Dov Borovsky

Mosquito oostatic factor: a novel decapeptide modulating trypsin-like enzyme biosynthesis in the midgut.

A peptide that inhibits egg development in mosquitoes (oostatic factor) has been purified from the ovaries of female Aedes aegypti. The factor is a decapeptide with a molecular mass of 1047.6. The primary sequence has been determined as NH2‐Tyr‐Asp‐Pro‐Ala‐Pro‐Pro‐Pro‐Pro‐Pro‐Pro‐COOH from mass spectra recorded on a quadrupole Fourier transform instrument. The amino acid sequence exhibits sequence correlation to mammalian, plant, and several viral proteins. Injection of synthetic analogs into mosquitoes, biting midges, flies, and fleas inhibited proteolytic enzyme biosynthesis in the midgut. Binding studies with [3H]oostatic factor indicated that the midgut epithelial cells have a factor‐specific receptor.— Borovsky, D.; Carlson, D. A.; Griffin, P. R.; Shabanowitz, J.; Hunt, D. F. Mosquito oostatic factor: a novel decapeptide modulating trypsin‐like enzyme biosynthesis in the midgut. FASEB J. 4: 3015‐3020; 1990.

Sequencing and characterization of trypsin modulating oostatic factor (TMOF) from the ovaries of the grey fleshfly, Neobellieria (Sarcophaga) bullata

Injection of crude extracts of late vitellogenic ovaries into staged females of the grey fleshfly Neobellieria (Sarcophaga) bullata inhibited oocyte development and biosynthesis of trypsin-like enzymes in the gut. Trypsin synthesis in N. bullata is cyclic and is correlated with egg development, which is discontinuous. A trypsin modulating oostatic factor (Neb-TMOF) was purified from 10,000 vitellogenic ovaries and sequenced by mass spectrometry. Neb-TMOF is a hexapeptide (NH2-NPTNLH-COOH). Injection of the hormone at physiological concentrations (10(-9) M), inhibited trypsin-like synthesis by the midgut of liver-fed female flies, and caused a reduction of the vitellogenin concentration in the hemolymph and of oocyte growth. The role of Neb-TMOF in controlling egg development and the physiological similarities with Aedes-TMOF are discussed.

De novo biosynthesis of juvenile hormone III and I by the accessory glands of the male mosquito

The role of the male accessory glands (MAG) in reproduction was investigated in the mosquito Aedes aegypti. MAG incubated with [14C]acetate synthesized radioactively labeled JH III, JH III bisepoxide and methyl farnesoate. MAG incubated with L-[methyl-3H]methionine synthesized [3H]JH III and a molecule that chromatographed on HPLC with JH I. Analysis of MAG and whole males extract by glass capillary combined gas-chromatography-selected ion monitoring mass spectrometry identified JH III and I as the main analogs that were synthesized by male mosquitoes. MAG of Culex nigripalpus, Anopheles rangeli and Anopheles trinkae also synthesized JH III from L-[methyl-3H]methionine, which indicates that the male mosquito has a complete JH III biosynthetic pathway. Unfed and unmated Culex quinquefasciatus do not develop their ovaries to the resting stage. Females injected with one MAG extract equivalent or implanted with A. aegypti MAG developed their ovaries to the resting previtellogenic stage, whereas females that were injected with saline did not. These results indicate that MAG synthesize and secrete JH III. The corpora allata (CA) of the male Aedes aegypti also synthesize JH III from L-[methyl-3H]methionine. This observation may suggest that JH synthesized by the males CA is used for internal regulation, whereas JH synthesized by the MAG is transferred with the sperm into the female.

