Purification, Stereoisomeric Analysis and Quantification of Biologically Active Compounds in Extracts from Pine Sawflies, African Butterflies and Orchid Bees

Thesis for the degree of Doctor of Philosophy, Sundsvall 2011

PURIFICATION, STEREOISOMERIC ANALYSIS AND QUANTIFICATION OF BIOLOGICALLY ACTIVE COMPOUNDS IN EXTRACTS FROM PINE SAWFLIES, AFRICAN BUTTERFLIES AND ORCHID BEES

Joakim Bång

Supervisor: Erik Hedenström

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

ISSN 1652-893X Mid Sweden University Doctoral Thesis 116 ISBN 978-91-86694-58-6

Akademisk avhandling som med tillstånd av Mittuniversitetet i Sundsvall framläggs till offentlig granskning för avläggande av filosofie doktorsexamen fredag 28 oktober 2011, klockan 10:15 i sal O111, Mittuniversitetet Sundsvall. Seminariet kommer att hållas på svenska.

PURIFICATION, STEREOISOMERIC ANALYSIS AND QUANTIFICATION OF BIOLOGICALLY ACTIVE COMPOUNDS IN EXTRACTS FROM PINE SAWFLIES, AFRICAN BUTTERFLIES AND ORCHID BEES

Joakim Bång

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, 2011

i PURIFICATION, STEREOISOMERIC ANALYSIS AND QUANTIFICATION OF BIOLOGICALLY ACTIVE COMPOUNDS IN EXTRACTS FROM PINE SAWFLIES, AFRICAN BUTTERFLIES AND ORCHID BEES

Joakim Bång

Department of Natural Sciences, Engineering and Mathematics Mid Sweden University, SE-851 70 Sundsvall, Sweden ISSN 1652-893X, Mid Sweden University Doctoral Thesis 116; ISBN 978-91-86694- 58-6

ABSTRACT

Stereochemistry plays an important role in nature because biologically important molecules such as amino acids, nucleotides and sugars, only exist in enantiomerically pure forms. Semiochemicals carry messages, between the same species (pheromones) and between different species (allelochemicals). Both pheromones and allelochemicals can be used as environmentally friendly pest management. Many semiochemicals, i.e. behaviour modifying chemicals, consist of pure or well-defined mixtures of stereoisomers, where some of the other stereoisomers can be repellent. It is therefore important to be able to separate them to produce a synthetic pheromone in a mixture that is attractive.

Pine sawflies are a family of insects that in some cases can be severe defoliators of conifer trees. Diprion pini, Diprion similis and Neodiprion sertifer are severe pests for these trees and have got the most attention in pine sawfly pheromone studies. The pheromone precursors are stored in the female body as long-chain secondary alcohols, which, when released, are esterified to acetates or propionates. The alcohols are chiral, and normally one of the stereoisomer is the main pheromone component, sometimes possible together with other stereoisomers as essential minor components.

Bicyclus is a genus of African butterflies, and especially Bicyclus anynana has become a popular model for the study of life history evolution, morphology, mating choice and genetics. The wing pattern of Bicyclus differs depending on the season, with large eyespots during the rain-season and small or absent spots during the dry season.

ii Euglossa is one of the genera among the orchid bees in the Neotropics that does not produce its own pheromone. Instead, the males collect fragrances from orchids and other sources and store them in a pocket in their hind legs. Both Bicyclus and Euglossa use semiochemicals similar to pine sawflies, and thus can be analysed by the same methods.

Pheromones and other semiochemicals in insects are often present in low amounts in a complex matrix, and purification of the sample before chemical analysis is often required. A common method is gradient elution on a solid phase silica column. Separation of stereoisomers can be achieved either by using a column with a chiral stationary phase (CSP) or with pre-column derivatisation using a column with an achiral stationary phase (ASP) or a combination of both, with mass detection as the dominant detection method. The purpose of this work has been to improve the purification method, find suitable methods to separate the stereoisomers of secondary alcohols, and to apply this on extracts of insects.

By selecting the right fractions to collect during gradient elution the purification method was optimised. To reduce plasticizer contamination from ordinary columns, solid phase columns of Teflon or glass were used. For pre-column derivatisation of different chiral alcohols various acid chlorides were tested. For the pine sawfly pheromone precursors enantiopure (2S)-2-acetoxypropionyl chloride was the best choice. To separate some of the stereoisomers achiral 2- naphthoyl chloride was used. For derivatisation of 6,10,14-trimethylpentadecan-2- ol (R)-trans-chrysanthemoyl chloride was the best choice. The derivatised alcohols were separated on different columns, both chiral and non-chiral. Varian FactorFour VF-23ms was chosen as a general-purpose column, the Agilent HP-88 column was the best column with an ASP of those tested, and the Chiraldex B-PA column (CSP) was the only one that could separate all eight stereoisomers of derivatised 3,7- dimethylundecan-2-ol, 3,7-dimethyldodecan-2-ol, and 3,7-dimethyltridecan-2-ol.

