Studies towards the sex pheromone of the green capsid bug

<p>The green capsid bug, <em>Lygocoris pabulinus</em> (L.) (Heteroptera: Miridae) is a serious pest in fruit orchards, which is difficult to control. Because it is difficult to determine the actual population density, fruit growers apply insecticides against the green capsid bug on regular times to reduce the risk of crop damage (calendar spraying). Previous studies have shown that males are attracted to females and this might open the possibility to monitor this mirid pest with its sex pheromone. This ultimately would lead to a reduction in the usage of insecticides. Therefore this study to unravel the chemical structure and composition of the sex pheromone of this mirid was undertaken.</p><p>Several methods were used to collect and identify volatiles emitted by male and female <em>L. pabulinus.</em> First of all, a thermodesorption system (TDS) was used. Intact females, males or pheromone glands were placed in the oven part of the TDS, which was subsequently heated, thereby stripping volatile compounds from the insect. With this method the composition of the known sex pheromones of <em>Adoxophyes orana</em> (Lepidoptera: Tortricidae) and <em>Campylomma verbasci</em> (Heteroptera: Miridae) were confirmed, using only a single insect per analysis. The advantages of this rapid method are its high sensitivity and the low degree of degradation and contamination of the stripped off compounds. This technique was effective in analysing volatiles from small insects by gas chromatography without prior manipulation, such as solvent extraction or distillation.</p><p>Secondly, two similar methods were used to extract compounds from the headspace of female <em>L. pabulinus</em> . These methods, Solid Phase Microextraction (SPME) and Stir Bar Sorptive Extraction (SBSE) were both successful in trapping volatiles from <em>L. pabulinus</em> . SBSE was found to be much more sensitive than SPME, but SPME was easier to operate.</p><p>Bioassays play an important role to determine the biological activity of extracts or compounds. A disadvantage of the methods described above is that no material can be gathered to use in any bioassay. Therefore samples from both sexes of <em>L. pabulinus</em> were collected in two other ways. Firstly, headspace extracts were obtained by trapping volatiles from males and females on Tenax and subsequently eluting the Teanx with an organic solvent. Secondly, extracts were made from different body parts of the green capsid bug.</p><p>In order to determine the biological activity of these extracts and of the pure compounds, two different bioassays were used, 1) a vibration bioassay and 2) a Y-track olfactometer.</p><p>In the so-called vibration bioassay the specific courtship behaviour, i.e. a vibration of the abdomen, of male <em>L. pabulinus</em> was used. When both live and dead females were offered to males in this bioassay, vibration behaviour was elicited. When females were dissected into separate body parts, heads, wings and legs elicited equal responses, while thorax plus abdomen gave a much lower response. When these separate body parts were extracted with an organic solvent, the leg extracts elicited significantly stronger responses than any other extract. This suggests that female <em>L. pabulinus</em> legs are either the source of a (close-range) sex pheromone, or that female <em>L. pabulinus</em> accumulates the pheromone on the legs by grooming behaviour. Live and dead males or male legs did not elicit any vibration behaviour in males. Substrates on which females had walked elicited similar responses in males as female legs, indicating that the female deposits the pheromone on the substrate. This occurs passively as no depositing behaviour was observed in the females.</p><p>In the Y-track olfactometer, males had to choose between two different sources of volatiles: the male or female extract to be tested and the solvent used to make these extracts as a control. Headspace extracts from male and female <em>L. pabulinus</em> , as well as male and female leg extracts were tested in the Y-track olfactometer. Both the female leg and headspace extracts attracted males. Male extracts had no activity at all.</p><p>All the extracts were analysed by Gas Chromatography/Mass Spectrometry (GC/MS) and coupled Gas Chromatography-Electroantennography (GC-EAD). Male and female headspace had an almost similar profile, except for a small amount of mono-alkenes, sometimes present in female headspace extracts. Chemical analysis of the leg extracts showed that these contained several hydrocarbons such as <em>n</em> -alkenes, <em>n</em> -alkanes and some methylalkanes. Female leg extracts contained more ( <em>Z</em> )-9-pentacosene while male leg extracts contained more ( <em>Z</em> )-9-heptacosene. Furthermore, two alkenes were present in different ratios in males and females. ( <em>Z</em> )-9-pentacosene and ( <em>Z</em> )-7-pentacosene were present in the ratio of 5:1 in females, but in about 1:5 in males. ( <em>Z</em> )-9-pentacosene and ( <em>Z</em> )-7-pentacosene were also the two alkenes observed in female headspace extracts.</p><p>GC-EAD recordings with the headspace extracts from both males and females revealed that three compounds were consistently EAD-active. These were hexyl butyrate, ( <em>E</em> )-2-hexenyl butyrate and ( <em>E</em> )-4-oxo-2-hexenal. These compounds were also found with the thermal desorption system (TDS) in males and females. Besides these compounds, sometimes EAG responses were obtained for 1-hexanol, hexyl acetate, nonanal and ( <em>Z</em> )-3-hexenyl butyrate. In a different GC-EAD set-up the female leg extracts as well as the female headspace extracts were analysed. These recordings showed that also ( <em>Z</em> )-9-pentacosene and ( <em>Z</em> )-7-pentacosene were EAD-active.</p><p>Although female headspace and female leg extracts differ much in composition, both attracted males in the Y-track olfactometer. On the contrary, male leg extracts did not attract males in this bioassay. The only difference between male and female leg extracts was the ratio of various alkenes. A mixture of ( <em>Z</em> )-9-pentacosene and ( <em>Z</em> )-7-pentacosene in the ratio 5:1 elicited vibrational behaviour in males. The results indicate that these alkenes are important cues for male <em>L. pabulinus</em> and are probably also responsible for the attraction of males in the Y-track olfactometer by female headspace extracts.</p><p>Male antennae reacted strongly to hexyl butyrate, ( <em>E</em> )-2-hexenyl butyrate and ( <em>E</em> )-4-oxo-2-hexenal whereas female antennae gave little or no response, suggesting that these compounds may be important chemical signals as well for male <em>L. pabulinus</em> in their communication with conspecifics. These three compounds, with or without any of the other compounds giving irregular EAG-responses, together with the alkenes are probably used by male green capsid bugs to locate females. Further research is needed to determine exactly which compounds are needed in a particular ratio to attract male <em>L. pabulinus.</em></p>