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The angiotensin converting enzyme inhibitor captopril reduces oviposition and ecdysteroid levels in lepidoptera

Archives of Insect Biochemistry and Physiology 57:123–132 (2004) The Angiotensin Converting Enzyme InhibitorCaptopril Reduces Oviposition and Ecdysteroid Levelsin Lepidoptera L. Vercruysse,1,2* D. Gelman,3 E. Raes,1 B. Hooghe,1 V. Vermeirssen,2 J. Van Camp,2 and
G. Smagghe1

The role of angiotensin converting enzyme (ACE, peptidyl dipeptidase A) in metamorphic- and reproductive-related events inthe Egyptian cotton leafworm, Spodoptera littoralis (Lepidoptera, Noctuidae) was studied by using the selective ACE inhibitorcaptopril. Although oral administration of captopril had no effect on larval growth, topical administration to new pupaeresulted in a large decrease of successful adult formation. Oviposition and overall appearance of adults emerging from treatedlarvae did not differ significantly from those emerging from non-treated larvae. In contrast, topical or oral administration ofcaptopril to newly emerged adults caused a reduction in oviposition. By evaluating the effect of captopril on ecdysteroid titersand trypsin activity, we revealed an additional physiological role for ACE. Captopril exerted an inhibitory effect on ecdysteroidlevels in female but not in male adults. Larvae fed a diet containing captopril exhibited increased trypsin activity. A similarcaptopril-induced increase in trypsin activity was observed in female adults. In male adults, however, captopril elicited re-duced levels of trypsin activity. Our results suggest that captopril downregulates oviposition by two independent pathways, onethrough ecdysteroid biosynthesis regulation, and the other through regulation of trypsin activity. Apparently, fecundity isinfluenced by a complex interaction of ACE, trypsin activity, and ecdysteroid levels. Arch. Insect Biochem. Physiol. 57:123–132, 2004.
KEYWORDS: angiotensin converting enzyme; captopril; larval growth and development; metamorphosis; oviposi-tion; egg viability; ecdysteroids; trypsin; Spodoptera littoralis INTRODUCTION
thus generating vasoconstricting angiotensin II.
ACE also degrades and inactivates bradykinine, a Angiotensin converting enzyme (ACE, peptidyl vasodilatory peptide (Erdös and Skidgel, 1897; dipeptidase A) is a Zn2+ metallopeptidase associ- Johnston, 1992). In mammals, ACE exists as two ated with the regulation of blood pressure in mam- isoforms, somatic ACE (sACE) with a molecular mals. It increases blood pressure by removing a weight of 140–180 kDa and two highly homolo- dipeptide from the C-terminus of angiotensin I, gous domains (N- and C-domains) that both are 1Laboratory of Agrozoology, Department of Crop Protection, Faculty of Agricultural and Applied Biological Sciences, Ghent University, Ghent, Belgium2Department of Food Technology and Nutrition, Faculty of Agricultural and Applied Biological Sciences, Ghent University, Ghent, Belgium3Insect Biocontrol Laboratory, USDA-ARS, Beltsville, Maryland Abbreviations used: ACE = angiotensin converting enzyme; Aea-TMOF = Aedes aegypti trypsin modulating oostatic factor; Neb-TMOF = Neobellieria bullatatrypsin modulating oostatic factor; sACE = somatic ACE; tACE = testicular ACE; 20E = 20-hydroxyecdysone Contract grant sponsor: Institute for the Promotion of Innovation by Science and Technology in Flanders (IWT); Contract grant sponsor: Special Research Fund ofGhent University; Contract grant number: 01102703.
Commercial products used in this study are not endorsed by the USDA.
*Correspondence to: L. Vercruysse, Laboratory of Agrozoology, Faculty of Agricultural and Applied Biological Sciences, Ghent University, Coupure Links 653,B-9000 Ghent, Belgium. E-mail: lieselot.vercruysse@ugent.be Received 12 March 2004; Accepted 7 July 2004 2004 Wiley-Liss, Inc.
