Insect Trypsin Modulating Oostatic Factor (Neb-TMOF) and Its Analogs: Preliminary Structure/Biological Function Relationship Studies

Neb-TMOF, the trypsin modulating oostatic factor of gray fleshfly Neobellieria bullata, is a hexapeptide with the following sequence: H-Asn-Pro-Thr-Asn-Leu-His-OH. It has been isolated from vitellogenic ovaries in 1994. TMOF, the newly discovered insect peptide, inhibits trypsin biosynthesis in the gut, lowers yolk polypeptide concentration in the hemolymph and strongly inhibits ecdysone biosynthesis by larval ring glands. It is interesting that this short non-protected peptide contains in its molecule two Asn residues at positions 1 and 4 and His at its C-terminus. To obtain information about the role of the His-6 and Asn-4 residues we synthesised two series of Neb-TMOF analogs, modified: (1) in position 6 by D-His (I), His(Bzl) (II) and Phe(p-X) derivatives, where X = NH₂ (III), NO₂ (IV), OEt (V) and OH (VI) and (2) in position 4 by such amino acid residues as Ser (VII), Thr (VIII), Gly (IX), Asp (X), Glu (XI) and D-Asn (XII). The influence of these peptides on trypsin biosynthesis in N. bullata was determined in vivo. In preliminary investigations, we found that Neb-TMOF, [Phe(NH₂)⁶], and [Phe(NO₂)⁶]-Neb-TMOF inhibited trypsin biosynthesis, whereas [D-His)⁶]- and [D-His(Bzl)⁶]-Neb-TMOF were inactive. In further biological studies performed in vitro on heart of Tenebrio molitor we found that Neb-TMOF and [Phe(p-NH₂)⁶-Neb-TMOF showed weak cardioexcitatory activity, about 30% of the cardioexcitatory activity of proctolin, an insect neuromodulating peptide.     

Insect Trypsin Modulating Oostatic Factor (Neb-TMOF) and Its Analogs: Preliminary Structure/Biological Function Relationship Studies

Summary Neb-TMOF, the trypsin modulating oostatic factor of gray fleshflyNeobellieria bullata, is a hexapeptide with the following sequence: H-Asn-Pro-Thr-Asn-Leu-His-OH. It has been isolated from vitellogenic ovaries in 1994. TMOF, the newly discovered insect peptide, inhibits trypsin biosynthesis in the gut, lowers yolk polypeptide concentration in the hemolymph and strongly inhibits ecdysone biosynthesis by larval ring glands. It is interesting that this short non-protected peptide contains in its molecule two Asn residues at positions 1 and 4 and His at its C-terminus. To obtain information about the role of the His-6 and Asn-4 residues we synthesised two series of Neb-TMOF analogs, modified: (1) in position 6 byd-His (I), His(Bzl) (II) and Phe(p-X) derivatives, where X=NH₂ (III), NO₂ (IV), OEt (V) and OH (VI) and (2) in position 4 by such amino acid residues as Ser (VII), Thr (VIII), Gly (IX), Asp (X), Glu (XI) andd-Asn (XII). The influence of these peptides on trypsin biosynthesis inN. bullata was determinedin vivo. In preliminary investigations, we found that Neb-TMOF, [Phe(NH₂)⁶], and [Phe(NO₂)⁶]-Neb-TMOF inhibited trypsin biosynthesis, whereas [d-His)⁶]- and [d-His(Bzl)⁶]-Neb-TMOF were inactive. In further biological studies performedin vitro on heart ofTenebrio molitor were found that-TMOF and [Phe(p-NH₂)⁶]-Neb-TMOF showed weak cardioexcitatory activity, about 30% of the cardioexcitatory activity of proctolin, an insect neuromodulating peptide.     

Proteolytic breakdown of the Neb-trypsin modulating oostatic factor (Neb-TMOF) in the hemolymph of different insects and its gut epithelial transport

