The folliculostatins of two dipteran insect species, their relation to matrix proteins and prospects for practical applications

The first insect folliculostatic peptide was isolated from vitellogenic ovaries of the mosquitoAedes aegypti. This decapeptide directly inhibits trypsin biosynthesis in the gut, and indirectly ovarian development. The factor was named Trypsin Modulating Oostatic Factor or TMOF by its discoverers. From the fleshfly Neobellieria bullata 2 folliculostatins have been isolated, the hexapeptide Neb-TMOF and the 19-mer Neb-colloostatin. The available data suggest that at least 2 of the 3 folliculostatins originate from matrix (like) proteins present in the ovary, a hitherto unknown source of hormones. Furthermore, one of the folliculostatins (Neb-TMOF) is a potent inhibitor of ecdysone biosynthesis by larval ring glands of fleshflies. The discovery of the dipteran folliculostatins, which do not show any resemblance to inhibins of vertebrates, may significantly contribute to a better understanding of the hormonal control of growth in insects and perhaps, in other animals as well. None of the 3 folliculostatins is blocked at its N- or C-terminus. This, in combination with the pleiotropy of their effects and the narrow species specificity make such peptides prime candidates for, testing their potential in insect pest control by means of molecular biological methods.     

The NMDA receptor antagonist MK-801 inhibits vitellogenesis in the flesh fly Neobellieria bullata and in the desert locust Schistocerca gregaria

We found that in the flesh fly Neobellieria bullata, vitellogenesis can be inhibited in a dose-dependent way by two injections of 60 microg MK-801/g body mass. In the desert locust Schistocerca gregaria, vitellogenesis can also be fully inhibited but only by repeated injections of 200-400 microg/g body mass. In this species, the inhibition can be overruled by coapplication of juvenile hormone. Vitellogenin bands remained visible in electropherograms of hemolymph of MK-801-treated female locusts, but vitellogenin did not accumulate as might be expected when only its uptake by the oocytes, and not its synthesis by the fat body, would be affected. Whether MK-801 acts by inhibiting juvenile hormone synthesis by the corpora allata remains to be investigated.     

Formation and dynamic alterations of horizontal microdomains in sperm membranes during progesterone-induced acrosome reaction

A peptidomics approach was applied to determine the peptides in the larval central nervous system of the grey flesh fly, Neobellieria bullata. Fractions obtained by high performance liquid chromatography were analysed by MALDI-TOF and ESI-Q-TOF mass spectrometry. This provided biochemical evidence for the presence of 18 neuropeptides, 11 of which were novel Neobellieria peptides. Most prominently present were the FMRFamide-related peptides: 7 FMRFamides, 1 FIRFamide, and Neb-myosuppressin. The three putative capa-gene products Neb-pyrokinin and the periviscerokinins Neb-PVK-1 and -2 were detected, as well as another pyrokinin. This Neb-PK-2 was also present in the ring gland along with corazonin, Neb-myosuppressin, and Neb-AKH-GK, an intermediate processing product of the adipokinetic hormone. Furthermore, the central nervous system contained Neb-LFamide, proctolin, and FDFHTVamide, designated as Neb-TVamide. With this study, we considerably increased our knowledge of the neuropeptidome of the pest fly N. bullata, which is an important insect model for physiological research.     

Isolation and characterization of an angiotensin converting enzyme substrate from vitellogenic ovaries of Neobellieria bullata

Vitellogenic ovaries of the gray fleshfly Neobellieria bullata contain a variety of unidentified substances that interact, either as a substrate or as an inhibitor, with angiotensin converting enzyme (ACE). We here report the isolation and characterization of the first ACE interactive compound hereof. This 1312.7 Da peptide with the sequence NKLKPSQWISL, is substrate to both insect and human ACE. It is a novel peptide that shows high sequence similarity to a sequence at the N-terminal part of dipteran yolk polypeptides (YPs). We propose to call it N. bullata ovary-derived ACE interactive factor or Neb-ODAIF. Both insect and human ACE hydrolyze Neb-ODAIF by sequentially cleaving off two C-terminal dipeptides. K(m) values of Neb-ODAIF and Neb-ODAIF(1-9) (NKLKPSQWI) for human somatic ACE (sACE) are 17 and 81 microM, respectively. Additionally, Neb-ODAIF(1-7) (NKLKPSQ) also interacts with sACE (K(m/i)=90 microM). These affinity-constants are in range with those of the physiological ACE substrates and suggest the importance of Neb-ODAIF and its cleavage products in the elucidation of the physiological role of insect ACE. Alternatively, they can serve as lead compounds in the development of new drugs against ACE-related diseases in humans.     