DOI: 10.1002/arch.20023Published online in Wiley InterScience (www.interscience.wiley.com) catalytically active, and testicular ACE (tACE) with concentrations. Vandingenen et al. (2001) also re- a single active site and a molecular weight of 90– ported that captopril fed to female flies caused an 110 kDa (Corvol et al., 1995). sACE is expressed increase in the liver meal-induced trypsin peak in in many different tissues, while tACE is unique to the midgut and elevated levels of protein-induced the testis. Whereas the role of sACE in the regula- yolk polypeptides in the hemolymph, but oocyte tion of blood pressure and water and electrolyte balance is well understood, the exact function of From other previous work, it is known that tACE is unknown (Turner and Hooper, 2002).
TMOF inhibits ecdysone biosynthesis in N. bullata Recently in several insects, a peptidyl dipepti- and Lymantria dispar (De Loof et al., 1995; Gelman dase that has very similar enzymatic properties to and Borovsky, 2000); however, the direct effect of mammalian ACE has been found (Lamango and captopril treatment on ecdysteroid biosynthesis has Isaac, 1994; Cornell et al., 1995; Wijffels et al., 1996; Schoofs et al., 1998). Two genes that code It appears that the effect of TMOF on trypsin for ACE homologues, AnCE and Acer, were identi- biosynthesis occurs independently of its effect on fied in Drosophila melanogaster. Since insects have ecdysteroid biosynthesis in the grey fleshfly. This an open circulatory system, the discovery of insect follows from observations made by Bylemans et ACE homologues has led to speculations about al. (1995), where injection of ecdysone together new physiological roles for this enzyme. In the with Neb-TMOF did not significantly counteract housefly Musca domestica, a soluble 67-kDa ACE the effect of TMOF on the inhibition of trypsin has been purified, and its low molecular weight suggests that it only has one active site (single do- In addition to influencing egg production, in main form). The physiological role for this enzyme the silkmoth, Bombyx mori, ACE was found to be is not known. At present, captopril (D-3-mercapto- active at the time in metamorphosis when wing 2-methyl-propionyl-L-proline), a strong and spe- formation was observed (Quan et al., 2001). More cific inhibitor of ACE, is often used in the treatment evidence in support of a role for ACE in metamor- of hypertension, and it has been reported that phosis was provided by Siviter et al. (2002). Dur- captopril displays the same potency for the inhi- ing pupal development of D. melanogaster, ACE-like bition of AnCE as for the inhibition of mamma- activity increased 3-fold at a mid-pupal stage, be- fore declining to larval levels at the time of adult Recent studies that were conducted with dipteran insects suggest a role for ACE in insect reproduc- In this report, we explore in a lepidopteran spe- tion. Results from studies in which ACE inhibitors cies, the Egyptian cotton leafworm, Spodoptera were fed to adult male mosquitoes (Anopheles littoralis, the effects of the phenotypic knockout of stephensi) suggested that AnCE has an important ACE activity by its selective inhibitor captopril. S. influence on male fertility and that this effect could littoralis is one of the major pest insects in the world be mediated through the regulation of neuropep- and many populations of this insect have devel- tide activity. Females that had been mated with oped high levels of insecticide resistance (Oerke these ACE-inhibited males showed a dramatic re- et al., 1994). In a first series of experiments, vari- duction in fecundity (Ekbote et al., 2003a). In ad- ous developmental stages were tested by direct and dition, Vandingenen et al. (2001, 2002) treated residual treatment with captopril. For larval and female adults of the grey fleshfly Neobellieria bullata pupal stages, we evaluated feeding, growth, and with captopril and studied the in vivo relationship development with particular attention given to between Neb-TMOF (trypsin modulating oostatic molting and metamorphosis. Oviposition and egg factor) and Neb-ACE. Since Neb-TMOF is an in vivo viability were also followed in treated male and substrate for Neb-ACE, the captopril treatment had female adults. Captopril was used at 10 µg/µl or a direct effect on trypsin activity and vitellogenin dosed at 50 µg, as in vitro tests showed that Archives of Insect Biochemistry and Physiology captopril completely inhibited ACE at 0.2 µM treatment. The phenotypes of treated and control (Vermeirssen et al., 2002). In a second series of ex- insects were evaluated to the larval-pupal molt.
periments, we determined for the first time the effect For pupae, the effects of captopril on metamor- of captopril on ecdysteroid titers in the hemolymph phosis and adult formation were evaluated. New of these different stages. Then, to address the mecha- (0–6 h) pupae were topically treated with captopril nism responsible for the negative effects of captopril (50 µg in 5 µl acetone), and two replicate groups on oviposition, we measured its effects on trypsin of 20 pupae each were used. Controls were treated activity in vivo and in vitro. Our objective was to only with acetone. The phenotype of treated and test whether captopril downregulates oviposition by control groups was followed to adult eclosion.
