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Trypsin Modulating Oostatic Factor from the mosquito, Aedes aegypti, (Aea-TMOF) inhibits juvenile hormone (JH) - stimulated egg chorionation and patency in the follicular epithelium cells of Heliothis virescens. Aea-TMOF exhibits highest inhibitory effect on oocytes or follicular epithelium cells when it is administered together with JH I rather than with JH III. These results indicate that Aea-TMOF specifically inhibits JH I-dependent events during egg maturation in Heliothis virescens. Preliminary pharmacological analysis of the Aea-TMOF effect on patency suggests that the decapeptide hormone acts upstream of the protein kinase-dependent step during the JH activated cellular signaling pathway.
Seven field isolates of Bacillus thuringiensis from the Lower Silesia, region of Poland, the Osola plain and phylloplane niches and soil samples from the Karkonosze National Park were tested in vitro for insecticidal activity against mosquito larvae Aedes aegypti. Both the spore/crystal mixture and pured crystals from B. thuringienis strains KpCl, KpF3 and OpQ3 (belonging to the first physiological group including the subspecies japonensis, yoso, jinghongiensis) proved to be the most active against insects (61-65% of corrected mortality). The lowest toxicity (7-28% mortality) was caused by B. thuringiensis wratislaviensis strains (PO12 and 13).
The present study larvicidal activities of hexane, chloroform, ethyl acetate, acetone and methanol extracts of Halimeda macroloba, Decsne, Caulerpa racemosa (Frosk) Weber-Van-Bosses and Ulva lactuca Lin, (Chlorophyceae) against Aedes aegypti. The marine macro green algae extracts were tested against early 4 th instar larvae of A. aegypti using WHO protocol and concentrations of 200, 400, 600,800 and 1000 ppm. The observed mortality was made 24 and 48 h after treatment, data was subjected to probit analysis to determine the lethal concentration (LC₅₀ and LC₉₀) of the treated larvae of the tested species. Among the tested extracts the maximum efficacy was observed in the ethyl acetate extracts. The ethyl acetate extracts of the seaweeds showed the presence of terpenoids, tannins and phenolic compounds stronger than the other extracts. The results revealed that all the extracts showed varied levels of larvicidal activity against A. aegypti tested. However, the ethyl acetate extract of C. racemosa showed remarkable larvicidal activity against A. aegypti (LC₅₀=579.9 and LC₉₀=1255.4 ppm values at 24 h and LC₅₀=495.4 and LC₉₀=1073.9 ppm at 48 h) followed by U. lactuca (LC₅₀=588.1 and LC₉₀=1290.7 ppm values at 24 h, and LC₅₀= 530.8, and LC₉₀= 1160.0 ppm at 48 h), respectively. The lowest larval mortality was observed with hexane extract of H. macroloba against A. aegypti with values of LC₅₀=1116.8 and LC₉₀= 1824.5 ppm (after 24 h) and LC₅₀=1059.9 and LC₉₀=1768.3 ppm (after 24 h). The present studies indicate that the larvicidal activity and phytochemicals derived from the ethyl acetate extract of C. racemosa have the potential to be used as an ideal eco-friendly approach and effective mosquito vector control agent.
The binding of TMOF to its gut receptor was followed by incubating guts removed from male and female Aedes aegypti. TMOF at physiological concentrations, in the presence of [γ32P]ATP, causes phosphorylation and release of gut-membrane protein (45 kDa) that is further processed by proteolysis. In the presence of protease inhibitors only the 45 kDa protein was released. The phosphorylation and processing of the 45 kDa protein does not happen in the absence of TMOF. Both larvae and adult guts release the protein in the presence of TMOF. Male Ae. aegypti do not synthesize trypsin in their gut and do not release the 45 kDa protein in the presence of TMOF because a TMOF receptor is probably absent. Homogenized guts do not release the 45 kDa protein, indicating that the protease processing or the ecto-protein kinase activity is probably reduced after breaking the tissue. The 45 kDa phosphorylated protein can be dephosphorylated by alkaline phosphatase and protein phosphatase, indicating that the phosphate group is covalently linked to either a serine or a tyrosine moiety. This is the first report that shows that in insects, binding of a peptide hormone activates its receptor by proteolysis.
The three-dimensional conformation of Aea-TMOF (Aedes aegypti Trypsin Modulating Oostatic Factor), a decapeptide (YDPAPPPPPP) isolated from mosquito ovaries that inhibits the translation of many trypsin-like serine proteases, e.g. in mosquitoes, flies and lepidopterans, was used as a model for the synthesis of 10 aromatic and aliphatic organic acid and ester analogues. The organic TMOF analogues were tested against herbivorous pest lepidopterans and larval Ae. aegypti. The compounds administered to microtiter plates or to leaf disks caused mortality to mosquito larvae and the diamondback moth, Plutella xylostella, larvae 3-6 days after treatment. The surviving diamondback moth larvae were sluggish, immobile and stopped feeding. A biochemical analysis showed that in larval P. xylostella the trypsin activity was low and correlated with the observed mortality. No activity was found against Heliothis virescens when four of the synthetic compounds that affected P. xylostella and Ae. aegypti were tested, although the decapeptide, TMOF, does affect trypsin biosynthesis in H. virescens.
Based on the 3D conformation of the N-terminus of TMOF in solution, six aromatic derivatives of enoic acid: 7-biphenyl-4-yl-hept-4-enoic acid (BPHE), 7-(4-butyl-phenyl)-hept-4-enoic acid (BuPHE), 7-cyclohexyl-hept-4-enoic acid (CyHE), 10-phenyl-dec-7-enoic acid (PDE), 7-p-tolyl-hept-2-enoic acid ethyl ester (THEEE) and 7-(4-methoxy-phenyl)-hept-2-enoic acid ethyl ester (MPEEE) were computer-modeled and synthesized. Treating first instar Aedes aegypti larvae with different concentrations of the TMOF mimics showed that addition of butyl to the benzyl ring, use of p-tolyl or converting the benzyl ring into cyclohexane increased the biological activity of the mimics by 5.2, 5.0 and 3.8-fold, respectively. Esterifying the carboxyl terminus into ethyl ester and addition of a methoxy group to the benzyl ring also increased, by 2-fold, the biological activity of the derivative. The position of the double bond in the aliphatic chain is important for enhanced biological activity. Aea-TMOF and CyHE fed to mosquito larvae equally inhibited trypsin biosynthesis in larvae for the first 24 h. The biological activity of CyHE, however, rapidly declined 2-3 days later, whereas TMOF activity stayed stable. These results indicate that TMOF organic mimics, although potent, need to be formulated in order to be more stable for future field applications.
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