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Iiiiiiiiiiiiiiiiiii L' I p /}'->{ ~(3, v-Ca q c I qo:r~o FINAL REPORT I I Covering Period: August 15. 1991 - February 28, 1996 I Submitted to the Office of the Science Advisor U.S. Agency for International Development I I BIORATIONAL PESTICIDES BASED ON PHEROMONE I ANALOGUES I I Principal investigator: Jan Vrkoc Grantee Institution: Institute of Organic Chemistry and Biochemistry Department of Natural Products I Academy of Sciences of the Czech Republic Flemingovo nam. 2, 16610 Praha 6. Czech Republic I Collaborator: Glenn D. Prestwich Institution: Depa1iment of Chemistry I State University of New Yoriv Stony Brook. N. Y. I 11 794-3400, U.S.A. I Project Number: 936-5600 I Grant Number: DHR-5600-G-00-1051-00 I A.I.D. Grant Project Officer: Dr. Phil Warren I Project Duration August 14. 1991 to February 28, 1996 m f:i ~i I R~c~D IN RIDJR JU~J ? f 199~ I BEST AVAfLABLE COPY ~f~~~Tf~ - ...... ~ ..···'" '-·- {~ : ' I L' _-:;rt:''•r ---- I I I 2. Table of Contents I 3. Executive Summary 2 ,.., I 4. Research Objectives .) 5. Methods and Results 4 I 5.1. Synthetic work 4 5.2. Vapor pressures determinations 4 I 5 .3. Entomological studies 4 I 5 .3 .1. Methods 5 5.3.2. Results 7 I 5.3.3. Discussion 10 I 6. Impact, Relevance and Technology Transfer 13 7. Project Activities I Outputs 13 I 8. Project Productivity 14 9. Future work 14 I 10. Literature Cited 15 I Appendix I Table 1 16 I Table 2 i7 I Table 3 18 Table 4 19 I Figure 1 - 12 Cydia molesta 20 Figure 1 - 18 Ostrinia nubilalis -'-"? I Publications 1 - 9 so I I I . I# I I I 3. Executive Summary I All new compounds were chemically characterized and biologically tested. The data from I electrophysiological and behavioral test suggested that inhibitory properties of the pheromone analogs are not entirely connected with their mimicking capability of the I pheromone. Some of new inhibitors ( chloroformates, sulfur analogs and 4-membered lactones) may prove useful as tools in further biochemical as well as field studies. Pest I control is a dynamic field, and changes in its technology come about frequently. New chemical compounds that are continually being made available usually displace older I materials. In past decades attention is given to volatile natural chemicals (semiochemicals) produced by arthropods for their chemical communication among or between species. Sex I pheromones, one of many semiochemicals. have been used often to effectively monitor or control a number of pests. One of the control strategy is based on the disruption of the I communication between sexes by permeating the atmosphere of the area under treatment by synthetic pheromone. The second strategy of mating disruption has been proposed which I relies on chemical inhibition of insect olfaction by irreversibly activating a receptor cell or I blocking pheromone recognition. For this strategy some synthetic pheromone analogs were suggested and for biological tests electrophysiological and behavioral test were used. I Eleven analogs were prepared for Cydia molesra and thirteen for Ostrinia nubilalis. Both species are worldwide serious pests and occur also in Czech Republic. All new compounds I were chemically characterized and biologically tested. The data from electrophysiological and behavioral test suggested that inhibitory prope1ties of pheromone analogs are not entirely I connected with their mimicking capability of the pheromone. Some of new inhibitors ( chloroformates. sulfur analogs and 4-membered lactones) may prove useful as tools in I fmther biochemical as well as field studies. I I I I I I -.)- I I I 4. Research Objectives: Chemical communication is the most important information channel in insects. Pheromones can be used as biorational means for pest control and they already found I his place in different areas of Integrated Pest Management. Hygienically safe, selective biorational pesticides based on pheromones need further development including their methods of application. In our research program we studied one class I of prospective biorational pesticides - analogs of insect sex pheromones which could have a potential to disrupt premating communication between the sexes and thus to I impair reproduction of populations. The searching of new methods in the strategy of pest control based on natural compounds or their analogs is in agreement with public aversion to chemical insecticides. As in other countries also in Czech Republic there I exists a need for the development of alternatives in existing pest management. The main aim of our proposal was to synthesize new types of pheromone mimics. provide physiological and behavioral testing on laboratory colonies. and in case of I finding an active analog to start preliminary investigation of antennal proteins and mating disruption field trials in small scale. The strategies of sensory disruption by analogs were supported by published data that some pheromone mimics already I known before this project started have shown significant inhibitory activity on premating behavior. From the