two independent pathways, one through ecdysteroidbiosynthesis regulation, and the other through in- Effect of Captopril on Oviposition and
Egg Viability Assay
MATERIALS AND METHODS
The effect of captopril on egg production was measured by two different methods. In one proto- Chemicals
col, L1–L6 larvae were fed on diet containing 10 µg/ µl captopril. After adult emergence, oviposition was Captopril (D-3-mercapto-2-methyl-propionyl-L- followed. In parallel, newly emerged (0–6 h) adults proline) was purchased from Sigma Co. (Bornem, that had been fed on control diet during larval de- Belgium). All other chemicals were of analytical velopment were topically treated on the abdomen grade or were obtained as described in the text.
with 50 µg captopril (in 5 µl acetone). Captopril treatment was either administered once, at the timeof adult eclosion, or, in a separate assay, every 2 All stages of a continuous colony of S. littoralis days for 10 days. In addition, adults were continu- were maintained under standard conditions of 23 ously treated with captopril at 10 µg/µl by adding ± 1°C, 70 ± 5% RH and a light:dark (16:8) photo- ACE inhibitor to the honey-water diet. To assessperiodic regimen as described previously (Smagghe the effects of captopril on oviposition, groups of et al., 2002). Larvae were fed on an agar-based ar- 10 newly emerged adults (sex ratio 1:1) were placed tificial diet that had been placed in multiwell cul- in a plastic box (10 × 10 × 15 cm) and the inside ture plates, and adults were fed a 20% honey water walls were covered with paper to provide oviposi- tion sites (Smagghe and Degheele, 1994). After thefirst oviposition, the number of eggs laid per fe- Assay to Assess the Effects of Captopril on
male was daily recorded for 8–10 days. Afterwards, Larval Growth and Development
egg viability was scored as a mean percentage ±SEM of first-instar larval emergence.
For larval bioassays, newly molted (0–1 d) lar- Trypsin Assay
lected and transferred to control diet or to artificialdiet containing captopril. Captopril (75 µl; 10 µg/ Trypsin activity was measured by monitoring the µl in methanol) was uniformly distributed on the digestion of casein, commonly used as a trypsindiet surface of the experimental group, and after sol- substrate (Bickerstaff and Zhou, 1993). Although vent evaporation, captopril was present as a film on casein is not a trypsin specific substrate, it is used the surface of the diet (Smagghe et al., 1999). Con- to measure trypsin activity in S. littoralis as trypsin trols were treated only with methanol. Equal num- is the major digestive proteolytic enzyme in the bers of larvae were placed on the treated and control cotton leafworm (De Leo et al., 1998). Briefly, diet. There was a minimum of 2 replicate groups/ casein was dissolved in sodium phosphate solu- tion (50 mM, pH 8.5) and boiled gently for 10 (Gelman et al., 1997). The concentration of ecdy- min. The casein solution was diluted to 300 µg/ steroids was expressed as pg equivalents/µl hemo- ml with sodium phosphate buffer (50 mM, pH 7.5). To construct a standard curve, several tubes,each containing 400 µl of casein, were placed in a water bath at 30°C for 5 min. To each tube, 100 µl Effect of Captopril-Containing Diets on Larval
of the diluted trypsin solution was added and the Growth and Development
mixture was incubated for 30 min. Protein con-tent was measured using the Bradford assay (Brad- Feeding of captopril at 10 µg/µl to first-sixth ford, 1976) with BSA standard and Coomassie blue.
(last) instar larvae on a continuous basis did not The effect of captopril on trypsin activity was fol- inhibit food consumption, larval weight gain, or lowed in vitro by adding 100 µl of different con- molting (data not shown). However, in last larval centrations of captopril to the incubation mixture instars pupal molt was significantly delayed by a containing a constant concentration of trypsin. To measure the effect of captopril on trypsin activity Although captopril had no effect on larval in vivo, larvae and S. littoralis adults of were fed growth, the percentage of successful adult forma- captopril. Adults were fed honey water containing tion was significantly reduced from 77.5 ± 2.5% 1% captopril for 3 days and larvae were fed artifi- in controls (acetone-treated) to 30.0 ± 6.2 % after cial diet containing 1% captopril for 4 days. Fol- lowing feeding, both larvae and adults werehomogenized and centrifuged in Tris/HCl buffer Effects of Captopril-Containing Diets on Oviposition
(50 mM, pH 7.4). After centrifugation, 100 µl of and Egg Viability
the diluted supernatant was added to 400 µl caseinand trypsin activity was measured.