Exploiting host molecules to augment mycoinsecticide virulence

35 that participates in critical host physiological processes, thereby minimizing nontarget effects. Insect diuretic hormones participate in the regulation of water balance, and MSDH belongs to the corticotropin-releasing factor– related family of peptides. Synthetic MSDH has been shown to stimulate fluid excretion in vivo, resulting in pronounced loss of fluid through the gut and the epidermis, decreased feeding and ultimately insect death8–10. TMOFs are unblocked decaand hexa-peptides that terminate trypsin biosynthesis in the insect gut and are found in the ovaries of insects that include both mosquitoes and flies. Aea-TMOF circulates in the hemolymph, binds to gut receptors on the hemolymph side of the gut and inhibits trypsin biosynthesis by exerting a translational control on trypsin mRNA11. Because TMOF resists proteolysis in the gut and easily traverses the gut epithelial cells into the hemolymph in adults and larvae, it was fed to different species of mosquito larvae in which it caused inhibition of food digestion and anorexia, ultimately leading to starvation and death12. TMOF is currently under development as an insecticide and appears to be very specific against mosquitoes with minimal nontarget effects13. Indeed, TMOFs from different insects have different peptide sequences (e.g., the AeaTMOF sequence is YDPAPPPPPP, whereas the gray flesh fly, Sarcophaga bullata, SbTMOF sequence is NPTNLH14). MSDH-Gly (42 amino acids) and the A. aegypti Aea-TMOF peptides were expressed in B. bassiana by transformation of expression vectors containing a constitutive B. bassiana-derived gpd promoter and the nucleotide sequence corresponding to the insecticides has resulted in substantial damage to ecosystems and the emergence of insecticide-resistant agricultural pests. Mosquitoes are vectors of many human and animal infectious diseases that cause death and economic hardship. World Health Organization (WHO; Geneva) recommendations suggest the use of different control strategies as part of integrated vector management control to prevent the emergence of insecticide-resistant mosquitoes. Effective strategies, however, for long-term reduction of mosquito populations remain elusive2. Entomopathogenic fungi are virulent to a wide range of Lepidoptera as well as mosquitoes and have been considered as possible candidates for reducing disease transmission of vectorborne infectious agents3,4. Methods have been developed for delivery of these agents in agricultural settings as well as to adult and larval mosquitoes, and the fungi appear to be equally (or more) effective against insecticide-resistant strains as compared with their insecticide-susceptible parental strains5–7. To test whether host molecules can be used to increase the virulence of entomopathogenic fungi, we engineered two insect peptides—Manduca sexta diuretic hormone (MSDH) and A. aegypti trypsinmodulating oostatic factor (Aea-TMOF)—in B. bassiana, which expresses and secretes these hormones as it infects its host. Our idea was that the exogenously produced host peptide hormone would disrupt the normal endocrine or neurological balance of the host, making it more susceptible to the invading fungus. As candidates, we chose a potential broad host range target (MSDH) as well as a more host-specific peptide (TMOF) target Exploiting host molecules to augment mycoinsecticide virulence

Mass spectrometry and characterization of Aedes aegypti trypsin modulating oostatic factor (TMOF) and its analogs

Trypsin modulating oostatic factor (TMOF), a decapeptide that directly inhibits the biosynthesis of trypsin- and chymotrypsin-like enzymes in epithelial cells of mosquito midgut and indirectly inhibits vitellogenesis in anautogenous females, has been sequenced by Fourier transform mass spectrometry analysis. The peptide has a primary amino acid sequence of NH2-Tyr-Asp-Pro-Ala-(Pro)6-COOH and probably exhibits left-handed helical conformation as was shown by computer stereoview simulation. The factor is metabolized very rapidly (half-life of 1.6 h) in intact mosquitoes when injected after the blood meal. Inhibition of trypsin biosynthesis was followed in ligated abdomens, which synthesize trypsin but do not metabolise TMOF. At concentrations of 3 x 10(-9) M and 6.8 x 10(-6) M, TMOF inhibited 50 and 90% of trypsin-like enzyme biosynthesis, respectively. Several analogs of varying chain lengths were synthesized and evaluated for biological activity using dose-response curves. Switching the positions of Tyr and Asp at the N-terminus reduced the activity of the hormone, indicating that the N-terminus is important for biological activity. Removal of two to five prolines at the C-terminus also reduced activity, indicating that both the N- and C-termini are important. Synthesis of trypsin-like isozyme was followed in several insect species using [1,3-3H]diisopropyl-fluorophosphate (DFP) in the presence of tosylamide-2-phenylethyl chloromethyl ketone. Marked reduction of [1,3-3H]diisopropyl-phosphoryl-trypsin-like derivatives was noted after TMOF treatment, as assessed by polyacrylamide gel electrophoresis. These results indicate that the biosynthesis of trypsin-like enzyme in mosquitoes and other insects may be regulated by sequence-related TMOFs.

