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The red bud borer Resseliella oculiperda (Rübs.) is a pest insect of apple trees when rootstocks are grafted with scion buds by shield budding. The female midges are attracted to the wounds of the grafted buds where they lay their eggs. The larvae feed on the cambium and destroy the buds completely or partially, leading to bad union of the buds with the rootstocks. Budding strips are used very often by growers to bind scion buds to rootstocks. These strips cannot prevent midges from reaching the damaged tissue. Chemical treatments applied to the grafts and other types of strip do not provide better protection against the pest and may cause other risks for growers. In orchard experiments in 2000 and 2001, the authors evaluated the repellent action provided by three essential oils and five compounds of plant origin against the midges by impregnating budding strips with them. The essential oils of lavender, Lavandula angustifolia (P. Mill.), and -terpineol decreased the infestation of buds by more than 95 and 80% respectively. The other potential repellents tested [the essential oil of Juniperus virginiana (L.), citronellal, the essential oil of Cinnamomum camphora (L.) J. Presl, R-carvone, linalool and R-fenchone] decreased infestation by 67, 66, 51, 45, 37 and 25% respectively. The formulation and commercial development of budding strips impregnated with lavender oil is discussed.

The present paper describes a novel Y-track olfactometer for the analyses of olfactory responses of insects which are easily disturbed and then feign to be dead. The olfactometer has been demonstrated to be useful for the analyses of olfactory responses of the vine weevil, Otiorhynchus sulcatus F.

The oral toxicity of excretion products of several Photorhabdus and Xenorhabdus strains was tested on two thrips species: Frankliniella occidentalis and Thrips tabaci. Out of 46 Photorhabdus isolates and 6 Xenorhabdus isolates only 6 North American P. temperata isolates were toxic to the thrips species. After 7 days of drinking from P. temperata supernatant a mortality of 90% could be reached. Thrips were also killed after sucking from leaves covered with the toxins. Possibilities of using P. temperata in the control of thrips will be discussed

Headspace extracts from female Lygocoris pabulinus ( L.) ( Heteroptera: Miridae) as well as female leg extracts attracted male L. pabulinus in Y-track olfactometer bioassays. In contrast, only female leg extracts were active in the vibration bioassay. Male extracts had no activity at all in either bioassay. When the female leg extract was analysed by coupled gas chromatography-electroantennography (Z)-9-pentacosene and (Z)-7-pentacosene were EAD-active. (Z)-9-Pentacosene and sometimes (Z)-7-pentacosene were also observed in female headspace extracts. EAG responses could be obtained for (Z)-9-pentacosene. A mixture of (Z)-9-pentacosene and (Z)-7-pentacosene in the ratio 5 : 1 elicited vibration behaviour in males. The results indicate that these alkenes are important cues for male L. pabulinus in mate location behaviour.