The degradation of the unblocked hexapeptide, trypsin modulating oostatic factor of the flesh fly Neobellieria (Sarcophaga) bullata (Neb-TMOF) was studied in vitro in the hemolymph of the lepidopteran Spodoptera frugiperda, the orthopteran Schistocerca gregaria and the dictyopteran Leucophaea maderae. The half-life in the different species varied from approximately 3min in L. maderae to approximately 25min in S. gregaria. Purification of the degradation products and ESI-Qq-oa-Tof mass spectrometry revealed the fragments Asn-Pro-Thr-Asn, Leu-His and Asn-Pro, which were the same in the hemolymph of all species. Except in Leucophaea, Neb-TMOF was cleaved in dipeptides starting from the C-terminus and the reaction could be, at least partially, inhibited by captopril. These observations suggest that a dipeptidase, which has very similar enzymatic properties as mammalian angiotensin converting enzyme (ACE) and which circulates in the hemolymph, apparently is involved in the breakdown of Neb-TMOF and might be a common but not a universal enzyme in insect hemolymph.The introduction of Neb-TMOF into the gut of S. gregaria with the help of a capillary tube (intubation) demonstrated that the intact peptide is able to cross the gut epithelium and to appear in the hemolymph compartment. Since [3H]-inulin, which is too large to cross cell membranes, was found to penetrate the gut walls at a measurable rate, the paracellular pathway might be also permeable to smaller peptides. There was indeed a clear correlation between the molecular weight of inulin, Neb-TMOF, and inositol and the rate of penetration of these compounds through the gut epithelium to the hemolymph. These are promising findings in view of a potential use of such peptides for insect control purposes.     

The putative‐farnesoic acid O‐methyl transferase (FAMeT) gene of Ceratitis capitata: characterization and pre‐imaginal life expression

Farnesoic acid O‐methyl transferase (FAMeT) is the enzyme involved in the penultimate step of insect juvenile hormone (JH) biosynthesis and is thus a key regulator in insect development and reproduction. We report the characterization of the putative‐FAMeT in the medfly or Mediterranean fruit fly, Ceratitis capitata. This gene was identified by suppressive subtractive hybridization and completely sequenced by the screening of a medfly cDNA library. The obtained sequence was analyzed for conserved protein domain identification and its expression profile was evaluated by quantitative Real‐Time PCR in medfly pre‐imaginal life. The tissue expression of the isolated gene was verified by in situ hybridization on third instar larvae sections. The characterization of the isolated gene pointed out several typical features of methyl transferase genes. The pre‐imaginal putative‐FAMeT expression levels were consistent with JH titer change in Diptera. As recognized in some crustaceans, this gene seems to be widely expressed in the medfly as well. Ceratitis capitata is one of the most relevant agricultural pests against which insecticides and the sterile insect technique (SIT) are extensively used in spite of the well‐known limitations of these approaches. Although results are not conclusive for the physiological role of the isolated gene, they suggest the characterization of a new gene in the Mediterranean fruit fly potentially involved in JH biosynthesis and may, therefore, have implications for pest control. © 2010 Wiley Periodicals, Inc.     

Captopril, a specific inhibitor of angiotensin converting enzyme, enhances both trypsin and vitellogenin titers in the grey fleshfly Neobellieria bullata.

A strong and constitutive angiotensin converting enzyme- or ACE-like activity was demonstrated in the hemolymph of the adult grey fleshfly Neobellieria bullata. In a competition assay, the N. bullata trypsin modulating oostatic factor (Neb-TMOF) was confirmed to be an in vitro substrate for this circulating Neb-ACE. Oral uptake of captopril, a selective and specific inhibitor of ACE, resulted in a complete phenotypic knockout of circulating ACE activity. When compared with control animals, captopril-fed female flies showed an increase in the liver meal-induced trypsin peak in the midgut and elevated levels of protein meal-induced yolk polypeptides in the hemolymph. The latter effect was not due to a slower vitellogenin uptake by the ovaries, because oocyte growth was not affected by the captopril treatment. The apparent synergism between the demonstrated ACE functionality and the previously reported effects of the oostatic peptide Neb-TMOF are discussed in the context of our recent finding that Neb-TMOF represents a prime candidate for being the first known in vivo substrate for circulating insect ACE. Arch.     

昆虫神经肽研究进展

近年来鉴定了化学结构的昆虫神经肽数目呈快速上升趋势, 家蚕滞育激素和性信息素合成激活肽被分离纯化.三种近年出现的研究方法对寻找新型昆虫神经肽起到重要作用,已经成功地鉴定了数个新型神经肽.昆虫神经肽cDNA或基因组DNA克隆显示了新的结构信息和神经肽间的相互关系.     

