopping trypsin biosynthesis in the gut. Thus, injecting Aea-TMOF alone did
not affect trypsin biosynthesis in the gut. Inhibiting ACE with captopril (90 nmol) allowed AeaTMOF to bind to its gut receptor and decreased
Archives of Insect Biochemistry and Physiology December 2008
_______________________________
Effect of Ace Inhibitors and TMOF on S. littoralis
trypsin activity in the gut (Table 4). Alternatively,
in the presence of both TMOF and captopril ACE is
greatly inhibited causing a marked reduction in
weight and in trypsin activity in the gut as an indirect effect, as was shown in the feeding experiments when ACE inhibitors were fed ad lib. This is
supported by the fact enalapril and captopril injections were responsible for changes in feeding
behavior.
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