Oviposition by adults that emerged from capto- pril-treated larvae (continuously treated with 10 µg/ Ecdysteroid Titers
µl from the 1st through the 6th instar), was notsignificantly different from that of controls. After Larval and pupal hemolymph ecdysteroid lev- els and adult whole body ecdysteroid levels weredetermined 24 h after topically treating last-instarlarvae, pupae, and adults with captopril (50 µg, in5 µl acetone) and controls with acetone (Smaggheet al., 1995). Briefly, hemolymph from anaesthe-tized larvae and pupae was collected and trans-ferred to 500 µl of ice cold 75% aqueous methanol.
After the removal of antennae, wings, and legs,adults were homogenized in 1 ml of ice cold 70%aqueous methanol. All samples were centrifugedfor 10 min at 21,460g, and the supernatant wastransferred into ice cold tubes. The precipitate waswashed with 500 µl of ice cold 75% aqueous Percentage of successful adult formation in methanol. After a third wash and centrifugation, Spodoptera littoralis after topical treatment with 50 µg combined supernatants were lyophilized and captopril per new pupa. Data are expressed as means ± stored in the freezer until analysis.
SEM based on 2–7 replicates, and (*) indicates a signifi- Ecdysteroid content was determined using RIA, cant difference by a Student’s t-test (P < 0.01) between and tritium labeled ecdysone (63.5 Ci per mmol) the experimental and control groups.
Archives of Insect Biochemistry and Physiology Effect of ACE inhibition with captopril on ovipo- Data are expressed as means ± SEM based on 3 indepen- sition of Spodoptera littoralis adults (A) after oral feeding
dent measurements, and (*) indicates a significant differ- captopril at 10 µg/µl continuously from the first to the ence by a Student’s t-test at P < 0.15 between experimental last larval instar, (B) when 50 µg captopril was repeatedly
administered topically at 2-day intervals in the adult stage.
9 days, cumulative egg-laying per female was activity that varied between 33.11 and 159.29 ng/ 1,576.3 ± 344.5 in the treated group and 1,682.7 ml. In contrast, when captopril was fed to larvae ± 177.0 in the control group (Fig. 2A). As stated and adults, a significant difference was observedpreviously, captopril had no effect on larval devel- between experimental and control groups, 2–4 days opment, nor on the overall appearance (e.g., size after feeding on artificial diet (Table 1). After 2, 3, and condition of appendages), of adults that de- and 4 days of treatment, trypsin activity increased by 1.62-, 1.67-, and 2.22-fold, respectively. Feed- In contrast, when newly emerged adults were ing female adults for 2 days with honey water con- treated with captopril, a single topical application taining captopril resulted in a 1.74-fold increase on the thorax caused a decrease in oviposition of in trypsin activity (Table 2). In contrast, male adults 33 ± 10% as compared to control adults (data not fed honey water containing captopril for 2 days shown). Repeated topical applications at 2-day in- exhibited lower levels of trypsin activity.
tervals resulted in a dramatic decrease in egg lay-ing as shown in Figure 2B. Similarly, when adults Ecdysteroid Titer Reduction Using Captopril
were treated with captopril dissolved in the honey-water diet, a significant reduction in oviposition As shown in Figure 3A, treatment of last instar (L6) larvae and pupae with captopril did not sig- Treatment of either larvae or adults with cap- nificantly affect hemolymph ecdysteroid levels. In topril had no effect on egg hatch. In both controland experimental groups, percent hatch was greater TABLE 1. Trypsin Activity (ng/mg Protein) After Feeding Larvae of Spodoptera littoralis during the First Four Days of the Last Instar WithArtificial Diet Containing 1% Captopril Effect of Captopril on Trypsin Activity In Vivo and
6.73 ±7.06aA 15.39±0.64bB 14.80±3.90bB 22.10±2.46bC In the in vitro assay, captopril at a concentra- *Data are expressed as means ± SEM based on 2 independent measurements. Pertreatment, significant differences by ANOVA at P = 0.05 between means in rows are tion of 1 nM to 1mM had no effect on trypsin indicated with lowercase letters (a and b) and in columns with capital letters (A–C).