昆虫海藻糖酶的基因特性及功能研究进展

海藻糖酶(Treh)是昆虫能量代谢必不可少的一类酶, 亦是昆虫体内几丁质合成通路的第一个酶。其基因表达和酶活性直接与正常发育、 蜕皮、 变态以及繁殖等昆虫重要生理过程密切相关。目前已有多种昆虫的海藻糖酶基因被成功克隆, 从而发现昆虫海藻糖酶基因家族由多个成员组成。海藻糖酶基因所编码的蛋白大多数具有一个信号肽前导区, 部分蛋白拥有1~2个跨膜结构域, 根据是否具有跨膜结构, 可将其分为可溶性海藻糖酶(Treh1)和膜结合型海藻糖酶(Treh2)两类, 膜结合型海藻糖酶具有2个特有的标签序列, 即“PGGRFREFYYWDSY”和“QWDYPNAWPP”。海藻糖酶的主要功能是将胞外和胞内的海藻糖降解成葡萄糖, 为昆虫的生命活动提供能量。具体表现为两个方面, 一是参与昆虫几丁质合成途径, 从而调控表皮、 中肠等处的几丁质合成; 二是通过与激素的协同作用, 调控昆虫体内海藻糖和葡萄糖等糖类物质的浓度变化, 从而有效保护体内细胞的适应并渡过相应的逆境环境, 并提高其抗逆能力。鉴于海藻糖酶的重要功能, 其已成为害虫控制的潜在新靶标。不同类型海藻糖酶的功能研究及酶抑制剂的研发与应用将进一步推动害虫生物防治的发展。     

Gonadoinhibitory effects of Neb-colloostatin and Neb-TMOF on ovarian development in the mealworm, Tenebrio molitor L

The gonadostatic action of the peptides Neb-colloostatin (SIVPLGLPVPIGPIVVGPR) and Neb-TMOF (NPTNLH) from Neobellieria bullata was studied in female mealworm Tenebrio molitor. Both peptides potently inhibit ovarian development and terminal oocyte maturation of mated females during their first reproductive cycle. Injection of 4 mug of Neb-colloostatin or Neb-TMOFNeb-TMOF reduced, at day 4 of the cycle, the size of the terminal oocytes to about half or one third of the normal size in saline-injected controls. In addition, follicular patency was arrested. The injections of Neb-colloostatin and Neb-TMOF also caused a delay to the first ovulation and oviposition as well as a reduction of the number of eggs by about 50% in the first 3 days of the oviposition period. At 4 days after adult emergence, none of the peptides had caused significant changes in protein concentration or composition of the haemolymph. However, both peptides reduced total protein content in ovaries and induced qualitative changes in ovarian protein patterns. Electrophoretic analyses indicated that Neb-colloostatin and Neb-TMOF caused a loss of two proteins (150, 180 kDa) and a drastic reduction of 4 others (39, 43, 47, 130 kDa), which are the most abundant ones in ovaries of control females. On the other hand, they increased the concentration of 2 other polypeptides (65, 70 kDa), which normally occur in insignificant quantities in ovaries. Our results indicate that both peptides have a very similar mode of action despite the differences in their amino acid sequence. They seem to interfere with vitellogenin production by the fat body as well as with vitellogen uptake by the oocytes through modification of patency.     

Alterations in regulation of insulin biosynthesis in pregnancy and starvation studied in isolated rat islets of Langerhans

1. Insulin biosynthesis in isolated rat islets of Langerhans was determined by the incorporation of [(3)H]leucine into newly synthesized islet proteins. Anti-insulin serum covalently coupled to a solid phase (CNBr-activated Sepharose 4B) was used to separate the immunoreactive proinsulin and insulin from other islet proteins. This method was applied to a study of the regulation of insulin biosynthesis in isolated rat islets of Langerhans during pregnancy, and immediately after a period of food deprivation. 2. Islets isolated from pregnant rats showed an increased basal rate of synthesis compared with the non-pregnant controls. In addition, they showed a significant increase in biosynthesis of proinsulin and insulin in comparison with the normal islets over a range of glucose concentrations of 2-20mm. 3. Addition of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine significantly increased the insulin-synthetic response of normal islets over the glucose range 5-20mm, so that their glucose response approached that of islets from pregnant rats. 4. Normal female rates were injected with a long-acting progesterone derivative (hydroxyprogesterone hexanoate), to investigate the role of progesterone on the increased insulin biosynthesis observed in islets in pregnancy. There appeared to be no marked difference in insulin biosynthesis between the islets from the progesterone-injected and control rats in the presence of 2mm- or 6mm-glucose alone. However, in the presence of 4mm- or 6mm-glucose and 3-isobutyl-1-methylxanthine there was a significant increase in insulin biosynthesis in the progesterone-treated animals. 5. Total islet protein biosynthesis was determined by the incorporation of [(3)H]leucine into trichloroacetic acid-precipitable islet proteins. Islets isolated from normal rats showed a 1.6-fold increase in incorporation over the glucose concentration range 2-20mm, and this value remained unchanged during starvation; however, rates of incorporation were significantly raised in islets isolated from pregnant rats in the presence of 20mm-glucose. 6. Islets from starved and fed control rats were incubated in the presence of increasing concentrations of glucose or glucose+3-isobutyl-1-methylxanthine. The islets isolated from the starved animals showed a diminished insulin-synthetic response to glucose as compared with the controls; this response was partially restored to normal values by elevation of cyclic AMP concentrations by using 3-isobutyl-1-methylxanthine. 7. It is suggested that the alterations in glucose-stimulated insulin biosynthesis observed in islets during pregnancy and after a period of starvation could be attributable, at least in part, to a long-term alteration of the cyclic AMP system, and in pregnancy to a direct or indirect effect of progesterone on beta-cell function.     

