Licentiate Thesis, Sundsvall 2009

Identification and Syntheses of Semiochemicals Affecting privata and Trioza apicalis

Anna Nilsson

Department of Natural Sciences, Engineering and Mathematics Mid Sweden University, SE-851 70 Sundsvall, Sweden

ISSN 1652-8948, Mid Sweden University Licentiate Thesis 43 ISBN 978-91-86073-61-9

i Akademisk avhandling som med tillstånd av Mittuniversitetet i Sundsvall framläggs till offentlig granskning för avläggande av filosofie licentiatexamen fredag den 18 december, 2009, klockan 13:00 i sal O111, Mittuniversitetet Sundsvall. Seminariet kommer att hållas på svenska.

Identification and Syntheses of Semiochemicals Affecting and Trioza apicalis

Anna Nilsson

Cover Description Cover picture: Front side on the left and back side: The autumn gum , Mnesampela privata (: Geometridae). Photo: Merlin Crossley. Front side on the right: The carrot psyllid, Trioza apicalis (Homoptera: Psylloidea). Photo: Jarmo Holopainen

© Anna Nilsson, 2009

Supervisor: Erik Hedenström

Department of Natural Sciences, Engineering and Mathematics Mid Sweden University, SE-851 70 Sundsvall Sweden

Telephone: +46 (0)771-975 000

Printed by Kopieringen Mid Sweden University, Sundsvall, Sweden, 2009

ii Identification and Syntheses of Semiochemicals Affecting Mnesampela privata and Trioza apicalis Anna Nilsson

Department of Natural Sciences, Engineering and Mathematics Mid Sweden University, SE-851 70 Sundsvall, Sweden ISSN 1652-8948, Mid Sweden University Licentiate Thesis 43; ISBN 978-91-86073-61-9

ABSTRACT

The Autumn gum moth, Mnesampela privata (Lepidoptera: Geometridae) is an endemic Australian moth whose larvae feed upon species of . The favorite host plants are E. globulus and E. nitens which are the most important species used in commercial plantations of the Australian pulpwood industry. The autumn gum moth has become one of the most significant outbreak of eucalyptus plantations throughout Australia. As a consequence great financial losses to the forest industry occur. Today insecticides such as pyrethroids are used for control of eucalyptus defoliators as M. privata. The carrot psyllid, Trioza apicalis (Homoptera: Psylloidea), is one of the major pests of carrot () in northern Europe. The psyllid causes curling of the carrot leafs and reduction of plant growth. Today the carrot crops are protected with the pyrethroid insecticide cypermethrin, which is toxic to aquatic organisms and is, from 2010, prohibited for use in Sweden by the Swedish Chemicals Inspectorate. An alternative to insecticides is to protect the seedlings with semiochemicals, a chemical substance or mixture of them that carries a message. This thesis describes the identification and the syntheses of semiochemicals from the above mentioned species. From analysis of abdominal tip extracts of M. privata females from Tasmania a blend of (3Z,6Z,9Z)-3,6,9-nonadecatriene and (3Z,6Z,9Z)-3,6,9-heneicosatriene was identified as the sex pheromone of this species. The identification of the C19- and C21-trienes was confirmed by synthesis. In the analysis of carrot leaf extracts we found a compound, α-cis-bergamotene, that induces antennal response in the carrot psyllid. This is just the beginning of

iii the studies of trying to manipulate this psyllid with semiochemicals instead of insecticides.

Keywords: Mnesampela privata, semiochemicals, pheromones, (3Z,6Z,9Z)-3,6,9- nonadecatriene, (3Z,6Z,9Z)-3,6,9-heneicosatriene, carrot psyllid, Trioza apicalis, α-cis-bergamotene.

iv SAMMANFATTNING

Mnesampela privata (Lepidoptera: Geometridae) är ett endemiskt australiskt mott, vars larver livnär sig på blad från arter av släktet Eucalyptus. Den Australiensiska massaindustrins viktigaste trädarter är E. globulus och E. Nitens, vilka också är mottets favoritvärdväxter. M. privata är en av de värsta skadeinsekterna i Australien och orsakar stora ekonomiska förluster för skogsindustrin. Idag besprutas utsatta Eukalyptus-områden med insekticider som pyretroider, som samtidigt dödar andra arter i området. Morotsbladloppan, Trioza apicalis (Homoptera: Psylloidea), är en allvarlig skadegörare på morot, (Daucus carota), i norra Europa. Angripna plantor får en blast som liknar kruspersilja, och angreppen leder till en minskning av tillväxten. Idag skyddas morotsodlingarna mot bladloppan med en insekticid, pyretroiden cypermetrin, som samtidigt är giftig för bland annat vattenlevande organismer. Efter ett beslut av Kemikalieinspektionen kommer användningen av cypermetrin att förbjudas i Sverige fr.o.m. 2010. För att skydda odlingar från skadeangrepp och samtidigt undvika besprutning med insekticider kan alternativet vara att använda ett naturligt ämne eller en blandning av ämnen, som förmedlar ett budskap s.k. semiokemikalier, vilka ofta är doftsignaler. Denna avhandling beskriver identifiering och syntes av sådana doftsignaler. Efter analys av körtelextrakt från M. privata honor insamlade i Tasmanien identifierades en blandning av (3Z,6Z,9Z)-3,6,9-nonadecatriene and (3Z,6Z,9Z)- 3,6,9-heneicosatriene som M. privatas sexualferomon. Identifieringen av C19- and C21-trienerna bekräftades vidare genom syntes av de aktiva föreningarna. Vid analys av morotsblads extrakt hittades en förening, α-cis-bergamotene, som kan uppfattas av morotsbladsloppan. Detta är bara inledningen av en studie för att försöka manipulera loppan med doftsignaler istället för insekticider.

v

ABBREVIATIONS

ACT Australian Capital Territory AGM Autumn gum moth CoA coenzyme A E entgegen, trans, Latin: across EAG electroantennogram EAD electroanntennographic detector Et2O diethyl ether GC gas chromatography h hour ha hectare IPM integrated pest managment LC liquid chromatography m-CPBA 3-Chloroperoxybenzoic acid min minutes µg microgram MS mass spectrometry ng nanogram NMR nuclear magnetic resonance spectrometry PDC pyridinium dichromate pg picogram rt room temperature SCR single-cell recording SPME solid phase micro extraction THF tetrahydrofuran Z zusammen, cis, Latin: on this side

vi TABLE OF CONTENTS

Abstract ...... iii Sammanfattning ...... v Abbreviations ...... vi List of papers ...... ix 1.Introduktion ...... 1 1.1 Semiochemicals ...... 1 1.1.2 Pheromones ...... 2 1.1.3 Allelochemicals ...... 4 1.2 Insect damages and pest management...... 5 2. Identification of semiochemicals ...... 7 2.1 Collection and purification ...... 7 2.2 Identification and synthesis ...... 8 3. PART – A: Mnesampela privata (Lepidoptera: Geometridae) ...... 12 3.1 Lepidopteran - Moth ...... 12 3.1.1 Mnesampela privata ...... 12 3.2 Lepidoptera pheromones ...... 13 3.2.1 Biosynthesis of Lepidopteran pheromones...... 14 3.3 Results ...... 17 3.3.1 Identification of the extract A, 1-2 day old virgin females ...... 17 3.3.2 Identification of extract B, 1-2 day old virgin females ...... 20 3.3.3 Identification of extract C, females of different ages ...... 24 3.4 Syntheses of reference substances ...... 28 3.5 Conclusions and future work ...... 30 4. PART - B: Trioza apicalis (Homoptera: Psylloidea) ...... 32 4.1 Homoptera ...... 32 4.1.1 Trioza apicalis ...... 32 4.2 The damage of carrots ...... 33

vii 4.3 Methods to control the carrot psyllids ...... 33 4.3.1 Pesticides ...... 33 4.3.2 Repellent ...... 34 4.3.3 Fibre Cloth protection ...... 34 4.3.4 Trap crops ...... 34 4.3.5 Semiochemicals ...... 35 4.4 Results ...... 36 4.4.1 EAG ...... 37 4.5 Conclusion and future work ...... 38 Acknowledgement ...... 39 References ...... 41

viii LIST OF PAPERS

This thesis is mainly based on the following papers, herein referred to by their Roman numerals:

Paper I Identification, synthesis and activity of sex pheromone gland components of the autumn gum moth (Lepidoptera: Geometriadae), a defoliator of Eucalyptus Steinbauer, M. J., Östrand, F., Bellas, T. E., Nilsson, A., Andersson, F., Hedenström, E., Lacey, M., J., Schiestl, F., P. Chemoecology 14:217-223 2004.

Paper II Identification, synthesis and field testing of (3Z,6Z,9Z)-3,6,9- heneicosatriene as a second bioactive component of the sex pheromone of the autumn gum moth, Mnesampela privata (Lepidoptera: Geometridae) Walker, P. W., Allen, G. R., Davies, N., W., Smith, J., Molesworth, P., Nilsson, A., Andersson, F., Hedenström, E. Accepted for publication in J. Chem. Ecol.