Characterization and localization of mosquito-gut receptors for trypsin modulating oostatic factor using a complementary peptide and immunocytochemistry.

The gut receptor of trypsin‐modulating oostatic factor (TMOF), a decapeptide hormone that regulates trypsin biosynthesis in the mosquito gut, has been characterized. The binding of TMOF to mosquito gut membranes reached maximum at pH 7.4 and 24°C. No binding was observed at pH 2.5 and the binding to the membranes declined rapidly at pH 8.0. At equilibrium, maximum binding to the receptor was observed at 60 min and 24°C. A synthetic complementary decapeptide NH2‐Ile‐Leu‐Gly‐Arg‐Gly‐Gly‐Gly‐Gly‐Gly‐Gly‐COOH (FOMT) for TMOF successfully competed with the gut receptor, and specifically bound TMOF (Kd = 4 μm and Kassoc = 2.5 × 105 m−1). TMOF binding to gut membranes was characterized with FOMT and a specific ELISA to the hormone at 24 and 72 h after blood feeding. Two classes of binding sites were found on the gut membrane; high affinity (Kd1 = 4.6 ± 0.7 × 10−7 m; Kassoc = 2.2 × 106 m−1 Bmax = 0.1 pmol/gut) and low affinity (Kd2 = 4.43 ± 1 × 10−6 m; Kaggoc = 2.3 × 105 m−1; Bmax = 0.2 pmol/gut). The total binding sites for high and low affinity classes of TMOF per gut were estimated as 6.3 × 1010 and 1.1 × 1011 sites, respectively. Specific binding sites on the gut increased after the blood meal and were visualized by immunocytochemical staining. These results suggest that TMOF regulates trypsin biosynthesis by binding to specific receptor sites that are located on the mosquito gut, and that this receptor can be studied using a complementary peptide approach.—Borovsky, D., Powell, C. A., Nayar, J. K., Blalock, J. E., Hayes, T. K. Characterization and localization of mosquito‐gut receptors for trypsin modulating oostatic factor using a complementary peptide and immunocytochemistry. FASEB J. 8: 350‐355; 1994.

Feeding the mosquito Aedes aegypti with TMOF and its analogs ; effect on trypsin biosynthesis and egg development

Female Aedes aegypti that were given a blood meal by enema deposited yolk in their oocytes and synthesized trypsinlike enzymes in their midgut. When females were given an enema of Aea-TMOF (Trypsin Modulating Oostatic Factor) (NH2-YDPAPPPPPP-COOH) and blood both egg development and trypsin biosynthesis were inhibited. Similar results were observed if TMOF was mixed with the blood meal and fed to female mosquitoes through a membrane. Renin inhibitor (NH2-PHPFHFFVYK-COOH) or poly proline given by enema with the blood meal did not affect egg development or trypsin biosynthesis. Feeding of TMOF analogs P1 (NH2-YDPAP-COOH) or P4 (NH2-YDPAPPPP-COOH) inhibited trypsin biosynthesis in the midgut. Injecting or giving an enema of an amidated peptide (NH2-WRPGPPPPPP-CONH2) of HIV-2 X-ORF protein also inhibited egg development and trypsin biosynthesis in the mosquito gut. When [3H]TMOF was purified by high performance liquid chromatography (HPLC) and fed with the blood meal through a membrane to female mosquitoes, [3H]TMOF outside the gut increased linearly for the first 24 h and 28% of the hormone was found outside the gut at 72 h. These results suggest that TMOF and its active analogs traverse the gut epithelial cells into the hemolymph, bind TMOF gut receptor(s) and modulate trypsin biosynthesis.