Recent advances in the biochemistry of spinosyns

Spinosyn and its analogs, produced by Saccharopolyspora spinosa, are the active ingredients in a family of insect control agents. They are macrolides with a 21-carbon, 12-membered tetracyclic lactones that are attached to two deoxysugars, tri-O-methylrhamnose and forosamine. Labeling studies, analysis of the biosynthetically blocked mutants, and the genetic identification of the spinosyn gene cluster have provided detailed information concerning the mechanism of spinosyn biosynthesis and have enabled combinatorial biosynthesis of a large group of new spinosyns. The following developments have recently impacted the field of spinosyn biology: (1) A second-generation spinosyn called spinetoram (XDE-175) was launched in late 2007; it is a semisynthesized spinosyn derivative produced through the modification of 3′-O-methyl group of rhamnose and the double bond between C5 and C6 of spinosyn J and L. This molecule was shown to have improved insecticidal activity, enhanced duration of control, and an expanded pest spectrum. (2) A new class of spinosyns, the butenyl-spinosyns, was discovered from Saccharopolyspora pogona. The butenyl-spinosyns are similar to spinosyns, but differ in the length of the side chain at C-21. In addition to structural similarities with the spinosyns, the butenyl-spinosyns exhibit a high level of similarity in insecticidal activity to spinetoram. (3) Spinosyn analogs, 21-cyclobutyl-spinosyn A and 21-cyclobutyl-spinosyn D were generated by metabolic engineering of the spinosyn biosynthetic gene cluster. They showed better insecticidal activities against cotton aphid and tobacco budworm than that of spinosyn A and D. Future progress toward the development of more potent spinosad analogs, as well as enhancements in production yields will likely result from these recent advances in the genetics and biochemistry of spinosyns.     

Biosynthesis of vitamin B-12 Part I. Role of the ribosomal proteins in vitamin B-12 biosynthesis

1. 70 S ribosomes isolated from strains of Escherichia coli 113-3, K₁₂ and B take part in vitamin B-12 biosynthesis from AdoCbi-GDP, NAD and dimethylbenzimidazole in the presence of enzymes of the cytosol fraction. 2. 70 S ribosomes from E. coli 113-3 bind Ado[⁵⁸Co]Cbi-GDP. This reaction is independent of fusidic acid. 3. Proteins from 5 S RNA complex as well as L2 protein isolated from E. coli 113-3 ribosomes catalyze vitamin B-12 biosynthesis. The main catalytic function in this reaction is performed by protein L18.4. Vitamin B-12 biosynthesis proceeding in the presence of isolated ribosomal proteins is inhibited by fusidic acid, chloramphenicol and vernamycin but not by erythromycin. 5. Vitamin B-12 synthesized in the presence of isolated ribosomal proteins is biologically active.     