TABLE 2. Trypsin Activity (ng/mg Protein) in Male and Female Adults of adults, captopril had no significant effect on ecdy- Spodoptera littoralis After 2 and 3 Consecutive Days of Oral TreatmentWith 1% Captopril in Honey Water Compared to Untreated Controls steroid levels in male adults; ecdysteroid titers intreatment and control groups were 11.32 ± 2.55 pg/ mg and 9.62 ± 3.85 pg/mg, respectively (Fig. 3B).
DISCUSSION
*Data are expressed as means ± SEM based on 3 replicates. For males as well as This is a first report on the effect of ACE-inhi- females, significant differences by ANOVA at P = 0.05 between means in rows areindicated with lowercase (a and b) and in columns with capital letters (A and B).
bition on ecdysteroid titers in the hemolymph oflarvae and pupae and in whole body extracts of contrast, when female adults of S. littoralis adults adults of a lepidopteran species, the cotton leaf- were treated with captopril, there was a 5-fold sig- worm S. littoralis. In addition, we report the effect nificant (P < 0.05) decrease in whole body ecdy- of captopril treatment on larval growth and devel- steroid levels. In captopril-treated females, the opment and on oviposition and egg viability. We ecdysteroid titer was 45.43 ± 11.58 pg/mg body also determined the effect of captopril on trypsin weight, whereas in controls it was 275.95 ± 99.96 activity in vitro and in vivo in larvae and adults.
pg/mg (Fig. 3B). Although inhibitory in female When administered orally to S. littoralis larvae, captopril did not affect larval development. Ourresults agree with those reported by Seinsche et al.
(2000) who tested the effect of ACE-inhibitors onthe development of Heliothis virescens larvae. Theyfound that larvae injected with captopril, enalapril-maleate, and lisinopril, three inhibitors of ACE, Ecdysteroid titers of Spodoptera littoralis after topical treatment with 50 µl captopril (A) of last-instar larvae and pupae, and (B) of male and
female adults. Data are expressed as means ± SEM based on 2–6 replicates,
and (*) indicates a significant difference by a Student’s t-test at P < 0.05
compared with the untreated control.
Archives of Insect Biochemistry and Physiology grew normally. On the other hand, combined ap- observed (Isaac et al., 1999). In another study in plication of ACE-inhibitors and helicokinins caused which A. stephensi females were fed a blood meal a reduction in weight gain and higher mortality containing either captopril or lisinopril, the pres- rates in last instar H. virescens larvae. As a result of ence of the ACE-inhibitors did not affect feeding ACE inhibition, which, in turn, prevented the hy- and mating behavior, but reduced fecundity in a drolysis of helicokinins (by ACE), diuretic activity dose-dependent manner (Ekbote et al., 2003a).
increased due to the elevated kinine titers. Our re- Since treated insects displayed normal blood di- sults show that application of captopril to S. gestion and a normal development of oocytes, it littoralis also did not significantly affect larval de- is possible that ACE-inhibitors interfere with oo- cyte transfer along the oviducts. The report that The large decrease in successful adult formation ACE-like activity has been localized in the repro- of S. littoralis after topical treatment of new (0–6 ductive organs of both male and female insects pro- h) pupae with captopril shows that ACE has a role vides additional evidence supporting a role for ACE in metamorphosis of holometabolous insects.
in reproduction (Isaac et al., 1998; Loeb et al., Siviter et al. (2002) previously suggested such a 1998). In Lacanobia oleracea, the highest level of role for ACE based on their findings that larval- ACE activity was found in the reproductive tract.
pupal transition of D. melanogaster was accompa- Almost all of the enzyme was found in the acces- nied by a 3-fold increase in ACE-activity. This sory glands of the male and in the spermatheca increase was attributed to the strong induction of and bursa copulatrix of the female (Ekbote et al., Ance expression in the imaginal cells by 20E.