analysis of the known structures of sensory disruptants and taking into account inconsistency of some results obtained in different species it I was concluded that further research in this field was needed. Mating disruption control strategy by permeating the area under the treatment by synthetic pheromone is still complicated and the mechanisms involved in achieving I this effect have not yet been recognized. Mechanisms for interrupting long distance communication even by natural pheromone are very complex and probably more than I one are involved at the same time and some of them may act synergistically. The ignorance of the mechanisms slow down the development of optimal design of this method and its practical implementation. I In contrast chemical inhibition of insect olfaction is based on irreversibly activating a receptor cell (hyperagonism ), or blocking pheromone recognition in the receptor cell (antagonism). Both modes are based on disruption of biochemical events in the I sensilla by limited quantities of a chemical compounds and in effect a selective anosmia is resulting. I The problem of our project was at the same time studied in several research teems in USA, France, Spain. Russia and Switzerland. The topics of research were mainly fluorinated analogs as for example mono-, di- and tri- fluoro analogs of Zl 1-14:0Ac I (l ), fluorinated ESE 10-12:Ac (2), fluorinated analogs of esters components of Diparopsis castanea (3) and trifluormethyl ketones of Spodoptera littoral is (4 ). I Chlorinated analogs of ESE 10-12 :OH ( 5 .6) have been found as biologically active and halogen acetate analogues of Sesamia nonagrioides are inhibitors (7). 2.6-Dimethyloctyl formate was found as an attractant of Tribolium confusum (8). I Oxime ether analogs of sex pheromone components of A.gratis segetum are I I I -.+- I pheromone mnmcs (9). Electrophysiological and morphological characteristics of pheromone receptors and the behavioral activity of analogs have been studied m I Diprion pini ( 10). The insects chosen for our study was one of key orchard pests, the Oriental fruit I moth ( (vdia moles ta), and a serious pest on maize in Europe and North America European com borer ( Ostrinia nubilalis). While Oriental fruit moth was taken as a model of pest species where mating disruption with pheromone was successfully used I both in Australia and United States. mating disruption strategy for 0. nubilalis has not been so far studied in detail. Our approach to the mating disruption by pheromone mimics was based on the use of new reactive mimics of both known and new I structural types. All our studies were done on the chemical and biological level, where mainly electroantennographical (EAG) and electrosensillographical (ESG) investigation of new pheromone analogues were studied. Both the most EAG active I pheromone mimics and some of EAG inactive compounds were then used for flight-tunnel behavioral investigations. Taking into account EAG responses and inhibitory effect of the selected analogs interesting structure-activity relationship was I found. I 5. Methods and Results: I 5.1. Synthetic work All pheromone mimics were synthesized by procedures reporting in project-funded I publications. All structures of the synthesized compounds were proved by MS, IR and NMR spectra. Eleven compounds were synthesized for Cydia molesta tests and thirteen I compounds for Ostrinia nubilalis tests. The chemical structures of the compounds are summarized in Table 1 and Table 2. From the list of compounds we wanted to prepare I we did not achieve the success only with azaanalogs. 5.2. Vapor pressures determinations The above prepared analogs show high differences in volatility. For dose response I electrophysiological studies the saturated vapor pressures of all compounds were determined using a method based on gas chromatographic retention data. The data are I summarized in Table 3. 5.3. Entomological studies I Oriental fruit moths, Cydia molesta, and European Com Borer, Ostrinia nubilalis. originated from a colony maintained under laboratory conditions. Larvae were reared on an semiartificial diet under a 16:8 light:dark regimen. Pupae were sexed and males I were kept separately from females under the same light and temperature conditions. Newly emerged adults were collected daily and provided with water and sugar solution. 2-4 days old males were used for EAG experiments, 3-4 days old males for I wind tunnel observation. I I I I - ..,_ I I Electroantennograph_1 · I Two glass AgiAgCl rnicroelectrodes filled with physiological saline were used for EAG recordings: the ground electrode was placed into the head capsule of an intact male moth and the recording electrode was connected with the distal end of the male antenna. tip I of which had been cut off. Ante1mal responses were amplified (signal conditioner CyberAmp 320, Axon Instruments), digitized (Metrabyte DAS-16 AID. sample period 250 msec) and analysed by a PC 486 computer (Stand Alone Acquisition System, Run Teclmologies).
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