i
Licentiate Thesis, Sundsvall 2009
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
ii
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 Mnesampela privata and Trioza apicalis
Anna Nilsson
Cover Description
Cover picture: Front side on the left and back side: The autumn gum moth,
Mnesampela privata (Lepidoptera: 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
iii
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 Eucalyptus. The moths
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 insects 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 (Daucus carota) 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
insect 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
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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.
v
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.
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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
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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
viii
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
ix
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.
1
1. INTRODUCTION
Many animals 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).
2
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).
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).
N
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).
Pheromones
Repellent Alarm Trail Sex Aggregation
3
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).
O
O
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).
O
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).
4
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).
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).
Allelochemicals
Allomones Kairomones Apneumones Synomones
5
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).
OH
O
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
6
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).
7
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
8
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
9
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 animal 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.
10
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
11
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”.
12
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
13
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).
Cl
Cl
OO
O
N
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).
OH
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 Noctuidae, 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.
14
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).
15
CoA
O
SCoA
O
SCoA
O
SCoA
O
O
O
Z9-14:acyl
Z11-16:acyl
Z7-12:OAc
SCoA
O
Z7-12:acyl
16-acyl
Fatty acid synhtesis
Z11 desaturation
-Oxidation
-Oxidation
Reduction of the acyl group followed by acetylation
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
16
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).
OH
O
Figure 17. Biosynthetic pathways to produce polyene hydrocarbons and epoxide
pheromones from linoleic and linolenic acid precursors. Picture from Millar 2000.
Reductive decarboxylation
Epoxidation
Reduction, epoxidation
or reduction, oxidation
C17- hydrocarbones, epoxides C18- hydrocarbons,
epoxides and aldehydes
Chain extension by 2-carbon units
C20- C22- C24- COOH
Reductive decarboxylation
Epoxidation
Reductive Epoxidation
C19- C21- C23- hydrocarbons
and epoxides
C20 - C22- hydrocarbons
and epoxides
17
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.
18
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.
19
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.
20
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).
21
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).
22
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.
A
B
23
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)
0
100
200
300
400
500
600
1 2 3 4 5 6 7
Sample number
Mean +
SE
response (
% o
f contr
ol)
Figure 23. Compound 3: (3Z,6Z,9Z)-3,6,9-nonadecatriene; 4: monoepoxide from C18
triene; 6: monoketone from C18 triene.
24
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).
25
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
numbera
Compound Peak
numbera
Compound
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 aNumbers correspond to the peaks in chromatogram in figure 24.
26
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
27
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
28
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).
29
OMe
O
C18 - C21
C18 - C21
C18 - C21
C18 - C21
O
O
O
C18 - C21
C18 - C21
C18 - C21
O O
O O
O O
O
OO
OO
O
Figure 27. Syntheses of reference substance.
a
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
mCPBA, CH2Cl2, 0 °C
Mixture of mono, di and triepoxides
Easily separated by flash chromatography
1) LiAlH4, Et2O, 0 °C
2) HCl
3) PDC, CH2Cl2, rt
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 %).
30
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.
31
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.
32
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
33
(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).
Cl
Cl
OO
O
N
Br
Br
OO
O
N
F3C
Cl
OO
O
N
OP
SN
H
O
S
O
DeltametrinDimethoate
Lambda -cyhalotrin Cypermetrin
Figure 28. Commonly used pesticides in Sweden.
34
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.
35
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.
36
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).
37
OX
O
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).
Kulkarnis methodAlizadehas methodOurs method
O
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.
38
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.
39
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
40
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.
41
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