Appendix to part A: Synthesis of potential pheromone components of the Autumn Gum moth Mnesampela privata. Nilsson A., Andersson F.

Appendix to part B: Trioza apicalis the synthesis of β-sesquiphellandrene and α-bergamoten. Nilsson A., Andersson F.

Paper I was reprinted with kind permission from Springer-verlag Heidelberg.

ix

1. INTRODUCTION

Many and plants use chemical signals to obtain information regarding the environment and to communicate with each other. Chemical ecology is the study of the chemicals involved in the interactions of living organisms. It focuses on the production of and response to signalling molecules (i.e. semiochemicals) and toxins. Chemical communication is of particular importance among social insects - including bees, wasps, and termites - as a mean of communication essential to social organization. In addition, this area of ecology deals with studies involving defensive chemicals, which are utilized to deter potential predators, which may attack a wide variety of species. Organic chemistry is the science dealing with the compounds containing carbon, which play a vital role in all living organisms. The carbon atoms have the ability to bind four other atoms, and the number of possible combinations grows like an avalanche for each extra carbon atom a molecule has. The majority of chemical substances are organic, so whether we are interested in process chemistry in the chemical industry or medicinal chemistry the scientific basis for controlling chemical processes are, in most cases, based on the theory of organic chemistry. Understanding how organic chemistry works on the molecular level is therefore fundamental to most of those, who are trying to develop products and processes based on scientific principles, because it gives the opportunity to manage, control and change the properties. The importance of organic chemistry to modern society is enormous, whether we like it or not we live in a world that uses chemicals in all possible contexts, and our society would not work without them. To obtain a more environmentally friendly pest management it is important that biologists and chemists cooperate closely in the development of biological methods including the use of synthetically prepared naturally occurring compounds.

1.1 Semiochemicals Chemicals in the environment of an insect mediate much of its behavior. By turning these chemicals to our own advantage, it is often possible to attract pest insects to traps or baits, or to repel them from our homes, our crops, or our domestic animals. Behavioral messages are delivered by a wide array of chemical compounds. As a group, these compounds are known as semiochemicals (semeon means a signal in Greek) and it is a generic term used for a chemical substances or mixtures that carry a message. Semiochemicals can be divided into two main groups, pheromone and allelochemicals (Nordlund et al. 1981).

1 1.1.2 Pheromones The term "pheromone" was introduced by Peter Karlson and Martin Lüscher in 1959, based on the Greek word pherein (to transport) and hormone (to stimulate). These chemical messengers are transported outside of the body and elicit a reaction in another organism of the same species. Soon after Butenandt characterized the first such chemical, Bombykol (a chemically well-characterized pheromone released by the female silkworm to attract males), Karlson and Lüscher proposed the term, pheromone, to describe chemical signals from conspecifics which elicit innate behaviors. Nowadays pheromones are divided into subgroups (Figure 1) such as alarm pheromones, repellent pheromones and sex pheromone (Nordlund et al. 1981).

Pheromones

Repellent Alarm Aggregation Trail Sex

Figure 1. Pheromone are divided into subgroups.

One example from the repellent group is the male of desert locust, Schistocerca gregaria, which in the gregarious phase releases phenylacetonitrile (Figure 2) as a courtship inhibition pheromone (repellent) to ensure his reproductive success by preventing the female from mating with competing males (Seidelmann et al. 2002).

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Figure 2. Phenylacetonitrile, courtship inhibition pheromone of male of the desert locust.

When attacked by a predator, some species release a volatile substance that can trigger flight or aggression in members of the same species. In response to attack by natural enemies, most aphid species release an alarm pheromone that causes nearby conspecifics to cease feeding and disperse. The primary component of the alarm pheromone of aphid species studied is (E)-β-farnesene (Figure 3) (Verheggen et al. 2008).

2

Figure 3. (E)-β-farnesene, a primary component of the alarm pheromone of aphid species.

Male-produced sex attractant have been called aggregation pheromones. They usually result in the arrival of both sexes at a calling site, increase in density of conspecifics surrounding of the pheromone source. The banded alder borer, Rosalia funebris, is a long horned beetle and the (Z)-3-decenyl(E)-2-hexenoate (Figure 4) has been identified as a male produced aggregation pheromone (Ray et al. 2009).

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Figure 4. (Z)-3-decenyl-(E)-2-hexenoate a male produced aggregation pheromone.

Trail pheromones are common in social insects. For example, ants mark their paths with this type of pheromone (Figure 5). Certain ants lay down an initial trail of pheromones as they return to the nest with food. This trail attracts other ants and serves as a guide. As long as the food source remains, the pheromone trail will be continually renewed because it evaporates quickly (Suckling et al. 2008).

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Figure 5. The Argentine ant uses (Z)-9-hexadecenal as its trail pheromone.

In animals, sex pheromones indicate the availability of the female for breeding. Male animals may also emit pheromones that convey information about their species and genotype. Many insect species release sex pheromones to attract a mate, and many lepidopterans (moths and butterflies) can detect a potential mate from as far away as ten kilometres. Moths and elephants are unlikely mates, so scientists and the general publics were surprised when it was discovered that the Asian elephant, Elephas maximus, shares its female sex pheromone, (Z)-7-dodecen-1-ylacetate (Figure 6), with some 140 species of moth (Rasmussen et al. 1997).

3 O

O

Figure 6. The Asian elephant, Elephas maximus, female sex pheromone (Z)-7-dodecen-1-ylacetate.

1.1.3 Allelochemicals Allelochemicals act as chemical signals between members of different species (Nordlund et al. 1981) and can be divided into several categories (Figure 7).

Allelochemicals

Allomones Synomones Kairomones Apneumones

Figure 7. The subgroups of allelochemiclas.

The flowers of certain orchids emit allomones that mimic the sex pheromones of their bee or wasp pollinator. Males of the respective insect species attempt to copulate with the orchid flower, and pollinate it in the process, thus benefiting the orchid, while the cost to the deceived male insect is minimal. For example, in the extracts of Andrena morio females and the flower extract of the orchid Ophrys iricolor the same patterns of alkanes, alkenes and aldehydes (Figure 8) were found to release responses in the antenna of male A. morio bees (Stoekl et al. 2007).

Figure 8. Tricosane a component that were found in extract from the female A. morio and from the orchid O. iricolor.

Synomones are beneficial to both producer and recipient. Nezara viridula’s (L.) (Heteroptera: Pentatomidae) feeding and oviposition induce leguminous plants (Vicia faba L. and Phaseolus vulgaris L.) to produce blends of volatiles that are characterized by increased amounts of (E)-β–caryophyllene (Figure 9). These blends attract female Trissolcus basalis (Wollaston) (Hymenoptera: Scelionidae), an egg parasitoid (Colazza et al. 2004).

4

Figure 9. (E)-β-caryophyllene produced by Leguminous plan.

Kairomones benefit the receiving organism but cause disadvantage to the producer. An example is Anopheles gambiae sensu stricto, a malaria mosquito that uses human volatiles such as lactic acid (Figure 10), ammonia and several carboxylic acids for host orientation (Smallegange et al. 2005).

O OH

OH

Figure 10. Lactic acid is one of the components of the complex odors that the human body produces and that the malaria mosquito use for host orientation.

Apneumones are chemicals derived from a non-living source that benefit the receiver. Examples are scarce, but Thorpe and Jones (1937) reported that the ichneumonid parasitoid Venturia canescens is attracted to the odor of oatmeal, its host’s food.

1.2 Insect damages and pest management Some insects can cause considerable damage to forests, plantations, crops etc., which leads to reduced revenues. Today it is common to combat these pests with chemical pesticides, which also harm other species. An attempt to reduce the use of chemical pesticides is to just focus on the insect that causes injury, by identifying the chemical signals that occur between the insects and try to use these to prevent harmful attacks. Integrated Pest Management (IPM) is an effective and environmentally sensitive approach to pest management that relies on a combination of common-sense practices. IPM programs use current, comprehensive information on the life cycles of pests and their interaction with the environment. This information, in

5 combination with available pest control methods, is used to manage pest damage by the most economical means, and with the least possible hazard to people, property, and the environment (Radcliffe et al. 2008). The principl use of insect semiochemicals as for example with sex pheromones is to attract insects to traps for detection and determination of temporal distribution. In most instances, it is the male, who respond to female-produced sex pheromones. Trap baits, therefore, are designed to closely reproduce the ratio of chemical components and emission rate of calling females. Ideally, trap bait should uniformly dissipate its pheromone content over time and not permanently retain or degrade the pheromone in the process. Trap baits of many designs have been tested over the years, but the hollow polyvinyl plastic fibre (emit from open ends), closed hollow fibre and bag (emit through walls) and laminated plastic flake (emit through walls and exposed edges) are commonly used today. Trap design is also critical to effective use of traps for monitoring insect populations. Traps vary in design and size dependent on the behaviour of the target insects (Flint et al. 1996).