2003b). ACE activity was also localized in the tes- Houard et al. (1998) described a 2-fold increase tis of N. bullata, Leptinotarsa decemlineata, and in ACE-activity during the early stages of D. Locusta migratoria (Schoofs et al., 1998). melanogaster metamorphosis. Activity peaked be- The present study shows that there is no residual tween pupal stages P6 and P8, and 20E increased effect of captopril on oviposition. No significant the expression of an ACE-like gene in imaginal difference in fecundity was observed between wing disc cells of B. mori (Quan et al., 2001).
adults emerging from captopril-treated and non- Ekbote et al. (2003b) also reported that lepi- treated larvae. But, when captopril was adminis- dopteran insects display an increase in ACE activ- tered orally or topically to newly emerged adults, ity during metamorphosis. ACE activity increased a decrease in oviposition was observed. Therefore, approximately 4-fold during the last larval instar we may conclude that captopril can penetrate and early pupal stages of Lacanobia oleracea. It is through the gut epithelium layer as through the possible that during metamorphosis, ACE contrib- skin. These results are in agreement with those re- utes to the generation of biologically active pep- ported for A. stephensi (Isaac et al., 1999; Ekbote tides and/or signal termination of already active In contrast to these results are the reports of ACE is not only thought to have a role during Vandingenen et al. (2001, 2002) and Hens et al.
metamorphosis. Several studies suggest a physi- (2002) concerning the interaction between ACE, ological role for the enzyme in insect reproduc- ACE-inhibitors, and trypsin modulating oostatic tion. In D. melanogaster, null alleles of Ance were factor (TMOF). TMOF was first identified in the larval lethal and a hypomorphic allele resulted in mosquito Aedes aegypti and named Aea-TMOF sterile male insects. The spermatocytes of these ster- (Borovsky et al., 1990). A second TMOF-like hor- ile males failed to develop beyond the primary mone was purified from extracts of vitellogenic ova- spermatocyte stage (Tatei et al., 1995). When male ries of the grey fleshfly N. bullata (Neb-TMOF) Anopheles stephensi mosquitoes were treated with (Bylemans et al., 1994). Aea-TMOF as well as Neb- ACE-inhibitors and allowed to mate with blood- TMOF terminate protein meal-induced trypsin bio- fed females, a dramatic reduction in fecundity was synthesis in the midgut, thereby impairing blood digestion and causing a lack of amino acids neces- (Ekbote et al., 1999). Moreover, Loeb et al. (1998) sary for vitellogenin synthesis by the fat body. Neb- demonstrated that ACE-activity stimulates ecdy- TMOF also inhibits in vitro and in vivo ecdysone steroid synthesis, perhaps due to feedback effects.
biosynthesis. It has been suggested that Neb-TMOF The experiments showed that both bovine ACE and is activated by Neb-ACE (Vandingenen et al., 2001).
bovine angiotensin II stimulate the synthesis of When female grey fleshflies (2 days after adult eclo- ecdysteroids by testis of L. dispar larvae and pupae, sion) were fed on a diet containing captopril fol- and yet inhibit the action of testis ecdysiotropin, a lowed by a liver meal on day 4, an increase in neuropeptide reported to be responsible for stimu- trypsin levels of 19–36% and an increase in lating ecdysteroid production by testes. Vandingenen vitellogenin titer was observed. The captopril treat- et al. (2001) suggested the reverse. In N. bullata ACE- ment might have reversed the effect of TMOF on inhibition would increase ecdysteroid titers by in- trypsin and vitellogenin biosynthesis. In this sce- hibiting the activation of Neb-TMOF; therefore, ACE nario, ACE-inhibition should lead to an increase activity suppressed ecdysteroid production. Our re- in fecundity; however, neither a stimulatory nor sults showed no differences in ecdysteroid titers af- an inhibitory effect on egg-laying was observed by ter captopril treatment of larvae and pupae. Nor was there a significant effect on treated male adults. Only To have better insight into the negative effects when female S. littoralis adults were treated with of captopril on oviposition in S. littoralis, we mea- captopril were ecdysteroid titers reduced.