Occurrence of insect kinins in the flesh fly,stable fly and horn fly-mass spectrometric identification from single nerves and diuretic activity

MALDI-TOF mass spectrometric analysis of single lateral abdominal nerves (LANs) demonstrate the presence of the insect kinin Musdo-K in the housefly Musca domestica, and identify heretofore unknown insect kinins in two other Dipteran species as Musdo-K in the stable fly Stomoxys calcitrans and horn fly Haematobia irritans. The insect kinin native to the flesh fly Neobellieria bullata is identified as Drome-K. Musdo-K and Drome-K are identical save for the conservative substitution of Ser for Thr in position 2. The sequences of the insect kinins are, therefore, remarkably conserved throughout Dipterans. The in vitro Malpighian tubule fluid secretion activity of Musdo-K in the stable fly is similar to that in the housefly, whereas that of Drome-K is 30-fold more potent in the flesh fly than in the fruit fly. Given the structural identities of the kinins and CRF-like diuretic hormones of these Dipteran species, the housefly can serve as a model insect for the study of diuretic peptides and their functions in the stable fly and horn fly, both livestock pests.     

Characterization of four substrates emphasizes kinetic similarity between insect and human C-domain angiotensin-converting enzyme.

Angiotensin converting enzyme (ACE) was already discovered in insects in 1994, but its physiological role is still enigmatic. We have addressed this problem by purifying four new ACE substrates from the ovaries of the grey fleshfly, Neobellieria bullata. Their primary structures were identified as NKLKPSQWISLSD (Neb-ODAIF-1(1-13)), NKLKPSQWI (Neb-ODAIF-1(1-9)), SLKPSNWLTPSE (Neb-ODAIF-2) and LEQIYHL. Database analysis showed significant homology with amino acid sequence stretches as present in the N-terminal part of several fly yolk proteins. An antiserum raised against Neb-ODAIF-1(1-9) immunostained one out of three yolk protein bands of SDS/PAGE-separated fly haemolymph and egg homogenate, thus confirming that these peptides originate from a yolk protein gene product. Kinetic analysis of these peptides and of the peptides Neb-ODAIF and Neb-ODAIF-1(1-7) with insect ACE and human ACE show both similar and unique properties for insect ACE as compared with human C-domain ACE.     

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.     

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.     

Sulfakinins reduce food intake in the desert locust, Schistocerca gregaria

In vertebrates, the peptides cholecystokinin (CCK), neuropeptide Y, galanin, and bombesin are known to be involved in the control of food intake. We report here that insect sulfakinins, peptides which display substantial sequence similarities with the vertebrate gastrin/CCK peptide family, significantly inhibit food uptake in fifth instar nymphs of the locust, Schistocerca gregaria. Upon injection of Lom-sulfakinin, a neuropeptide present in the corpus cardiacum of locusts, food intake was significantly reduced in a dose-dependent manner within a fixed 20 min time period. The induced effect ranged from 13% inhibition (10 pmol of injected peptide) to over 50% inhibition at 1 nmol. Other naturally occurring sulfakinins from different insect species also elicited this satiety effect. Analogous to the satiety effect of CCK in vertebrates, the sulfate group is required for activity. No effect on the palptip resistance was found after injection with sulfakinin. Therefore it seems unlikly that sulfakinins reduce food intake by decreasing the sensitivity of the taste receptors.     

Isolation and primary structure of two sulfakinin-like peptides from the fleshfly,Neobellieria bullata

1. Two novel insect myotropic peptides termed neosulfakinin-I (Neb-SK-I) and neosulfakininII (Neb-SK-II) were isolated from the heads of 42 thousand fleshflies, Neobellieria bullata (Diplera, Sarcophagidae).2. A series of four, high-performance liquid Chromatographic (HPLC), fractionations performed on columns with different characteristic features yielded two purified biologically active, hindgut motility stimulating fractions, suitable for amino acid sequence analysis.3. The proposed sequences for the two peptides are: Phe-Asp-Asp-Tyr-Gly-His-Met-Arg-Phe-(NH₂), (Neb-SK-I) and X-X-Glu-Glu-Gln-Phe-Asp-Asp-Tyr-Gly-His-Met-Arg-Phe-(NH₂), (Neb-SK-II).4. These sulfakinins exhibit very high homology to putative drosulfakinin sequences which, however, have not yet been isolated, but were deduced from a cloned Drosophila gene encoding these peptides.5. Here we provide the first evidence for the expression of such peptides present in Dipterans.6. Insect sulfakinins show structural identities with the hormonally-active portion of vertebrate gastrin II-, cholecystokinin- and caerulin-related peptides and they share common carboxy terminal sequences with invertebrate/vertebrate peptides of the FMRFamide peptide family.     