The applicability of the plant vigor and resource regulation hypotheses in explaining Epiblema gall moth–Parthenium weed interactions

A basic question in insect–plant interactions is whether the insects respond to, or regulate plant traits, or a complex mixture of the two. The relative importance of the directions of the influence in insect–plant interactions has therefore been articulated through both the plant vigor hypothesis (PVH) and the resource regulation hypothesis (RRH). This study tested the applicability of these hypotheses in explaining the interactions between Parthenium hysterophorus L. (Asteraceae) and its stem‐galling moth, Epiblema strenuana Walker (Lepidoptera: Tortricidae). Parthenium plants exposed to galling were sampled at three sites in north Queensland, Australia, over a 2‐year period, and the relationship between gall abundance and plant vigor (plant height, biomass, flowers per plant, and branches per plant) was studied. To test the predictions of PVH and RRH, the vigor of parthenium plants protected from galling using insecticides was compared to galled plants and plants that escaped from galling. The vigor of ungalled plants was less than the vigor of galled plants. The higher plant vigor in galled plants was not due to galling, as was evident from insecticide exclusion trials. The insect seemed to preferentially gall the more vigorous plants. These findings support the predictions of the PVH and are contrary to those of RRH. Since gall abundance is linked to plant vigor, galling may have only a limited impact on the vigor of parthenium. This has implications for weed biological control. If the objective of biological control is to regulate the population of a plant by a galling insect, a preference for more vigorous plants by the insect is likely to limit its ability to regulate plant populations. This may explain the paucity of successes against biocontrol of annual weeds using gall insects.     

Salicylate-mediated suppression of jasmonate-responsive gene expression in Arabidopsis is targeted downstream of the jasmonate biosynthesis pathway

Jasmonates (JAs) and salicylic acid (SA) are plant hormones that play pivotal roles in the regulation of induced defenses against microbial pathogens and insect herbivores. Their signaling pathways cross-communicate providing the plant with a regulatory potential to finely tune its defense response to the attacker(s) encountered. In Arabidopsis thaliana, SA strongly antagonizes the jasmonic acid (JA) signaling pathway, resulting in the downregulation of a large set of JA-responsive genes, including the marker genes PDF1.2 and VSP2. Induction of JA-responsive marker gene expression by different JA derivatives was equally sensitive to SA-mediated suppression. Activation of genes encoding key enzymes in the JA biosynthesis pathway, such as LOX2, AOS, AOC2, and OPR3 was also repressed by SA, suggesting that the JA biosynthesis pathway may be a target for SA-mediated antagonism. To test this, we made use of the mutant aos/dde2, which is completely blocked in its ability to produce JAs because of a mutation in the ALLENE OXIDE SYNTHASE gene. Mutant aos/dde2 plants did not express the JA-responsive marker genes PDF1.2 or VSP2 in response to infection with the necrotrophic fungus Alternaria brassicicola or the herbivorous insect Pieris rapae. Bypassing JA biosynthesis by exogenous application of methyl jasmonate (MeJA) rescued this JA-responsive phenotype in aos/dde2. Application of SA suppressed MeJA-induced PDF1.2 expression to the same level in the aos/dde2 mutant as in wild-type Col-0 plants, indicating that SA-mediated suppression of JA-responsive gene expression is targeted at a position downstream of the JA biosynthesis pathway.     

New Bioactive Macrocyclic Diterpenoids from Euphorbia helioscopia

Three new macrocyclic diterpenoids, euphoscopoids A – C ( 1 – 3 ), including two new jatrophanes and a new lathyrane, were isolated from the whole plant of Euphorbia helioscopia. Their structures were elucidated by spectroscopic methods. Antifeedant and cytotoxic activities of these isolates were evaluated. All compounds showed significant antifeedant activity against a generalist plant‐feeding insect, Helicoverpa armigera, with EC₅₀ values ranging from 2.05 to 4.34 μg/cm². In addition, compound 2 showed moderate cytotoxicity against tumor cell lines NCI‐H1975, HepG2, and MCF‐2, while compounds 1 and 3 were not active at 80 μm . The results suggested not only the defensive function of macrocyclic diterpenoids in E. helioscopia against insect herbivores, but also their potential applications as new natural insect antifeedants.     

A cluster of genes for the biosynthesis of spinosyns, novel macrolide insect control agents produced by Saccharopolyspora spinosa

Spinosyns A and D are the active ingredients in a family of insect control agents produced by fermentation of Saccharopolyspora spinosa. Spinosyns are 21–carbon tetracyclic lactones to which are attached two deoxysugars. Most of the genes involved in spinosyn biosynthesis are clustered in an 74 kb region of the S. spinosa genome. This region has been characterized by DNA sequence analysis and by targeted gene disruptions. The spinosyn biosynthetic gene cluster contains five large genes encoding a type I polyketide synthase, and 14 genes involved in modification of the macrolactone, or in the synthesis, modification and attachment of the deoxysugars. Four genes required for rhamnose biosynthesis (two of which are also required for forosamine biosynthesis) are not present in the cluster. A pathway for the biosynthesis of spinosyns is proposed.     