sured the effect of captopril on trypsin activity in Our results and those of other researchers indi- vitro and in vivo. The in vitro tests revealed that cate that there is an extremely complex relationship there is no direct effect of captopril on trypsin ac- between fecundity (oviposition), vitellogenin pro- tivity. However, captopril treatment of S. littoralis duction, trypsin synthesis, and 20E, ACE, and TMOF larvae and female adults resulted in an increase in activity. Previous studies with A. aegypti demon- trypsin activity, whereas treatment of male adults strated that an ecdysteroid peak is necessary to ini- elicited a decrease in trypsin activity. Therefore, the tiate vitellogenesis in the primary follicle and results of the tests with female adults of S. littoralis separation of the secondary follicle (Beckemeyer and are in compliance with the results reported by Lea, 1980). In those insects in which ACE stimu- Vandingenen et al. (2001) using the grey fleshfly lates 20E biosynthesis, adding captopril, an ACE in- N. bullata, both in regard to captopril-induced hibitor, should correlate with a decrease in 20E trypsin activity and to the lack of stimulation of production probably at the site of biosynthesis in oviposition (N. bullata) or decreased levels of ovi- the ovaria, leading in turn to a blockage of vitello- position (S. littoralis) after treatment with captopril.
genesis. In addition, the effect of captopril is prob- And, although Isaac et al. (1999) reported that ACE ably indirect via peptides such as TMOF, as reduced male fertility, this decrease in fertility ac- Vandingenen et al. (2001) postulated that ACE ac- tually resulted from a decrease in oviposition. In tivates TMOF. Thus, ACE inhibition can lead to an contrast to the significant effect of captopril on fe- increase in trypsin activity, which, in turn, increases cundity, captopril had no effect on S. littoralis egg vitellogenin synthesis. Under these circumstances, an increase in oviposition could be expected. How- Several recent studies provide evidence for re- ever, in our experiments, a decrease in oviposition ciprocal interactions between ACE and ecdysteroid was observed. This agrees with Vandingenen et al.
production (Loeb et al., 1998; Quan et al., 2001; (2001) who reported a lack of stimulation of ovi- Vandingenen et al., 2001; Siviter et al., 2002). Quan position in N. bullata. We hypothesize that ACE has et al. (1998) reported that BmAcer expression is multiple modes of action, and that the exact mecha- ecdysone-inducible. A 20E-induced synthesis of nism of captopril’s activity is not clear. We suspect ACE-like activity was also observed in D. melano- that in our experiments, the stimulatory effect of gaster (Siviter et al., 2002) and in A. stephensi captopril on trypsin activity is counteracted by its Archives of Insect Biochemistry and Physiology negative effect on 20E, so vitellogenesis is blocked Cornell MJ, Williams TA, Lamango NS, Coates D, Corvol P, and oviposition is decreased. The question as to Soubrier F, Hoheisel J, Lehrach H, Isaac RE. 1995. Clon- whether there is a direct effect of 20E on trypsin or ing and expression of an evolutionary conserved single-domain angiotensin converting enzyme from Drosophila melanogaster. J Biol Chem 270:13613-13619.
In conclusion, our results suggest that there is an important role for ACE in metamorphic- and Corvol P, Michaud A, Soubrier F, Williams, TA. 1995. Recent reproductive-related events in the lepidopteran S. advances in knowledge of the structure and function of the littoralis. There appears to be a relationship between angiotensin I converting enzyme. J Hypertens 13:S3-S10.
ACE inhibition, trypsin activity, ecdysteroid titers, De Leo F, Bonadé-Bottino MA, Ceci LR, Gallerani R, Jouanin and oviposition levels, but further experiments are L. 1998. Opposite effects on Spodoptera littoralis larvae of needed to clarify the mechanisms of action/inter- high expression level of a trypsin proteinase inhibitor in action in these crucial life-cycle events.
transgenic plants. Plant Physiol 118:997-1004.
ACKNOWLEDGMENTS
De Loof A, Bylemans D, Schoofs L, Janssen I, Spittaels K, Vanden Broeck J, Huybrechts R, Borovsky D, Hua Y-J, Koolman J, This research is supported by a PhD grant for Sower S. 1995. Folliculostatins, gonadotropins and an model Lieselot Vercruysse from the Institute for the Pro- for control of growth in the grey fleshfly, Neobellieria(Sarcophaga) bullata. Insect Biochem Mol Biol 25:661-667.
motion of Innovation by Science and Technologyin Flanders (IWT) and by Project 01102703 from the Ekbote U, Looker M, Isaac RE. 2003a. ACE inhibitors reduce Special Research Fund of the Ghent University.
fecundity in the mosquito, Anopheles stephensi. CompBiochem Phys B 134:593-598.
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C domain of the mammalian homologue and does notrequire glycosylation for secretion and enzymic activity.
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