Isolation of the hypertrehalosaemic factors I and II from the corpus cardiacum of the Indian stick insect, Carausius morosus, by reversed-phase high-performance liquid chromatography, and amino-acid composition of factor II

The separation of two peptides with hypertrehalosaemic activity from the corpus cardiacum of the Indian stick insect, Carausius morosus, was achieved by reversed-phase high-performance liquid chromatography using a Nucleosil C-18 column with a trifluoroacetic acid/acetonitrile gradient. The eluant was monitored at 210 nm and the hypertrehalosaemic activity was detected in ligated stick insects using a bioassay. In addition, the isolated material was potent in causing hyperlipaemia in migratory locusts and also in raising blood carbohydrates in the American cockroach. The amino-acid composition of the major peptide, hypertrehalosaemic factor II, was determined after acid hydrolysis with HCl or methanesulfonic acid. The analyses suggest that factor II is a nonapeptide which contains the following amino-acid residues: Asp, 2 Thr, Glu, Pro, Gly, Leu, Phe and Trp. This composition is almost identical to that of locust adipokinetic hormone I, lacking only one Asp residue.     

Presence of angiotensin converting enzyme (ACE) interactive factors in ovaries of the grey fleshfly Neobellieria bullata

Angiotensin converting enzyme (ACE) activity, defined as a captopril-inhibitable dipeptidyl carboxypeptidase activity towards 3H-hippurylglycylglycine, was demonstrated in haemolymph, testes and ovaries of the grey fleshfly Neobellieria bullata, hereby suggesting a physiological role for ACE in these particular tissues. While the ACE activity in haemolymph and testes reached relatively high levels, only minute ACE activity could be detected in ovaries throughout the entire vitellogenic cycle. Ovarian extracts of Neobellieria bullata do contain, however, in addition to Neb-TMOF, the Neobellieria bullata trypsin modulating oostatic factor which is an in vitro and a putative in vivo substrate of ACE in circulation, several other heat-stable molecules which individually function either as an ACE substrate or ACE inhibitor. Presumably these ACE interactive factors mask ACE activity in the fly ovaries, as measured by a classic substrate-binding assay. Purification and characterisation of these ACE substrates/inhibitors is in progress and is likely to facilitate the elucidation of the enigmatic physiological relevance of ACE in insects.     

Synthesis and effect of shortened oostatic decapeptide (TMOF) analogs with isosteric structures on reproduction of Neobellieria bullata.

Oligopeptides 2a-2d derived from the oostatic decapeptide (TMOF) sequence, H-Tyr-Asp-Pro-Ala-Pro-Pro-Pro-Pro-Pro-Pro-OH (1a) and containing isosteric structures were synthesized and assayed to determine their effect during reproduction in the flesh fly Neobellieria bullata. The N-terminal linear tetra- and pentapeptides 2a, 2b containing the Pro-psi[CH2O]Ala isosteric linkage affect egg development in 80-90% of ovarioles resulting in some resorbed egg chambers, abnormal yolk deposition, the formation of large eggs with irregular yolk granules and proliferation of follicular epithelium. In comparison with their nonisosteric precursors 1b, 1c they exhibit even more accelerated oostatic activity. However, peptides 2c, 2d containing a Pro-psi[CH2S]Ala isosteric linkage are less active.     

Immunological evidence for an allatostatin-like neuropeptide in the central nervous system of Schistocerca gregaria,Locusta migratoria and Neobellieria bullata