Action of a juvenile hormone analogue on the activity of Periplaneta americana corpora allata in vitro and on juvenile hormone III levels in vivo

The effects of the juvenile hormone (JH) analogue fenoxycarb (ethyl[2-(4-phenoxyphenoxy)-ethyl]carbamate) on the activity of corpora allata (CA) from adult female Periplaneta americana have been investigated. The in vitro biosynthesis of JH III by isolated CA was inhibited by about 85% in the presence of a high concentration (1 × 10⁻⁴ M) of fenoxycarb. However, at lower concentrations (1 × 10⁻⁶ M and 1 × 10⁻⁸ M) no inhibition of JH biosynthesis was apparent. Topical treatment of adult female cockroaches with fenoxycarb (100 μg/insect) did not reduce the subsequent rate of JH III biosynthesis by CA in vitro. By contrast, the same treatment markedly reduced the titre of endogenous JH III in intact cockroaches. These results suggest that CA activity in adult female P. americana may be controlled by negative feedback, and that this system of control is dependent on the maintenance of contact between the CA and nervous or humoral factors in the intact insect. Alternatively, it is possible that treatment with fenoxycarb increases the rate at which endogenous JH is metabolized.     

Abscisic acid biosynthesis during corn embryo development

In this study we examined the biosynthesis of abscisic acid (ABA) by developing corn (Zea mays L.) embryos. Three comparisons were made: ABA biosynthesis in embryos isolated from kernels grown in vitro with those grown in the field; the developmental profile of ABA content with that of biosynthesis; and ABA biosynthesis in corn embryos lacking carotenoid precursors with ABA biosynthesis in normal embryos. Embryos were harvested at various times during seed development and divided into two groups. Endogenous levels of ABA were measured in one group of embryos and ABA biosynthetic capacity was measured in the other group. The ABA biosynthetic capacity was measured with and without tetcyclacis (an inhibitor of ABA degradation) in embryos from both field-grown and in-vitro-grown corn kernels. Reduced-carotenoid (either fluridone-treated or genetically viviparous) embryos were also included in the study. Corn kernels developing under field and in-vitro conditions differed from each other in their responses to tetcyclacis and in their profiles of ABA biosynthesis during development. Therefore, in-vitro kernel culture may not be an appropriate substitute for field conditions for studies of embryo development. The developmental profiles of endogenous ABA content differed from those of ABA biosynthesis in isolated embryos of both in-vitro-and field-grown kernels. This indicated that ABA levels in the developing embryos were determined by import from the maternal tissues available to the embryos rather than by in-situ biosynthesis. In embryos with reduced levels of carotenoids, either fluridone-treated or genetically viviparous embryos, ABA biosynthesis was low or nonexistent. This result is expected for the presence of an indirect pathway of ABA biosynthesis and in the absence of ABA precursors.Abbreviations ABA abscisic acid - DAP days after pollination     

In vitro degradation of the Neb-Trypsin modulating oostatic factor (Neb-TMOF) in gut luminal content and hemolymph of the grey fleshfly, Neobellieria bullata

The unblocked hexapeptidic Trypsin Modulating Oostatic Factor of the fleshfly, an inhibitor of both trypsin and ecdysone biosynthesis, resists very well proteolytic breakdown by enzymes present in the lumen of the gut of previtellogenic fleshflies. However, when incubated in hemolymph of adult flies, females and males, its half-life time is a mere 0.5 min. In hemolymph of last instar larvae, this value increases to about 1.5 min. Whereas PMSF, a potent inhibitor of serine proteases has no effect, captopril and lisinopril, both known to be specific inhibitors of mammalian angiotensin I converting enzyme (ACE), effectively inhibit TMOF breakdown in fly hemolymph. Digestion of Neb-TMOF by recombinant Drosophila AnCE on itself results in identical degradation products as with total hemolymph. In both cases ESI-Qq-oa-Tof mass spectrometry demonstrated the appearance of peptide fragments with the sequences NPTN, LH and NP. These observations not only confirm the reported presence of circulating ACE-like activity in flies but also strongly suggest that in flies this hemolymph ACE-like activity might be involved in the regulation of the oostatic activity as exerted by Neb-TMOF.