To determine the stereoisomeric purity of standard solutions used in field experiments and extracts of different species of insects the optimised methods were applied. For extracts from B. anynana, Euglossa and Neodiprion lecontei this work describe the first determination of the stereochemistry of some of their semiochemicals.

iii For the determination of the stereochemistry of chiral semiochemicals the methods for purification and separation presented herein have shown to be of great value. The results will hopefully contribute to a better understanding of the communication among insects, and ultimately to a more environmentally friendly pest control.

Keywords: Semiochemicals, sex pheromone, pine sawflies, Bicyclus, Euglossa, chiral separation, derivatisation, GC-MS.

iv SAMMANDRAG

Många naturligt förekommande kemiska ämnen finns som två spegelbilder av varandra, ungefär som höger och vänster hand. Dessa kan ha helt olika egenskaper och det är därför viktigt att kunna separera dem. Insekter och andra djur använder olika doftämnen för att kommunicera med varandra, om det är inom samma art kallas de för feromoner. De kan bestå av ett ämne eller en blandning av flera. Dessa doftämnen kan man även använda för att på ett miljövänligt sätt bekämpa skadeinsekter. En fälla med syntetiskt feromon för en viss insekt lockar endast till sig den arten, medan alla andra är opåverkade. Eftersom dessa ämnen ofta finns som spegelbilder där kanske bara den ena är aktiv och den andra rent av frånstötande, måste man kunna separera dem för att framställa ett syntetiskt feromon som är attraktivt.

Målet med detta arbete har varit att bestämma feromonet hos olika arter av tallsteklar som kan vara svåra skadedjur på tallskog. De metoder som tagits fram har även tillämpats på några arter av afrikanska fjärilar samt orkidébin från Centralamerika eftersom de använder snarlika doftämnen.

Att få fram feromonet från en insekt är lite som att leta efter in nål i en höstack eftersom de ofta bara innehåller några miljarddels gram per individ. Provet behöver först renas, och en del av arbetet i det här projektet har gått ut på att ta fram en lämplig reningsmetod. Huvudfokus har dock varit på att ta fram metoder som kan separera och identifiera det eller de ämnen, och spegelbilder av dessa, som doftämnena består av. När lämpliga metoder tagits fram har extrakt av olika insektsarter analyserats. I några fall är det första gången som deras feromon bestämts i detalj. Resultaten kan förhoppningsvis bidra till en ökad kunskap om insekters sätt att kommunicera, och i slutändan till miljövänligare bekämpning av skadeinsekter.

v TABLE OF CONTENTS

ABSTRACT ...... II SAMMANDRAG ...... V LIST OF PAPERS ...... VIII LIST OF ABBREVATIONS ...... IX 1. INTRODUCTION ...... 1 1.1. Stereochemistry ...... 1 1.2. Semiochemicals ...... 4 1.2.1. Pheromones ...... 5 1.2.2. Allelochemicals ...... 6 1.2.3. Semiochemicals in pest management ...... 6 1.3. Olfactory system of insects ...... 8 1.4. Pine sawflies ...... 9 1.4.1. Pine sawfly pheromone ...... 9 1.5. Bicyclus anynana (Squinting Bush Brown) ...... 16 1.6. Euglossa (Orchid bees) ...... 17 2. ANALYTICAL METHODS ...... 18 2.1. Antennal response ...... 18 2.2. Collection of volatiles ...... 18 2.3. Extraction and purification ...... 19 2.4. Separation of stereoisomers using GC ...... 21 2.4.1. GC columns with a chiral stationary phase ...... 21 2.4.2. Pre-column derivatisation ...... 23 2.5. Detection and identification ...... 25 3. OBJECTIVES ...... 28 4. RESULTS ...... 29 4.1. Method development ...... 29

vi 4.1.1. Purification ...... 29 4.1.2. Derivatisation ...... 31 4.1.3 GC separation ...... 36 4.2. Application of methods ...... 45 4.2.1. Purity of synthetic references ...... 45 4.2.2. Pine sawflies ...... 47 4.2.3. Bicyclus ...... 50 4.2.4. Euglossa ...... 52 5. CONCLUDING REMARKS ...... 54 ACKNOWLEDGEMENTS ...... 56 REFERENCES ...... 57

vii LIST OF PAPERS

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

Paper I The Male Sex pheromone of the Butterfly Bicyclus anynana: Towards an Evolutionary Analysis. C. M. Nieberding, H. de Vos, M. V. Schneider, J.-M. Lassance, N. Estramil, J. Andersson, J. Bång, E. Hedenström, C. Löfstedt and P. M. Brakefield. PLoS ONE, 3 (2008), e2751.

Paper II Field Response of Male Pine Sawflies, Neodiprion sertifer (Diprionidae), to Sex Pheromone Analogs in Japan and Sweden. O. Anderbrant, J. Löfqvist, E. Hedenström, J. Bång, A. Tai and H-E. Högberg. Journal of Chemical Ecology, 36 (2010), 969-977.