Methanolic brain extracts of Locusta migratoria inhibit in vitro juvenile hormone biosynthesis in both the locust L. migratoria and the cockroach Diploptera punctata. A polyclonal antibody against allatostatin-5 (AST-5) (dipstatin-2) of this cockroach was used to immunolocalize allatostatin-5-like peptides in the central nervous system of the locusts Schistocerca gregaria and L. migratoria and of the fleshfly Neobellieria bullata. In both locust species, immunoreactivity was found in many cells and axons of the brain-retrocerebral complex, the thoracic and the abdominal ganglia. Strongly immunoreactive cells were stained in the pars lateralis of the brain with axons (NCC II and NCA I) extending to and arborizing in the corpus cardiacum and the corpora allata. Although many neurosecretory cells of the pars intercerebralis project into the corpus cardiacum, only 12 of them were immunoreactive and the nervi corporis cardiaci I (NCC I) and fibers in the nervi corporis allati II (NCA II) connecting the corpora allata to the suboesophageal ganglion remained unstained. S. gregaria and L. migratoria seem to have an allatostatin-like neuropeptide present in axons of the NCC II and the NCA I leading to the corpus cardiacum and the corpora allata. All these data suggest that in locusts allatostatin-like neuropeptides might be involved in controlling the production of juvenile hormone by the corpora allata and, perhaps, some aspects of the functioning of the corpus cardiacum as well. However, when tested in a L. migratoria in-vitro juvenile hormone-biosynthesis assay, allatostatin-5 did not yield an inhibitory or stimulatory effect. There is abundant AST-5 immunoreactivity in cell bodies of the fleshfly N. bullata, but none in the CA-CC complexes. Apparently, factors that are immunologically related to AST-5 do occur in locusts and fleshflies but, the active protion of the peptide required to inhibit JH biosynthesis in locusts is probably different from that of AST-5.     

Mass spectrometric analysis of putative capa-gene products in Musca domestica and Neobellieria bullata

Neuropeptides of the capa-gene are typical of the abdominal neurosecretory system of insects. In this study, we investigated these peptides in two widely distributed and large pest flies, namely Musca domestica and Neobellieria bullata. Using a combination of MALDI-TOF and ESI-QTOF mass spectrometry, periviscerokinins and a pyrokinin were analyzed from single perisympathetic organ preparations. The species-specific peptide sequences differ remarkably between the related dipteran species. These differences could make it possible to develop peptide-analogs with group- or species-specific efficacy.     

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.     

Controlled transcriptional regulation in eukaryotes by a novel transcription factor derived from Escherichia coli purine repressor

Neb-LFamide or AYRKPPFNGSLFamide was originally purified from the grey flesh fly Neobellieria bullata as a myotropic neuropeptide. We studied the occurrence of this peptide and its isoforms in the central nervous system of different insect species by means of whole mount fluorescence immunohistochemistry, mass spectrometry, and data mining. We found that both sequence and immunoreactive distribution pattern are very conserved in the studied insects. In all species and stages we counted two pairs of immunoreactive cells in the pars intercerebralis. These cells projected axons throughout the ventral nerve cord. In the adult CNSs they formed a large number of immunoreactive varicosities as well. Mass spectrometry and data mining revealed that SIFamide exists in two isoforms: [G1]-SIFamide and [A1]-SIFamide. In addition, the SIFamide joining peptide is relatively well conserved throughout arthropod species. The conserved presence of two cysteine residues, separated by six amino acid residues, allows the formation of disulphide bridges.     

Inhibition of PK/PBAN-mediated functions in insects: discovery of selective and non-selective inhibitors

The antagonistic properties of a few linear and backbone cyclic (BBC) conformationally constraint peptide libraries and their analogs, were tested for the ability to inhibit pyrokinin/pheromone biosynthesis activating neuropeptide (PK/PBAN) mediated functions: sex pheromone biosynthesis in Heliothis peltigera female moths, cuticular melanization in Spodoptera littoralis larvae, pupariation in the fleshfly Neobellieria bullata and hindgut contraction in Leucophaea maderae, elicited by exogenously injected PBAN, pheromonotropin (PT), leucopyrokinin (LPK), myotropin (MT) or by the endogenous peptides. The data revealed differential inhibitory patterns within the same assay with different elicitors (in both the pheromonotropic and melanotropic assays) and among the different functions and disclosed selective antagonists, hinting at the possibility that the receptors that mediate those functions may differ from one another structurally.     

Fatty acid esters of juvenoid alcohols as insect hormonogen agents (juvenogens)

A series of 8 new juvenogens (3--10) was prepared starting from a pair of isomeric insect juvenile hormone bioanalogues ( and ). The biological activity of the juvenogens -- was tested for their effect on reproduction of the blowfly Neobellieria (Sarcophaga) bullata and for the juvenilizing activity on the termite Prorhinotermes simplex. Results of biological screening are important in structure--activity studies and promising for potential practical application of some of the juvenogens studied, especially against termites.