6

2. IDENTIFICATION OF SEMIOCHEMICALS The chemical methods used to identify pheromones depend on the properties of the compounds thought to be involved. Methods to investigate volatile hydrocarbon semiochemicals from insects involve several steps as collection, purification and identification (Wyatt 2003).

2.1 Collection of biological active compounds When observing insects behavior that indicates chemical communication between individuals of the same species or between species, the first step is to investigate what compounds that are responsible for the observed behavior and if these can be collected, purified and identified. There are some various techniques for collection of such compounds, for instance SPME and headspace analysis.

SPME One method to collect semiochemicals is the solid phase micro extraction technique (SPME), which allows solvent-free sampling and sample preparation. In SPME, an inert fibre coated with a thin layer of polymer (similar to the polymers used inside GC columns) is exposed to the sample. Compounds migrate into the polymer until equilibrium is reached, which occurs in minutes or less, as the polymer layer is very thin. The fibre, approximately just a millimeter in diameter, is mounted inside a syringe. When removed out of the syringe, the fibre retains the molecules until introduced into the GC injector port where the compounds are desorbed by heat and carried by the gas into the column (Wyatt 2003).

Headspace Analysis The simplest sampling method consists of direct sampling of headspace volatiles of an equilibrated sample, sometimes heated, in a closed container. This method is practical only for compounds, whose concentration and vapor pressure is sufficient to provide at least nanogram quantities in the sample aliquot. Volumes of 100 -1000 µl or in certain cases even larger is removed with a gas tight syringe and slowly injected directly into the GC, using splitless or on-column injection so that most of the volatiles are transferred on to the column (Millar et al. 1998).

Extraction technique Another way to isolate the compounds of interest is to make an extract from the insect, female or male. To prepare the extract you can use the whole body or just a gland and put it in a tube with organic solvent. The extract is now ready for

7 analysis directly or for further purification sometimes including fractionation or other techniques. One of the disadvantages of making crude extracts of pheromone glands is that many non-pheromone compounds are present in the blend, giving quite a “dirty” and complex mixture. Another approach is to separate the components by exploiting their chemical or physical characteristics, such as solubility in different solvents. The dual aim of this process is to both purify and concentrate the pheromone. Then the sample is shaken with water (polar) and organic solvent (non-polar) mixture (Wyatt 2003). Sometimes you want to make fractionation of the extract and for this liquid chromatography (LC) is often used.

LC Liquid chromatography (LC) is an analytical or preparative chromatographic technique that is useful for separating ions or molecules that are dissolved in a solvent. If the sample solution is in contact with a second solid or liquid phase, the solutes will interact with the other phase to different degrees due to variations in adsorption, ion-exchange, partitioning, or size. These differences allow components of the mixture to be separated from each other by exploiting their varying affinities for the solid phase resulting in different transit times of the solutes, when passed through a column (Scott 2009).

GC For identification of the components in the extract often gas chromatography (GC) is applied. To isolate a specific compound from the extract, this technique may also be used for preparative purpose. A mixture of several compounds is injected into a heated chamber (the injector) where the resulting sample vapor is mixed with a current of inert gas and is swept into the entrance of the column. As compounds continue to move through the column their separation from one another increases. At the end of the column, each isolated compound elutes sequentially into a detector. With the same column and GC operating conditions, a particular compound will always elute with the same retention time. Hence retention time is a diagnostic character that can be used to identify individual compounds in mixtures of unknowns (Sullivan).

After purification the extract or a pure compound from the extract can be used for test of biologic activity.

2.2 Identification and synthesis An electroantennographic detector (EAD, electrophysiological recordings of an insect antenna) and/or GC-EAD are often used to show that there are antennal responses of one or more substances. Absence of an EAG or EAD response can be

8 due to a very low level of compound reaching the antennal preparation. Single-Cell Recording (SCR) can also be used and this technique allows recording from just one sensillium.

EAG Compounds can be exposed to the antenna after being purified or synthesized in a separate process; this procedure for manually exposing an antenna to compounds one at a time is known as an electroantennogram (EAG), and has received wide use in entomological studies since the late 1960s (Sullivan). The EAG detects the stimulation of the insect antenna by chemical compounds, e.g. a semiochemical. Electrodes are placed at each end of the antenna and connected to a DC amplifier. The rapid depolarization when the sensory cells of the antenna are stimulated by pheromone is dose dependent and varies between chemicals depending on the number of sensory cells on the antenna, which are sensitive to them. An EAG will not detect pheromone components for which there are few receptors (Wyatt 2003).

EAD An important technique in elucidating semiochemicals of insects is the GC coupled with an EAD. The EAD consist of an antenna placed between two electrodes from which an amplified signal is recorded by computer software. After the injection of a sample into the GC, any flame ionization detector (FID) signal would indicate the elution of all chemical compounds. In contrast, an EAD response is only recorded from active compounds such as a semiochemicals would only give a response from the antennae. Then a synthetic compound can be compared by retention time and EAD signal (Byers 2004).

SCR Single-Cell Recording from olfactory receptors is a technique used in research to observe changes in voltage or current in a neuron. In this technique an anesthetized has one microelectrode inserted into its skull and one into a neuron in the antenna. The electrode measures the changes in charge as the neuron reaches its action potential, which is a self-regenerating wave of electrochemical activity that allows excitable cells (such as muscle and nerve cells) to carry a signal over distance. It is the primary electrical signal generated by nerve cells and arises from changes in the permeability of the nerve cell’s axonal membranes to specific ions (Millar et al. 1998).

When biological activity is found, the next step is to identify the structure of the active compound. This can be done with several techniques including synthesis.

There are many different ways to deal with the identification of semiochemicals.

9 But there are four main techniques and they are arranged in the order in which they are most commonly applied (Millar et al. 1998).

Mass spectrometry is usually used for two main purposes in chemical ecology. First, this is an analytical tool, whose sensitivity and specificity are unsurpassed, when the compounds under study are characterized and their mass spectra are known. The other use for MS in chemical ecology studies is in structure elucidation of either completely unknown compounds or compounds whose structures are unknown but that are related to known compounds. In either case, mass spectrometry can provide crucial structural information. In addition the semiochemical can be matched with natural product spectra contained in some MS-spectra libraries such as the NIST Library and the Wiley Registry of Mass Spectral Data. While searching in an MS-spectra library you must consider that different types of mass analyzers do produce slightly to considerably different spectra. Nuclear Magnetic Resonance spectrometry enables the determination of the structures of completely new compounds and the amount needed to carry out these NMR analyses is now approaching the nanogram level. The purity of the sample and lack of proton containing solvents and water is necessary to get successful NMR-spectra. Coupled Gas Chromatography – Infrared Spectroscopy provides a spectrum where it is possible to determine the presence of or absence of specific functional groups. Microchemical methods describe a wide variety of reactions that can be used to provide critical information on the presence or absence of specific functional groups, often with nanogram to subnanogram quantities (Millar et al. 1998). Usually reactions are epoxidation of double bonds and derivatization of acids and alcohols.

GC-MS The gas mixture travels through a GC column, where the compounds become separated as they interact with the column. The separated compounds then immediately enters the mass spectrometer which is based on a simple idea: a compound or mixture of compounds is ionized and broken into fragments, the ions are separated (or analyzed) on the basis of mass/charge ratio, and the relative abundance of each ion is recorded as a spectrum (Millar et al. 1998).

NMR Nuclear magnetic resonance is a property that magnetic nuclei have in a magnetic field and applied electromagnetic (EM) pulse, which cause the nuclei to absorb energy from the EM pulse and radiate this energy back out. The energy radiated back out is at a specific resonance frequency which depends on the strength of the

10 magnetic field and other factors. This allows the observation of specific quantum mechanical magnetic properties of an atomic nucleus (Friebolin 1998).

GC-IR The GC-IR interface blends the separation capability of gas chromatography with the identification power of FT-IR to provide an efficient tool for analyzing complex mixtures. Infrared spectroscopy is the subset of spectroscopy that deals with the infrared region of the electromagnetic spectrum. It covers a range of techniques, the most common being a form of absorption spectroscopy (Williams et al. 1995).

After identification and synthesis of biological active compounds the synthetic products are sent to the entomologist/biologist for test of their biological activity. More EAG, GC-EAD or SCR studies are made and if the activity of the synthetic compound is confirmed its time to test it in the field.

Field test Semiochemical-baited traps are placed in the area to verify that it is the right compound, stereoisomer or compound mixtures that have been produced. If insect catch is small, the stereoisomeric purity might be too low, some compound might be missing or the ratio between the compounds can be wrong.

