Analysis of the sex pheromones of Symmetrischema tangolias and Scrobipalpuloides absoluta

Sex pheromones are substances which are used by insects to attract a partner with the intention to mate. Pheromones are essential for the species survival because without them the partner cannot be located. When the chemical structures are known, the sex pheromones could be applied for pest control of that species. Sex pheromones are produced by the insect itself and they are attractive in very low concentrations. The development of resistance against sex pheromones, in contrast to pesticides, is considered unlikely. Owing to the species specificity and the low amounts necessary, the natural environment of the pest is less afflicted by the use of sex pheromones in comparison to conventional pesticides.This thesis describes the isolation, identification and possible applications of the sex pheromones of two South-American moths, Symmetrischema tangolias and Scrobipalpuloides absoluta . The research was carried out as a co-operative project between the Research Institute for Plant Protection (IPO-DLO), Wageningen, The Netherlands, the Department of Organic Chemistry of the Wageningen Agricultural University (OC-WAU) and the International Potato Center, Lima, Peru (CIP). The project was financially supported by the Netherlands' Minister for Development Co-operation.The moth Symmetrischema tangolias (Gyen), synonym: Symmetrischema plaesiosema (Turner), occurs mainly in the higher regions of the Andes in Peru and Bolivia where it is a severe pest of potatoes. The larva lives in the fields in the stems of potato plants and in storage places in the tubers. The harvested potatoes are stored in open facilities which are easy accessible to the insects. The potato growing is essential for the local food provision in the above-mentioned areas.The sex pheromone of Symmetrischema tangolias has been identified (chapter 3) as a 2: 1 mixture of (E,Z)-3,7-tetradecadienyl acetate ( 6 ) and (E)-3-tetradecenyl acetate ( 1 ). In the sex pheromone glands, two additional minor, to the sex pheromone related, compounds have been identified, namely (Z)-5-tetradecenyl acetate (25) and (Z)-7-tetradecenyl acetate ( 26 ) (figure 8.1). The ratio of these compounds in the sex pheromone gland is: 63 : 31 : 5 : 1 for 6 : 1 : 26 and 25 respectively. In a new wind tunnel at IPO-DLO, several mixtures of the identified and synthesised sex pheromone components have been tested in different ratios (chapter 6). A mixture of 6 and 1 appears to be more attractive than the two components separately. Mixtures of 6 and 1 in ratios of 1 : 1 and 2: 1 are equally attractive. The addition of small amounts of minor components 25 and 26 does not affect the attractiveness of the sex pheromone blend. The function of these two minor components therefore remains unclear.The amounts and ratios of the four gland constituents were measured during the 24-hrs dark- light cycle (chapter 4). A new approach was followed which involved the direct introduction of sex pheromone glands into the gas chromatograph (GQ by using a special temperature programmable GC-injector. With this method it was possible to examine glands without prior processing which provides a clear advantage over earlier described methods. It turns out that the total amount of sex pheromone in the glands varied strongly from individual to individual (3.8 - 350 ng/♀). The total sex pheromone amounts were significantly lower in the scotophase than in the photophase (62.2 and 101.5 ng/♀respectively). The ratios of the various gland constituents showed a symmetrical distribution which did not fluctuate during the 24-hrs dark-light cycle.The synthetic sex pheromone was highly attractive to male Symmetrischema tangolias in field tests conducted in potato fields and in storage facilities in Peru. The local farmers were exited about the results and wish to apply the sex pheromone as soon as it becomes available. Although the synthesis of the mono-unsaturated compound 1 is easy, large scale synthesis of the double-unsaturated compound 6 (which has been never reported before) is problematic.The moth Scrobipalpuloides absoluta (Meyrick), synonym: Scrobipalpula absoluta (Meyrick), lives in low altitude areas of South America. The larva is a leafminer of tomatoes and has developed into a devastating pest in tomato cultivation, especially in Brazil, Peru and Chile. In contrast to small-scale potato crops in higher parts of the Andes, the tomato growing is a large-scale and professional business. The harvested tomatoes are largely processed and exported. Therefore, tomato growing is of national economic interest for the involved countries.The sex pheromone of Scrobipalpuloides absoluta has been identified (chapter 5) as a 9: 1 mixture of (E,Z,Z)-3,8,11-tetradecatrienyl acetate ( 16 ) and (E,Z)-3,8- tetradecadienyl acetate ( 3 ) respectively (figure 8.2).The ratio of these two identified compounds in the sex pheromone gland is 92: 8 (for 16 : 3 respectively). On a Peruvian tomato farm, several mixtures of the identified compound were tested in ratios between 100 : 0 and 80 : 20, for 16 : 3 respectively. All tested mixtures turned out to be highly attractive to Scrobipalpuloides absoluta males, however, none of them appeared superior. Both the identified sex pheromone components 16 and 3 are unique to this species. The synthesis of the minor compound 3 can be increased easily, however, large-scale synthesis of 16 may provide more problems due to its three double bonds.Various techniques were used to identify the sex pheromones of Symmetrischema tangolias and Scrobipalpuloides absoluta. By means of GC, the chain lengths, functional groups and number of double bonds could be established. The active components were found in the gas chromatogram by means of electroantennography (EAG-detector). This detection technique is based on the application of the insect's antenna (olfactory organ) as a detector. From the gas chromatographical research it turned out that the sex pheromone candidates for both species belonged to the same group of linear unsaturated compounds all with a chain length of 14 carbons. The mono-unsaturated components 1 , 25 and 26 could be identified with related reference compounds which were all available at IPO-DLO. Of the double-unsaturated compound 6 of the Symmetrischema tangolias sex pheromone sufficient amounts could be isolated by means of preparative GC to unambiguously determine the (E)-3-double bond with NMR. Through EAG-recordings of all mono-unsaturated related compounds the (Z)-7-double bond was postulated as the other double bond in molecule 6 . The synthesis of 6 confirmed the postulated structure. By means of derivatisation of sex pheromone compounds with dimethyl disulphide (DMDS) subsequently followed by analysis of the derivatives obtained with mass spectrometry, the double bond positions in the mono- and double-unsaturated compounds could be determined directly. For compound 3 the two double bonds could be located at positions 3 and 8 with the DMDS method. All four possible (E/Z) isomers were synthesised, tested with EAG-recordings and compared to the analytical obtained data on the minor sex pheromone component of Scrobipalpuloides absoluta. Through this the identity of 3 could be confirmed. With the DMDS derivatisation technique two of the three double bonds in 16 could be unambiguously located at positions 3 and 8, similar to the minor component 3 . The obtained EAG results suggested the same E/Z configuration for these two double bond positions in 16 . GC-analysis of available mono- and polyunsaturated related compounds left three possibilities for the structure of 16 . The identity of 16 was determined through synthesis of all three possibilities and comparison of the analytical data with the data obtained for the sex pheromone compound.A closer investigation of the DMDS derivatives of these latter three triple-unsaturated compounds revealed the possibility for the direct determination of all three double bond positions solely through interpretation of mass spectra. This has not been reported before for these types of molecules. For this reason, and because of its importance for this kind of research, a separate chapter (chapter 2) was dedicated to it.The discussion contains a proposal for the biosynthetic formation of the identified sex pheromones. Also the price control in commercial synthesis of sex pheromones is mentioned and illustrated through an example. Both pheromones of Symmetrischema tangolias and Scrobipalpuloides absoluta contain a synthetically problematic compound. Considering the need for these sex pheromones it is recommended to continue the research for more efficient synthetic routes.