Folliculostatins, gonadotropins and a model for control of growth in the grey fleshfly, Neobellieria (Sarcophaga) bullata

The sequences of two folliculostatic peptides of the fleshfly Neobellieria bullata have been determined recently. The first peptide (Neb-TMOF: H-NPTNLH-OH), originates from a 75 kDa precursor protein found in vitellogenic oocytes. The hexapeptide directly inhibits the synthesis of trypsin-like enzymes in the gut, and thus lowers the concentration of yolk polypeptides in the hemolymph. It also inhibits the biosynthesis of ecdysone in the larval ring gland. Therefore, it could also be named prothoracicostatic hormone (Neb-PTSH). The second peptide (Neb-colloostatin: H-SIV-PLGLPVPIGPIVVGPR-OH) acts on previtellogenic follicles and is a cleaved product of a collagen-like precursor molecule. Our results indicate that peptides that are cleaved from matrix proteins could act as growth-inhibiting factors. Gonadotropin releasing hormone (GnRH)-immunolike peptides were not identified, but progress is being made in the isolation and characterization of factors which stimulate cAMP production by the ovary. Using these results, a novel model of growth control in which matrix proteins play an important role as a potential source of growth regulators has been developed.

Does ovarian ecdysone stimulate mosquitoes to synthesize vitellogenin

Abstract Physiological amounts of 20-hydroxyecdysone do not initiate vitellogenin synthesis in unfed, non-vitellogenic mosquitoes. Injecting more than 10,000 times the physiological amount induced synthesis, but considerably less than was induced by a blood meal. A dose of 20-hydroxyecdysone which exceeded the physiological level only several hundred times, did not sustain vitellogenin synthesis, when blood-fed mosquitoes were ovariectomized just prior to injection. Transplanting ovaries from vitellogenic to non-vitellogenic females did not initiate synthesis of vitellogenin in the recipient. In vitro, neither 20-hydroxyecdysone nor the ovaries of vitellogenic females were able to induce synthesis of vitellogenin in non-vitellogenic fat bodies. These experiments suggest that ecdysteroid, released by the ovaries, does not initiate ovarian development in mosquitoes.

Activities of natural methyl farnesoids on pupariation and metamorphosis of Drosophila melanogaster.

Methyl farnesoate (MF) and juvenile hormone (JH III), which bind with high affinity to the receptors USP and MET, respectively, and bisepoxy JH III (bisJH III) were assessed for several activities during Drosophila larval development, and during prepupal development to eclosed adults. Dietary MF and JH III were similarly active, and more active than bisJH III, in lengthening larval development prior to pupariation. However, the order of activity was changed (JH III>bisJH III>MF) with respect to preventing prepupae from eclosing as normal adults, whether administered in the larval diet or as topically applied at the white puparium stage. If endogenous production of all three larval methyl farnesoids was suppressed by a strongly driven RNAi against HMGCR in the corpora allata cells, most larvae did not attain pupariation. Farnesol (which has no demonstrated life-necessary function in larval life except in corpora allata cells as a precursor to methyl farnesoid biosynthesis) when incorporated into the diet rescued attainment of pupariation in a dose-dependent manner, presumably by rescuing endogenous production of all three hormones. A more mild suppression of endogenous methyl farnesoid production enabled larval attainment of pupariation. However, in this background dietary MF had increased activity in preventing puparia from attaining normal adult eclosion. The physiological relevance of using exogenous methyl farnesoids to block prepupal development to normally eclosed adults was tested by, instead, protecting in prepupae the endogenous titer of methyl farnesoids. JH esterase normally increases during the mid-late prepupal stage, presumably to clear endogenous methyl farnesoids. When JH esterase was inhibited with an RNAi, it prevented attainment of adult eclosion. Cultured adult corpora allata from male and female Aedes aegypti released both MF and JH III, and the A. aegypti nuclear receptor USP bound MF with nanomolar affinity. These A. aegypti data support the use of Drosophila as a model for mosquitoes of the binding of secreted MF to USP.