The challenges presented by the identification of semiochemicals have forced research teams to combine the expertise of both chemists and biologists but this is not always easy. As Albone said in 1984: “Biologists assume chemists will always find the answers, however small or contaminated the sample are; chemists sometimes do not appreciate how much the behavior of animals, even laboratory cultured insects, can vary unpredictably from day to day”.

11

3. PART – A: MNESAMPELA PRIVATA (LEPIDOPTERA: GEOMETRIDAE)

3.1 Lepidoptera - Moth Lepidoptera is the second largest insect group and includes nearly 150 000 described species (Ando et al. 2004). The name Lepidoptera is derived from the Greek, meaning “scaly winged,” and refers to the characteristic covering of microscopic dustlike scales on the wings and constituting of butterflies, moths and skippers. Because of their day-flying habits and bright colours, the butterflies are more familiar than the chiefly night-flying and dull-coloured moths, but the latter are far more varied and abundant. The skippers are a worldwide group intermediate between butterflies and moths (DeLong et al. 2009). Moths, butterflies, and skippers show great diversity in size and development rates. Some moths have wingspans as small as 4 mm, whereas the largest moths and butterflies measure nearly 30 cm. All lepidopterans undergo complete metamorphosis and the life cycle of lepidopterans consists of four stages (DeLong et al 2009): egg, larva (caterpillar), pupa (chrysalis), and adult (imago). Fast-developing species may complete their development in as little as three weeks, while slower ones may require as long as two or even three years. The larvae do most of the eating, with the majority feeding on foliage, although many species eat stems, roots, fruits, or flowers. A number of moths and a few butterfly larvae are serious pests in agriculture and forestry.

3.1.1 Mnesampela privata The autumn gum moth, Mnesampela privata (Guenée) (Lepidoptera: Geometridae), is a native eucalypt defoliator with a geographic range through southern and south-eastern Australia (Rapley et al. 2004). Among the most preferred eucalypt hosts of M. privata are the species Eucalyptus Globulus and E. Nitens (both are commonly referred to as blue gums). These species are among the most important species used in commercial plantations in Australia. There has been rapid, large scale and widespread planting of these two eucalypts in South-eastern and south- western Australia over the last decade. M. privata is only a pest problem in the first four years following plantation establishment, probably because larval performance is increased on juvenile compared to adult foliage. During development the larvae undertake two distinct feeding patterns; the young larvae skeletonise the leaf surface avoiding essential oil glands, while older larvae are able to consume foliage at a vastly increased tare by scalloping the leaf. The defoliation of young trees can result in poor tree form, reduced growth rates and in severe cases tree mortality. M. privata larvae are usually controlled using aerial

12 applications of alpha-cypermethrin (Figure 11), which is a highly active broad spectrum insecticide, effective by contact and ingestion against target pests. It is widely used in agricultural crops, forestry as well as in public and animal health (Neumann et al. 1997).

N O Cl O O Cl

Figure 11. Alpha cypermetrin a pyrethroid insecticide.

3.2 Lepidoptera pheromones Lepidopteran sex pheromones have been identified from more than 570 species (El-sayed 2008). The most predominant group of compounds found in these species is composed of unsaturated C10 – C18 alcohols and their derivatives (type I) (Ando et al. 2004). The first pheromone was identified by Butenandt in 1959 as (10E, 12Z)-Hexadeca-10,12-dien-1-ol (Bombykol) (Figure 12) in females of the silkworm moth (Bombyx mori).

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Figure 12. Bombykol was the first sex pheromone that was identified in females of silkworm moth.

In contrast, most females in the family Geometridae produce (3Z,6Z,9Z,)-3,6,9- trienes (Figure 13), (6Z,9Z)-6,9-dienes and their monoepoxides with a C17 – C23 straight chain (Type II). Some species in the families of , Arctiidae and Lymantriidae also utilize these compounds (Wei et al. 2003).

Figure 13. (3Z,6Z,9Z,)-3,6,9-nonadecatriene a common pheromone component in the family of Geometridae.

13

The Japanese giant looper Ascotis selenaria cretacea (Butler) is a serious defoliator of tea gardens in Japan and is closely related to M. privata (Witjaksono et al. 1999). The sex pheromone of A. s. cretacea is identified to (6Z,9Z)-6,9-cis-3,4- epoxynonadecadiene (Figure 14).

O

Figure 14. (6Z,9Z)-6,9-cis-3,4-epoxynonadecadiene sex pheromone of the Japanese Giant Looper, Ascotis selenaria cretacea.

Another closely related geometridae is the Giant Looper, Boarmia (Ascotis) selenaria (Schiffermüller). The giant looper is a serious pest of fruit and foliage in avocado orchards in various regions of Israel (Cossé et al. 1992). The sex pheromone for this looper is identified to be a mixture of (3Z,6Z,9Z)-3,6,9-nonadecatriene and (6Z,9Z)- 6,9-cis-(3S,4R)-epoxynonadecadiene (Figure 15).

O

Figure 15. (3Z,6Z,9Z)-3,6,9-nonadecatriene and (6Z,9Z)-6,9-cis-3S,R- epoxynonadecadiene identified as the sex pheromone of the Giant Looper, Boarmia (Ascotis) Selenaria.

3.2.1 Biosynthesis of Lepidopteran pheromones The variation of the lepidopteran pheromones results from differences in both their biosynthetic starting material and enzyme systems. The biosynthetic pathways of Type I pheromones (Figure 16) are well examined and they are biosynthesized from saturated fatty acids produced in de novo synthesis starting from acetyl-CoA (Ando et al. 2008). For example, (Z)-7-dodecenyl acetate is produced in the female pheromone glands of a Noctuidae species via the following successive reactions: desaturation of palmitic acid, chain shorting by β–oxidation, reduction of an acyl group and acetylation (Komoda et al. 2000).

14 O

CoA

Fatty acid synhtesis

O SCoA 16-acyl

Z11 desaturation O SCoA Z11-16:acyl -Oxidation

O SCoA Z9-14:acyl -Oxidation

O SCoA Z7-12:acyl

Reduction of the acyl group followed by acetylation

O O

Z7-12:OAc

Figure 16. Proposed biosynthetic pathway for the pheromone components of M.confusa.

Type II pheromones are expected to be produced from linoleic and linolenic acids [(9Z,12Z)-9,12-octadecadienoic and (9Z,12Z,15Z)-9,12,15-octadecatrienoic acids] (Figure 17) because these dietary acids are unsaturated at the same positions of the pheromones if the positions of the double bonds are counted from terminal methyl groups (Millar 2000). For examples, cis-3,4-epoxy-(6Z,9Z)-nonadecadiene a major pheromone component of A. s. cretacea might be produced by chain elongation of

15 linolenic acid to C20 trienoic acid, decarboxylation to C19 (3Z,6Z,9Z)-3,6,9-triene (a minor pheromone component) and epoxidation of the double bond at the 3- position (Ando et al. 2008).

O

OH

Reductive decarboxylation Reduction, epoxidation Epoxidation or reduction, oxidation

C17- hydrocarbones, epoxides C18- hydrocarbons, epoxides and aldehydes

Chain extension by 2-carbon units C20- C22- C24- COOH

Reductive decarboxylation Reductive Epoxidation Epoxidation

C19- C21- C23- hydrocarbons C20 - C22- hydrocarbons and epoxides and epoxides

Figure 17. Biosynthetic pathways to produce polyene hydrocarbons and epoxide pheromones from linoleic and linolenic acid precursors. Picture from Millar 2000.

16 3.3 Results This section is divided into three parts depending on the preparation of the extract and the age of the females. The first section (3.3.1) deals with an extract A, which was purified by distillation and prepared from the ovipositor of female that was 1- 2 days old. The second part (3.3.2) deals with an extract B, which was not purified at all and was prepared from the ovipositor of female that was 1-2 days old. In the last section (3.3.3) an extract C also this time without the purification step, was prepared from the abdominal tip of females of different ages.

3.3.1 Identification of the extract A, 1-2 day old virgin females Collection and extract preparation Female pupae were collected from plantation of E.grandis near Mildura, Victoria in 1999 and from plantation of E. nitens and E. globulus at Australian Capital Territory, Canberra in 1999-2003. Pupae were also reared from eggs collected at Cornelian Bay, Tasmania and Ginninderra Experiment Station, Australian Capital Territory in 1999-2003. Larvae were reared according to protocols in Steinbauer and Matsuki 2004. Sexed pupae were kept at 18 °C, 24 h scotophase and ambient humidity until ommatidia and wing pads were placed in individual containers in a controlled temperature cabinet set to a 12 h:12 h light cycle. The temperature in this cabinet stayed within 16-19 °C. One- to two-day old virgin female moths were placed in a freezer for 4 to 5 min. The ovipositor, with as little other tissue as possible, was removed and placed in a small tube with 20-30 L of HPLC-grade hexane. The tube was capped with aluminium foil and stored in a freezer. The extracts were refined by a mild thermal desorption technique (Whittle et al. 1991). The hexane extract was injected onto glass beads tightly packed in a tube and the hexane was evaporated in a stream of nitrogen, 20 mL/min, at room temperature. The tube was placed in a small oven arranged so that a stream of helium passed through the tube at 12mL/min. The oven was maintained at around 135 °C. The effluent from the tube was trapped in a capillary tube (150 mm x 1.6 mm) cooled with solid CO2. Desorption was conducted for 8 to 15 min and then capped with aluminium foil and stored in a freezer.