Epoxides are cyclic ethers that readily react with various nucleophilic compounds. Consequently, epoxides can be used in many chemical synthesis reactions. Two enantiomeric forms of an epoxide are possible if one of the carbon atoms is chiral. This means that the epoxide is actually a racemic mixture of its two enantiomers. Due to the universal presence of chirality in nature it is important to use the proper epoxide enantiomer in the synthesis of compounds such as pharmaceuticals and agrochemicals, which should affect biological processes. Therefore, enantiopure epoxides are valuable intermediates in the synthesis of biologically active compounds by the pharmaceutical and agrochemical industries.Epoxide hydrolases (EHs) catalyze the hydrolysis of an epoxide into its corresponding diol. Moreover, EHs can hydrolyze racemic epoxide mixtures in an enantioselective manner. This results in the hydrolysis of one enantiomer, while the other remains unaffected and thus enantiopure. Therefore, EHs might be valuable tools to obtain enantiopure epoxides from racemic mixtures.EHs from yeast species, Rhodotorula glutinis in particular, have been usedto hydrolyze various epoxides with high activity and enantioselectivity. Consequently, the EHs from yeast species are promising biocatalysts that can be used in the production of enantiopure epoxides.In chapter 1 the work that is presented in this thesis is introduced. The aims of the research project were to gain important fundamental knowledge on EHs from yeasts and to develop biotechnological processes based on the use of these enzymes for the production of enantiopure epoxides. To introduce EHs in general, a broad overview is given in chapter 2 dealing with the molecular biology, biochemistry and potential application of these enzymes.In order to determine the relationship of these yeasts EHs to other known EHs, the EH-encoding genes and cDNA sequences from the yeast strains Xanthophyllomyces dendrorhous and R. glutinis were isolated (chapters 3 and 4). The genes were denominated EPH1 . Whereas the X . dendrorhous EPH1 open reading frame (ORF) of 1236 bp was interrupted by 8 introns, the 1230 bp-large R. glutinis EPH1 ORF was interrupted by 9 introns. The genes encoded polypeptides of 411 and 409 amino acids respectively, with corresponding calculated molecular masses of 46 kDa. The deduced amino acid sequences were similar to that of mammalian microsomal epoxide hydrolases. These enzymes belong to thea/bhydrolase fold family of enzymes, which have similar enzymatic structures and mechanisms. The EPH1 cDNA sequences were expressed in Escherichia coli to demonstrate their function. The epoxides, 1,2-epoxyhexane and 1-methyl-cyclohexene oxide, were hydrolyzed in an enantioselective manner. The inactivation of the EPH1 gene of X. dendrorhous showed that it was not essential for growth in rich medium under laboratory conditions.The epoxide hydrolase of R. glutinis was overproduced in the heterologous host Escherichia coli BL21(DE3) in order to develop a highly effective epoxide hydrolysis system (chapter 5). A strong improvement in Eph1 activity was found in cell extracts of the recombinant E. coli when compared to cell extracts of Rhodotorula glutinis , despite the formation of inactive Eph1 inclusion bodies. Co-expression of genes encoding molecular chaperones (DnaK-DnaJ-GrpE, GroEL-GroES, and trigger factor) decreased the amount of Eph1 inclusion bodies. However, there was no equivalent increase in active soluble Eph1. An increase in the level of soluble Eph1 was demonstrated by lowering the cultivation temperature from 37ºC to 21ºC and by using a fermenter for cultivation. Compared to R. glutinis the total increase in Eph1 activity for the recombinant E . coli towards 1,2-epoxyhexane was over 200 times, without loss of enantioselectivity. The utility of this Eph1 overproduction system was demonstrated by the hydrolysis of 1-oxa-spiro[2.5]octane-2-carbonitrile, which is a new R. glutinis Eph1 substrate and a versatile building block in organic synthesis. Whereas the recombinant E. coli , expressing R. glutinisEPH1 , could be used to hydrolyze 1-oxa-spiro[2.5]octane-2-carbonitrile with high Eph1 activity in an enantioselective manner. This was not possible using R. glutinis itself.To explore the biological diversity and potential industrial use of EHs from yeasts the Eph1-encoding cDNA sequences were also isolated from the carotenoid producing yeast species Rhodosporidium toruloides CBS 349, Rhodosporidium toruloides CBS 14 and Rhodotorula araucariae CBS 6031. These cDNA sequences encoded polypeptides of 409, 409, and 410 amino acids large respectively with molecular masses of 46 kDa. The deduced amino acid sequences were similar to that of the epoxide hydrolases from R. glutinis , X. dendrorhous and Aspergillus niger , which all correspond to the microsomal epoxide hydrolase sequence. Consequently, these cloned Eph1s probably belong to thea/bhydrolase fold family of enzymes. The epoxide hydrolase encoding cDNAs of the Rhodosporidium and Rhodotorula species were expressed in Escherichia coli BL21(DE3). The recombinant strains were able to hydrolyze trans -1-phenyl-1,2-epoxypropane in an enantioselective manner. The recombinant counterpart of Rhodosporidium toruloides CBS 14 was found to be a highly active and enantioselective biocatalyst for this substrate, despite the low activity and enantioselectivity of Rhodosporidium toruloides CBS 14 itself.

Transgenic Spodoptera exigua are being developed by microinjection of a piggyBac vector. The vector expresses green fluorescent protein (GFP) under the control of the actin promoter. Forty percent of the first-instar larvae that hatched from the injected eggs were green fluorescent. However, after backcrossing none of the G1 first-instar larvae was fluorescent and a transgenic line could not be established. Several possibilities for the use of transgenic insects are discussed

Gewasbescherming publiceert een serie artikelen over de verschillende DLO-PO onderzoekprogramma's. In dit artikel aandacht voor signalering en beheersing van plaaginsecten, mijten en slakken. Rubrieken in dit artikel: signaalstoffen, biologische bestrijding en trangene resistente gewassen

By using a thermodesorption system (TDS) together with a programmable temperature vaporizer (PTV) injector, we confirmed the composition of the sex pheromone of Adoxophyes orana (Lepidoptera: Tortricidae) and Campylomma verbasci (Heteroptera: Miridae) from a single insect per analysis. Intact females and males or pheromone glands were placed in the oven part of the TDS, which was subsequently heated. The compounds released were transferred to the PTV, which was cooled to -150°C. Injection was on a dual-column GC by heating the PTV rapidly to 250°C. The major sex pheromone compounds of A. orana were found only in the pheromone gland of females. Male and female C. verbasci showed fingerprint-identical chromatograms, except for the two sex pheromone compounds, which were present only in females. No distinct differences were found in compounds released from female and male Lygocoris pabulinus (Heteroptera: Miridae). The advantages of this rapid method are the high sensitivity and the low degree of degradation and contamination. This technique was effective in analyzing small insects by gas chromatography-mass spectrometry (GC-MS) without prior manipulation, such as solvent extraction or distillation.

The amounts and ratios of the four constituents of the sex pheromone gland of the moth Symmetrischema tangolias were measured during a 24-hr dark–light cycle. A new approach was followed that involved the direct introduction of sex pheromone glands into the liner of a two-dimensional gas chromatograph (2D-GC) by using a special temperature programmable GC injector. With this method it was possible to examine individual glands without prior processing. This provides a clear advantage over other methods. The detection limit was 0.5 ng. The total amount of sex pheromone in the glands varied strongly from individual to individual (3.8–350 ng/female). The total sex pheromone amounts were significantly lower in the scotophase than in the photophase (62.2 and 101.5 ng/female, respectively). The ratios of the various gland constituents showed a symmetrical distribution, which did not fluctuate during the 24-hr dark–light cycle.