GC and GC-MS Extract A gave us about 7 interesting peaks (Figure 20) to identify and three of the peaks were found to arise from (3Z,6Z,9Z)-3,6,9-nonadecatriene, 1-hexadecanol and 1-octadecanol (paper I). The identification were established as the peaks coincided precisely with the synthetic references both in their EI mass spectra (Figure 18) and their GC retention times on columns of low and high polarities.

17 The other components were identified as a homologues series of saturated hydrocarbons.

Figure 18. 70 eV EI mass spectrum of (3Z,6Z,9Z)3,6,9-nonadecatriene from female extract.

EAG and GC-EAD

The identified alcohols and C19 triene were examined for activity in male antennae during EAG (Figure 19). The C19 triene elicited a response, whereas neither of the alcohols elicited an antennal response different from that of blank (Paper I).

Figure 19. Results of EAG recordings of male M. privata antennae.

18 Using GC-EAD on extract A, we observed a strong response of male AGM antennae to the peak eluting from the GC-column at 19.40 min and was identified as C19 triene (Figure 20). The other six compounds (1-hexadecanol, 1-octadecanol and saturated straight hydrocarbons, C21- C 27) in the extract did not elicit response on the male antennae (Paper I).

Figure 20. Gas chromatographic analyses (FID) of a female M.privata pheromone gland extract with electroantennographic detection (EAD) using male antennae.

Field test Field tests (Paper I) were performed in both low and high population density areas. In the low population density area (i.e. Lyneham Ridgeand Ginninderra,

ACT) the traps with synthetic C19 triene caught more AGM males than unbaited traps (up to a total of 4 males compared with 0 in the blank traps). The majority of virgin females (7 of 11) caught none or only one male during their time in the trap with the exception of one female, which attracted 48 males over three consecutive nights. The catches in the low density areas were too small to allow for any statistical comparisons.

In the high population density area (Mildura) the synthetic C19 triene proved attractive to AGM males. The traps baited with the C19 triene caught significantly more moths than unbaited traps. We only tested virgin females in the high-density area during one night and the only female that emerged caught one male during the first night. The next morning she was dead.

19

There was no indication in these field trials that either of the alcohols worked synergistically with the C19 triene, because the traps with the C19 triene and either of the alcohols did not catch more males than the C19 triene did by itself.

However, field trials did not give the catch that we expected. At this time we started to speculate that the AGM species probably uses more than (3Z,6Z,9Z)- 3,6,9-nonadecatriene as its sex pheromone. The extract that we used was refined by a mild thermal desorption technique and the additional component may be too fragile to survive the refinement process or the GC process or it may be formed enzymatically from the triene (or other components) on the surface of the gland before release as effluvium and therefore would be bare represented in solvent extracts. So the next step was to start investigate some new untreated extracts.

3.3.2 Identification of extract B, 1-2 day old virgin females Collection and extract preparation Female pupae were collected from plantation of E. grandis near Mildura, Victoria in 1999 and from plantation of E. nitens and E. globulus at Australian Capital Territory, Canberra in 1999-2003. Pupae were also reared from eggs collected at Cornelian Bay, Tasmania and Ginninderra Experiment Station, Australian Capital Territory in 1999-2003. Larvae were reared according to protocols in Steinbauer and Matsuki 2004. Sexed pupae were kept at 18 °C, 24 h scotophase and ambient humidity until ommatidia and wing pads were placed in individual containers in a controlled temperature cabinet set to a 12 h:12 h light cycle. The temperature in this cabinet stayed within 16-19 °C. One- to two- day old virgin female moths from female pupae collected from plantation of E. nitens and E. globulus were placed in a freezer for 4 to 5 min. The ovipositor, with as little other tissue as possible, was removed and placed in a small tube with HPLC-grade hexane. The tube was capped with aluminium foil and stored in a freezer until the analysis was done.

GC-MS This new extract gave between 20 (E. nitens) – 22 (E. globulus) GC peaks instead of the seven peaks (Figure 20) obtained from extract A (Figure 21). In the region ~38 and ~48 min was at least three peaks that showed MS spectra that reminded of a polyunsaturated compound (ie ions at m/z 67, 79 and 108).

20

Figure 21. GC-spectrum from extract of female feeding of E.globulus.

Most females in the family of Geometridae produce (3Z,6Z,9Z,)-3,6,9-trienes,

(6Z,9Z)-6,9-dienes and their monoepoxides with a C17 – C23 straight chain (Wei et al. 2003). There for we started to synthesize a mixture of monoepoxides from C18 -

C21-trienes and diens. The starting material used also gave us the monoepoxides from (9Z)-9- C18 - C21 (section 3.4). Two of the monoepoxides with the length of C21 overlapped with different peaks in the GC-spectrum from female extract (Figure 22).

21 A

B O

O

Figure 22. A. GC-spectrum: overlay female extract and synthetic C21-monoepoxide B. Structure of 9,10-epoxy-heneicosane and 3Z,9Z-cis-6,7-epoxy-heneicosadiene.

The first interesting compound from the extract that eluted at ~44.6 min is the epoxide from (9Z)-9-heneicosene, and is until now an unknown pheromone component according to pherobase (El-sayed 2008). The second overlay of peaks is at ~47.1 min and this is the epoxide from (3Z,6Z,9Z)-3,6,9-heneicosatriene. Interpretation of the MS spectrum from both the synthetic epoxide and from the natural extract (peak eluating at the time of ~47.1) suggests that it is the (3Z,9Z)-cis- 6,7-epoxy- heneicosadiene (Millar 2000). This compound is present in many of the exracts. In some GC-MS runs it is observed and in some others this epoxide is not present.

Neither of the other monoepoxides from C18 - C21 triene and diene did elute from the GC-column at a retention time that coincided with GC retention characteristics from the female moth extract.

22 To try to identify more of the peaks from the extract, we also wanted to compare different kinds of ketones and diepoxides. We already prepared the diepoxides as they were produced along with the the monoepoxides (Section 3.4). The ketones were easily synthesised from the monoepoxide (Section 3.4). The ketones are also known pheromone components (Tóth et al. 1987; Buser et al. 1985). The diepoxides are not yet reported as pheromone components (El-sayed 2008). Although we got

GC retention time overlay from the synthetic diepoxide from C18 triene, the mass spectra were not the same.

EAG Using EAG we wanted to find out if the male antenna gave any response to some of our synthesised reference substances (Figure 23). The tests showed strong response from the (3Z,6Z,9Z)-3,6,9-nonadecatriene but not much of response to other compounds tested. Although the results suggest that monoepoxide from C18 triene and also monoketone from C18 triene might have some biological activity. However, none of these two compounds eluted from the GC-column at retention times that coincided with GC peaks from the female moth extract. Although we think that this needs further testing (Walker 2006). The (3Z,9Z)-cis-6,7-epoxy- heneicosadiene was at this time not available for testing.

Male AGM response (N = 5 moths)

600

500

400

300

200

100 Mean +Mean SE (% response of control)

0 1 2 3 4 5 6 7 Sample number

Figure 23. Compound 3: (3Z,6Z,9Z)-3,6,9-nonadecatriene; 4: monoepoxide from C18 triene; 6: monoketone from C18 triene.

23

Field test In 2004 in Australian Capital Territory, Canberra eight lures with 0.1 or 1 mg of either mono- or diepoxide from C19 triene was tried in field. None of the lures did catch AGM males or other moths (Steinbauer 2004).

3.3.3 Identification of extract C, females of different ages Collection and preparation of extract Eggs and early instar larvae infesting E. globulus trees was collected near Cornelian Bay, Hobart, Tasmania during April and May 2005. Different this time was the preparation of the extract. Tips from females of different ages (one – nine days old) were placed into individual borosilicate vials with 150 μl inserts (Waters Corporation, Australia), soaked in 15 μl of dichloromethane containing 100 ng methyl stearate as an internal standard. After one hour at room temperature the tips was removed before storing the extract at -20 °C. The solutions were allowed to reach room temperature before injecting 1 μl into a GC-MS. The analyses were made by our collaborators in Australia.

GC-MS Interesting with the newly prepared extract (Paper II) was that in the GC region (Figure 24) that we focused on before (region between ~38 and ~48 min) there was not any peaks that had EI mass spectra with similar MS characteristics of a polyunsaturated compound (ie ions at m/z 67, 79 and 108). In this region the peaks were identified as arising from alkanes of different carbon chain length, C15-C18

(Table 1).

24

Figure 24. Representative GC-MS chromatogram of extract from a female M. privata abdominal tip. Number designations for peaks are as listed in Table 1. Insert shows magnified portion of chromatogram to indicate where compounds 7 (n-octadecanal) and 8 ((3Z,6Z,9Z)-3,6,9-heneicosatriene) eluted.

Table 1. Main compounds identified by GC-MS in abdominal tip extracts from female M. privata. Peak Compound Peak Compound numbera numbera 1 n-Hexadecanal 12 n-Eicosanal 2 (3Z,6Z,9Z)-3,6,9-nonadecatriene 13 n-Eicosanol 3 n-Hexadecane 14 n-Tricosane 4 n-Nonadecane 15 n-Tetracosane 5 Palmitic acid 16 n-Pentacosane 6 n-Hexadecyl acetate 17 3-Methylpentacosane 7 n-Octadecanal 18 n-Hexacosane 8 (3Z,6Z,9Z)-3,6,9-heneicosanetriene 19 2-Methylhexacosane 9 n-Heneicosane 20 n-Heptacosane 10 Methyl stearate (internal standard) 21 n-Octacosane 11 n-Docosane a Numbers correspond to the peaks in chromatogram in figure 24.

25

Most females in the family of Geometridae produce (3Z,6Z,9Z,)-3,6,9-trienes,

(6Z,9Z)-6,9-dienes and their monoepoxides with a C17 – C23 chain (Millar 2000).

The C21 (Z,Z,Z,)-3,6,9-trienes eluate from the GC-column (Paper II) at a retention time that coincided with GC retention characteristics from the female moth extract. Comparing the mass spectrum showed that they was indistinguishable. An co- injection of the synthetic C21 triene with a female moth extract also established the identity of the natural compound as (3Z,6Z,9Z)-3,6,9-heneicosatriene.

Levels of trienes were extremely low in some moths and it was not possible (even by SIM-MS) to determine the C19/C21 triene ratio in all individuals. The C21 triene was below the detection limit in 23 out of 41 extracts while the C19 triene was below detection limit in one female extract. Consequently the calculated C19/C21 ratio is based on 15 individual females of known age and 3 of unknown age in which both compounds could be measured. Mean estimated titres of trienes per female (calculated as internal standard equivalents) were 30.74 ng (+ 6.1 SE) (range = 604 pg to 143 ng) for C19 triene and 1.44 ng (+ 0.3 SE) (range = 368 pg to 3.8 ng) for C21 triene (Figure 25).

Figure 25. Mean (+ SE) estimated titres of C19 and C21 trienes (nanograms) in abdominal tip extracts of unmated female M. privata.

SIM-MS analysis of solvent extracts from the abdominal tips of nine male M. privata from Tasmania, detected C21 alkatriene in six individuals and putatively identified (3Z,6Z,9Z)-3,6,9-tricosatriene (C23 alkatriene) in all individuals. No C19

26 alkatriene was found in any of the male extracts. This interesting founding will be investigated further in the near future.

EAG and GC-EAD

Using EAG (Paper II) we showed that the antennal responses to 5 μg of C21 (3Z,6Z,9Z)-3,6,9-trienes were consistently higher than the blank stimulus (Figure

26) but significantly lower responses to 5 μg C19 (3Z,6Z,9Z,)-3,6,9-trienes (P < 0.05,

LSD). EAG response to C21 triene was significantly greater than that of 1- octadecanol and hexadecyl acetate (P < 0.05, LSD) which elicited an antennal response similar to the blank stimulus. When C21 triene was added to 5 μg C19 triene at a range of ratios, including similar to that found in female extracts, there was a slightly but not significantly enhanced antennal response (P > 0.05, LSD).

Figure 26. Mean (+ SE) EAG response of male M. privata antennae to 5 μg of C19 triene, C21 triene, 1-octadecanol (C18 alcohol), hexadecyl acetate (C16 acetate) and various blends of C19:C21 trienes dissolved in hexane, expressed as a percentage of the control stimulus (dashed line).

Using a GC-EAD we would confirm the result, but no antennal response or FID peak was detected in the region where the C21 triene was expected to have eluted. The absence of a FID peak was probably due the lack of sensitivity of the GC-FID used to detect the C21 triene at such low levels. Similarly, the absence of an EAD

27 depolarization in the region where the C21 triene was expected to elute was also probably due to the very low levels of C21 triene reaching the antennal preparation.

Field test Field trials in Tasmania (Paper II) demonstrated a strong synergistic effect of C21 triene, which when added in small amounts (1-6 %) to 1 mg C19 triene, gave up to 10 fold increase in the number of male M. privata caught in pheromone traps.

Addition of C21 triene to C19 triene also more than tripled the sensitivity of pheromone traps to detect low populations of M. privata in Eucalyptus plantations and significantly reduced by-catches of other geometrid species.

3.4 Syntheses of reference substances The pathway of synthesis (Figure 27) may be divided into three main stages (1) reduction of acid or ester by LiAlH4 to produced alcohol (Wang et al. 2007); (2) conversion of alcohol into tosylates (Kabalka et al. 1986,); (3) nucleophilic substitution reaction, addition of tosylate to a dimethylcuprate, (or diethylcuprate/ dipropylcuprate) generated from the appropriate alkylmagnesium bromide and CuI, a modified procedure to the one reported by Johnson and Dutra in 1973 leading to the target compound C 19 – C21-triene (Wang et al. 2007; Kabalka et al.

1986), for the target compound C18 –triene the third step is a reduction of the tosylate by LiAlH4 (Kabalka et al. 1986). Epoxidation of trienes with mCPBA gave epoxides and lithium aluminium hydride reduction of the epoxides followed by oxidation with pyridinium dichromate furnished ketones (Tóth et al. 1987; Buser et al. 1985).

28

a OMe O ° 1) LiAlH4 , Et2O, 0 C

2) p-Toluene sulfonyl chloride, Pyridine, CH2Cl2, rt.; 1,4-butanediol, Pyridine 5 °C ° 3) (C19-C21)-RMgBr, CuI, THF, -30 C to rt

(C18) LiAlH4, THF, rt

C - C 18 21

mCPBA, CH Cl , 0 °C 2 2

Mixture of mono, di and triepoxides Easily separated by flash chromatography

C18 - C21 C18 - C21 O O O

C18 - C21 C18 - C21 O O O

C18 - C21 C18 - C21 O O O

° 1) LiAlH4, Et2O, 0 C

2) HCl

3) PDC, CH Cl , rt 2 2 O

O O

O O

O

Figure 27. Syntheses of reference substance. aTo obtain even more compounds from the synthesis we sometimes used a starting material that was a mixture of (Linolenic acid ~70 %, Linoeic acid ~20 % and Oleic acid ~5 %).

29 3.5 Conclusions and future work

The level of the C21 triene (Paper II) was found to be too low to be determined in AGM females until they were at least three days old. Thus, it was not possible

(even by SIM-MS) to determine the C19/C21 triene ratio in extracts A (Section 3.3.1) and B (Section 3.3.2) as these extracts were prepared from females one to two days of age. Another big difference between the extracts was that these two extracts (A and B) were prepared from the whole ovipositors, whereas the extract C (Section 3.3.3) was prepared from females of different age and only from the abdominal gland tip. Surprisingly, the epoxide from C21 triene was not found in the abdominal tip extract even though the extract were prepared from females of different age.

The use of synthetic pheromone for a more sustainable method for the control of M. privata, by mating disruption or mass trapping methods, seems not economically feasible at present given the high costs associated with the used synthetic method for the C19 and C21 trienes. In the light of our new finding to include the C21 triene in the pheromone bait, the relationship between trap catch and subsequent M. privata egg and larval infestations needs to be re-examined as

Östrand et al. (2007) trapping studies were conducted with baits containing C19 triene alone.

We also recommend that traps used to monitor M. privata populations in Tasmania are baited with lures containing both C19 and C21 trienes, at a ratio of 16:1 (Paper II) to maximise trap captures. However, further research is needed to verify whether this is an optimal ratio of alkatrienes to use for monitoring M. privata in other geographical areas. Initial results from this study suggest that baits for monitoring

M. privata populations in Western Australia should contain a 4:1 ratio of C19 and

C21 trienes to closer approximate that found in female extracts from this area. We also recommend the Delta trap design for monitoring M. privata in preference to the Unitrap design due to lower costs and more efficient capture rates, although the sticky base needs to be replaced regularly to avoid underestimation of population size through trap saturation. In the future it could also be interesting to try the (3Z,9Z)-cis-6,7-epoxy-heneicosadiene together with the (3Z,6Z,9Z)-3,6,9- heneicosatriene and/or (3Z,6Z,9Z)-3,6,9-nonadecatriene in field tests to se if it has any effect on the catch of males AGM.

Whether the C19 triene also is a component of the sex pheromones of M. arida, M. heliochrysa D. amblopa, A. smithi and three moths of an unidentified species of geometridae which we caught in traps containing C19 triene, also remains to be determined (Paper one and two). A. smithi were also caught in traps baited with different ratio of C19:C21 triene.

30 The importance of C21 and C23 triene found in male M. privata abdominal tip remains to be investigated further. Heath et al. (1988) also found that abdominal tip extracts from males of the noctuid Anticarsa gemmatalis contained C21 alkatriene, as a major component of the female sex pheromone of this species. Heath et al. (1988) suggested that C21 alkatriene was released from extrudable hairpencils on the abdomen of male A. gemmatalis and used during courtship to evoke female acceptance.

31 4. PART - B: TRIOZA APICALIS (HOMOPTERA: PSYLLOIDEA)

4.1 Homoptera The members of this large group of sucking insects exhibit considerable diversity both in body size (1/2 mm - 20 cm) and number of species (32,000). All homopterans are plant feeders, with mouthparts adapted for sucking plant juices from a variety of plants. Many homopterans cause problems by destroying cultivated plants such as fruit trees and grain crops; other homopterans carry diseases. A few homopterans provide secretions or other products that are beneficial to humans. The most distinctive features of homopterans are the mouthparts and the wings. The rigid mouthparts consist of a two pairs of stylets: mandibles and the maxillae. The mandibles pierce the plant tissue while the maxillae form two conducting tubes, one for food and the other for saliva. The forewings are usually pigmented or transparent, and they can be slightly thickened and waxy in some species. The hindwings are always membranous. At rest, they are held over the body in a roof- like manner with the tips slightly overlapping. Also, homopterans tend to have extremely complex digestive tracts, which form filter chambers in most groups. Other features in homopterans are generally similar to those of other insects (DeLong et al. 2009). Most homopterans reproduce sexually and are egg-laying. Eggs are laid on or in the preferred food plant so that the hatching larvae have a readily available food source. Metamorphosis is typically gradual, with immature stages resembling the adults but lacking wings. Life cycles are usually short (Britannica, 1973).

4.1.1 Trioza apicalis The carrot psyllid (T. apicalis) is one of the major pests of carrot (Daucus carota) in northern Europe. Over-wintered adults cause damage to the crop by feeding on carrot seedling. The carrot psyllid is an economically important pest on carrots in northern and central Europe. It has one generation per year and overwinters in the adult stage on coniferous trees. As soon as carrot seedlings have sprouted in early summer, the psyllids invade the carrot fields where they feed and lay eggs during the summer months. The invading adult generation gradually dies off. The eggs hatch into nymphs in about 10 days and the nymphal period lasts for about six weeks and passes through five instars before they become adults. During this period, the nymps live rather sedentarily on the underside of the carrot leaves. The new generation emerges in late summer and early autumn, one to three days after the last moult they leave the carrots and start migrating to seek out winter habitats. The adults pass the winter in diapause on coniferous trees, mostly Norway spruce, Picea abies (L.) Karst. The damage to the carrots occurs mainly during the oviposition period. Both adults and nymphs cause damage to the carrot

32 (Kristoffersen 2006 and references therein; Nehlin et al. 1994 and references therein).

4.2 The damage of carrots Symptoms of the infested carrots include discoloration, dark green color, and curling of the leaves together with diminished and distorted root growth (Kristoffersen 2006). The carrots in turn are small and poorly developed (Forsberg et al. 1993) and in worst case the plant may die. After attack the plant can produce healthy leaves and visually it may appear that the plant also grows after the injury, but the roots are often affected. Carrots, which seem to grow after the attack may have large pith, become hard and a taste of wood develops. It is the saliva injected by adults during feeding that disturbs the metabolism of the plant and causes curling of the green parts. Five different components could be identified from the psylla saliva and these were, glucose, fructose, sucrose, inositol and an unknown amine, which may be of interest (Markkula et al. 1976). The nymphal feeding causes reduction of the root growth. Feeding by the adults is usually considered to cause the most serious damage. Very young seedlings are more damaged than older ones and this becomes obvious about 2 days after infestation. If the infestation is very light, the plant can recover and undamaged leaves will develop (Markkula et al. 1976).

4.3 Methods to control the carrot psyllids 4.3.1 Pesticides In Sweden, carrot psyllids are commonly controlled by spraying at 10-day intervals with pesticides such as dimethoate, deltametrin, lambda-cyhalotrin or cypermetrin (Figure 28). Up to eight sprayings may be needed (Nehlin et al. 1994; Jönsson et al. 2008). N O O O S Br O P N O O Br S H Deltametrin Dimethoate N N O O F C O Cl O 3 O O Cl Cl Cypermetrin Lambda -cyhalotrin

Figure 28. Commonly used pesticides in Sweden.

33

4.3.2 Repellent The use of fresh spruce and pine sawdust along the seedling rows in carrot fields reduce injury by the carrot psyllid (Apsits 1931). The use of sawdust has been investigated in Sweden (Rämert et al. 1993) during 5 weeks in 1987 – 1988. The sawdust was spread on the soil surface at various intervals and with varying doses. When the first egg was observed, treatment was started. The results showed that the number of eggs per seedling was reduced compared with the control. In 1994 Nehlin, et al. (1994) further explored the possibility of using sawdust and its volatile constituents as protecting agents against T. apicalis. In the test area, three weeks after the oviposition period started, all plants were injured. The use of sawdust at the highest level (0.5 l/m) reduced the injury level. Old sawdust treated with (-)-Limonene also showed a good protection. But the sawdust was effective only at very close range. Plants growing further than ten cm away from the edge of the fresh sawdust were damaged. A slight yellowing of the carrot foliage was observed in the sawdust treated area, indicating a deficit of nitrogen. Before use on a large scale the possible plant toxicity must be investigated. The effective high dose of sawdust is equivalent to a total dose of 15 l/m or 150 m3/ha; it would not be feasible to use such high application rates on a commercial scale.

4.3.3 Fibre Cloth protection To cover the ground with cloth can provide 100 % protection against the carrot psyllids (Rämert et al. 1989) provided the screen is used properly. It is important that the coverage is done at the right time, the edges are anchored well, a good crop rotation is applied, and the weeds are cleared quickly as unveiling is short, and that holes and tears in the cloth are avoided. A fibre cloth should be on from sowing to mid-June and after apparent attack of psyllids until harvest. Apart from protecting against harmful attacks the screens also protects against frost and provide a more balanced growing climate, and have also shown a beneficial effect on growth. The main drawbacks with cloth covering are that it is time consuming to work with, and that weeds benefit. The quality of carrots can also be adversely affected by elevated temperatures.

4.3.4 Trap crops Rows with a trap crop, in the form of appropriate carrot varieties, can reduce the pressure of carrot psyllids (Piirainen 1999)in the field. The trap crops should be sown in good time before the main sowing of carrots, so that psyllids in the close surroundings are tempted to fly out to trap crops.

34 4.3.5 Semiochemicals In 2006, Kristoffersen performed GC-SCR studies on extracts (female and male) from T. apicalis, from carrot leaves and from various conifers (in particular pine, spruce and juniper). The compounds that were identified and gave response were terpinolene, nonanal, terpinene-4-ol, cis-3-hexenal and one unidentified sesquiterpene. Nonanal was found and identified as an active compound in extracts from male and female T. apicalis. This indicates that there may be pheromonal activity in this psylloid. Pheromones have not been identified in psylloids before, although there have been some indications toward the presence of pheromonal activity in behavioural and electrophysiological experiments. The function and significance of nonanal need to be tested behaviourally, and any synergistic effects between host odours and potential pheromones also remains to be investigated (Kristoffersen 2006). In some carrot growing areas in the Swedish provinces Halland and Värmland the following potential kairomones were tested in 2006 (Anderbrant 2009): terpinolene, nonanal, terpinene-4-ol, cis-3-hexenal, β-sesquiphellandrene and 3-carene. Neither of the above mentioned components did affect the catch of carrot psyllids.

35 4.4 Results GC-MS analyses were carried out on extracts from carrots and demonstrated the presence of several components of which one gave EAG-response from a carrot psyllid. After considering the mass spectrum of the unknown compound from the extract we thought the kairomone compound could be β-sesquiphellandrene (Figure 29) and we synthesized this compound (see Appendix part 2). Its mass spectrum was nearly identical with that from the EAG-active compound from carrot. However, the GC retention times for the two compounds were not the same.

Figure 29. β-sesquiphellandrene

Another kairomone candidate was bergamotene. This is a sesquiterpene initially isolated by Herout and coworkers in 1950 and occurring in more than twenty-five different plants, e.g. black pepper oil (Russell et al. 1968), cumin (El-Sawi et al. 2002) and from carrot seeds (Zalkow et al. 1963). We started to synthesize the compound according to the method used by Kulkarni, et al. in 1987. If we choose the starting material to be nerylacetone the target molecule will be cis-bergamotene and if we choose geranylacetone the final product will be trans-bergamotene. The compound that we synthesized with this method showed similar mass spectrum as that of the active compound from the extracts, but their GC retention times were not the same. In the reduction of the ketone Kulkarni, et al. (1987) used anhydrous hydrazine and the reaction solution was heated at most to 155 °C and then KOH added and heated again to 190 °C. In Sweden the use of anhydrous hydrazine is forbidden. Therefore we used hydrazine hydrate instead, and maybe our choice of reactant affected the result. We changed our method for the synthesis of bergamotene and this time we applied the method of Alizadeha, et al. (2002). The difference in the two methods takes place after the ring closure of the acid chloride, until that step both pathways are the same (Figure 30).

36 O

O X

a,b,c d O

Figure 30. Reagents: a) NaH, triethylphosphonoacetate, X = OEt; b) 10 % NaOH, X = OH;

c) SOCl2, pyridine, benzene, X = Cl; d) Et3N, Toluene.

Anhydrous hydrazine is also prescribed in this method but the conditions of the reaction can be considered to be milder. Even here we used hydrazine hydrate instead of anhydrous hydrazine (Figure 31).

O

Kulkarnis method Alizadehas method Ours method

Figure 31. Reagents:

Kulkarni:1. N2H4, (CH3CH2O)2O 105°C 3h, 155°C, 0.5h2. KOH 190°C.

Alizadeha: 1. N2H4, AcOH, EtOH 30°C 48h, 2. t-BuOK DMSO 85°C 15h.

Our: 1.N2H4 - H2O, AcOH EtOH 30°C 48h, 2. t-BuOK DMSO 85°C 15h.

This method gave the desired product but in 26 % yield starting from the ketone. The product showed the same retention time and similar mass spectrum as those from the active compound found in the extract from carrot.

4.4.1 EAG New EAG tests are planned for autumn 2009.

37 4.5 Conclusion and future work In Sweden, the cultivated area of carrot was 1,804 ha in 2007 and the harvested production was 89,400 tons of carrot. The total value of carrots grown on open ground in 2006 is approximately 350 million kr (Jordbruksverket 2008). A desirable alternative sustainable control strategy would be to manipulate the behaviour of the insect. Today spraying with insecticides is applied. To avoid spraying carrot fields with chemical pesticides or at least reduce sprayings, some additional studies are need in order to find good techniques for how to best combat psyllids by using chemical signals such as pheromones and/or host plant scents. The organic farms try to manage the psyllid attacks by sowing late, moving crops between seasons, covering with fibre cloth and applying trap crops.

Where does mating occur? In order to understand the life cycle of the carrot psyllid and its approach to the carrots, this is an important question to answer. According to Lundblad in 1929 psyllids mate after they landed in fields and according to Piirainen in 1999 a female mates several times with different males in the field; this is considered likely as the female should be able to lay up to 900 eggs (Láska 1964). Can nymphs and the new generation of carrot psyllids cause damage to the carrots? The literature says that attacks of wintering males, nymphs and the new generation of psyllids are not as serious as the attacks caused by wintering females (Markkula et al. 1976; Nehlin 1992).

Our synthesis of bergamotene needs to be developed so that the last step, the reduction of the ketone, will be improved. One possibility is to reduce the ketone in the same way as that used in the synthesis of β-sesquiphellandrene, with n- butyllithium and methyltriphenylphosphonium bromide (Flisak et al. 1986). Bergamotene has also been synthesized by other groups. Thus, in their approach Corey and Desai 1985 also used anhydrous hydrazine. Earlier in 1971 Corey and Cane synthesized bergamotene in a 21 step synthesis and Larsen et al. 1977 used 13 steps in his synthesis. In 1972 Narasaka et al made a total synthesis of α-cis- Bergamotene using allyl 2-pyridyl sulfide derivatives and 9-iodo-α-pinene.

In 1988 Cane et al. isolated and identified a bergamotene synthetase, which catalyzes the cyclization of farnesylpyrophosphate (FPP) to β-bergamotene. The year after they report that they had established the absolute configuration of the β- bergamotene, isolated from mycelial extracts of Pseudeurotium ovalis and formed from FPP and bergamotene synthetase as (-)-β-trans-bergamotene, with (1S,5S,7R)- configuration, based on a novel combination of enzymatic and NMR spectroscopic techniques (Cane, 1989). The stereochemistry of bergamotene makes an enantioselective synthetic approach difficult and up until today just racemic bergamotene has been synthesized.

38 ACKNOWLEDGEMENT Jag vill tacka följande personer som under denna tid, på ett eller annat sätt, stått bakom mig och hjälpt mig att förvekliga denna avhandling:

 Erik Hedenström, i form av handledare. Tack för att du gav mig chansen en gång för länge sen och att du varit så positiv genom åren fast tvivlen och frågorna har varit många. Jag kan bara konstatera att du hade rätt när du sa ”det ordnar sig” gång på gång.

 Hans-Erik Högberg Tack för att du tog dig tid att läsa igenom manuskriptet till avhandlingen, dina synpunkter var värdefulla.

 Fredrik Andersson för all hjälp på lab dels med råd när det alltid skulle till och krångla och dels för all synteshjälp när det blev bråttom med kemikalier och jag ofta passa in dessa tillfällen med mammaledighet. Tack också för hjälpen med de sista bilderna.

 Martin J Steinbauer and Paul Walker for good collaboration.

 Olle Anderbrant för svar på alla frågor och för att du tagit dig tid att läsa igenom manuskriptet.

 Jarmo Holopainen and Merlin Crossley for the use of your photos and to Don Herbison-Evans for pleasant conversation and for the nice picture of the website http://www-staff.it.uts.edu.au/~don/larvae/enno/privata.html

 Alla nuvarande och tidigare medlemmar i organgruppen: Dan, Jimmy, Jonas, Micke, Staffan, Sunil, Jessica, Mona, Marica, Kristina, Palle, för trevliga fika och pratstunder och för att du tog dig tid att läsa igenom manuskriptet. Joakim för att du stod ut med alla frågor jag haft om diverse saker och för att du studerat manuskriptet under lupp, det var inte många ”cis, trans och punkter” som slapp igenom. Hoppas att du snart får värma dig permanent söderut! Carina tack för resesällskapet, Natalia för all den tid du lade ner för att hjälpa mig med allt från lösenord till kontroll läsning av utkast. Ditt vänliga sätt är som en frisk vind, lycka till med allt det ”nya”. Ida, (ett tag tillhörde du ju nästan denna grupp) tack för alla skratt på vinden, de pratglada luncherna och långa fikarasterna, men framför allt ”hästpratet” vad vore livet utan dessa besvärliga individer?

 Övriga och tidigare personal på NAT (gamla NTM och KEP) Siw, Viktoria, Veronika, Ingrid, Johnny, Kristina Hohweiler (bibl. Östersund) för din

39 hjälp när endnote krånglade, Christina Olsson (vaktmästeriet) för din professionella hjälp vid tryckningen, Håkan och Torborg som får våning tre att gå runt. Ett speciellt tack till Torborg som hjälpte mig med städningen av dragskåpet när lillflickan i magen gjorde sig påmind.

 Toppenkurskamraterna på grundutbildningen Jonna och Ulrika tänk det gick till slut, ni var suveräna att plugga med, utan er hade det varit betydligt jobbigare! Petra och Sofia för alla roliga fester och pluggstunder, för visst är linjär algebra roligt? Jag har många glada minnen fast det bara blev ett halvår plugg ihop, tur att vänskapen höll i sig längre.

 Mina underbara vänner, Kristin för att du är så klok, kommer aldrig att glömma de timslånga samtalen och de annorlunda festkvällarna det alltid blev. Catrin ”vi är bäst och har aldrig fel”. Både du och jag vet att inget mer behöver sägas eller betyder något! Ni är verkligen de vänner man kan önska sig, om än lite väl klarsynta ibland...

 Mamma för att du alltid stöttat mig på ditt egna lilla vis. Pappa för all hjälp med hus, el, ved, hästar ja med allt. Du anar inte så uppskattat det varit. Carina för att du är min kloka syster och för att du gjorde ett försök att läsa det jag skrivit. Farmor du har ställt upp så mycket genom åren, jag är lyckligt lottad med en farmor som du.

 Minos du var mitt allt under så många år, du är största anledningen till att jag är där jag är i dag. Tack för alla sannolika och osannolika turer du gett mig. Jag hoppas hovarna springer över kullarna och att magen får färskt grönt gräs hela dagarna.

 Min familj, Casper och Cornelia för att ni är ni och ger så mycket glädje. Joakim my love, my love, för att du alltid finns där hur det än stormar runt omkring, nu är det nog snart våran tid för det där lugna stabila du vet.

I am also grateful for the financial support from EU (European Regional Development Fund) and from Länsstyrelsen i Västernorrlands län.

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45