Paper III (6R,10R)-6,10,14-Trimethylpentadecan-2-one, a Dominant and Behaviorally Active Component in Male Orchid Bee Fragrances. T. Eltz, E. Hedenström, J. Bång, E. A. Wallin and J. Andersson. Journal of Chemical Ecology, 36 (2010), 1322–1326.

Paper IV Purification, Stereoisomeric Analysis and Quantification of Sex Pheromone Precursors in Female Whole Body Extracts from Pine Sawfly Species. J. Bång, E. Hedenström and K. Sjödin. Journal of Chemical Ecology, 32 (2011), 125-133.

Paper V Sex Pheromone of the Intruduced Pine Sawfly, Diprion Similis (Diprionidae), Revisited: no Activity of Earlier Reported Synergists. O. Anderbrant, B. Lyons, J. Bång, E. Hedenström and H-E. Högberg. Submitted.

Paper VI Stereoisomeric separation of derivatised 2-alkanols using GC-MS: Sex pheromone precursors found in pine sawfly species. J. Bång, E. Hedenström and O. Anderbrant. Submitted.

Not incl. Chemical ecology and insect conservation: optimising pheromone- based monitoring of the threatened saproxylic click beetle Elater ferrugineus. G. P. Svensson, C. Liedke, E. Hedenström, P. Breistein, J. Bång, and M. C. Larsson. Journal of Insect Conservation (in press).

Paper II-IV were reprinted with kind permission from Springer Science+Business Media.

viii LIST OF ABBREVATIONS

ASP Achiral stationary phase CI Chemical ionization CSP Chiral stationary phase EAD Electroantennographic detection EAG Electroantennogram ECD Electron capture detection EI Electron impact FID Flame ionization detection GC Gas chromatography GC-FTIR Gas chromatography - Fourier transform infrared spectroscopy GC-MS Gas chromatography - mass spectrometry Gk. Greek IUPAC International Union of Pure and Applied Chemistry MS Mass spectrometry m/z Mass-to-charge ratio NMR Nuclear magnetic resonance NPD Nitrogen phosphorus detection OBP Odorant-binding protein ORN Olfactory receptor neurons SCR Single cell recording SIM Selected ion monitoring SPE Solid phase extraction SPME Solid phase microextraction SSR Single sensillum recording

(2R*,3S*,7R/S) = (2R,3S,7R), (2S,3R,7R), (2R,3S,7S) and (2S,3R,7S), previously called threo. (2R*,3R*,7R/S) = (2R,3R,7R), (2R,3R,7S), (2S,3S,7R) and (2S,3S,7S), previously called erythro.

1. INTRODUCTION 1.1. Stereochemistry

Stereochemistry is the study of the three-dimensional shapes of molecules. Many molecules exists as different isomers, which is defined by IUPAC as “One of several chemical species (or molecular entities) that have the same stoichiometric molecular formula but different constitutional formulae or different stereochemical formulae and hence potentially different physical and/or chemical properties” (Moss 1996).

Isomers are divided into constitutional isomers and stereoisomers.

Constitutional isomers have different connectivity between the atoms:

• Positional isomers: different position of functional groups (Figure 1A). • Skeletal isomers: different carbon skeleton (Figure 1B). • Functional-group isomers: different functional groups (Figure 1C).

A. Positional isomers: B. Skeletal isomers: C. Functional group isomers:

H3C OH H3C CH3 H3C OH Hexan-1-ol Butane Ethanol

OH CH3

H3C O CH3 H3C CH3 H3C CH3 Hexan-2-ol Isobutane Methoxymethane

Figure 1. Constitutional isomers.

Stereoisomers have the same connectivity but differ in the arrangement in space:

• Enantiomers: a pair of molecules which are nonsuperposable mirror images of each other (Figure 2A). • Diastereomers: stereoisomers that are not mirror images of each other (Figure 2B). They have different physical properties and differ to some extent in chemical behaviour. One form of diastereomers is the E/Z isomers of alkenes. If the groups with the highest priority are on opposite sides of the double

1 bond, the alkene is designated E (from the German entegen), and if they are on the same side it is designated Z (from the German zuzammen) (Figure 2C).

A. Enantiomers B. Diastereomers C. E/Z isomers (diastereomers)

(Low) H Cl (High) H H COOH COOH HO COOH HOOC OH H2N H H NH2

(High) H C CH (Low) 3 E 3 CH3 CH3 HO H HO H (Low) H CH3 (Low) CH3 CH3

(High) H3C Cl (High) Z

Figure 2. Stereoisomers.

Enantiomers and diastereomers have normally one or more stereogenic centres, normally a carbon bonded to four different substituents. If a molecule has a plane of symmetry it must be superposable on its mirror image, and is hence achiral.

The stereogenic centres are assigned the letter R or S, depending of the order of the groups attached to it. With the group of the lowest priority (lowest atomic number) pointing back, the rest of the groups are counted from highest to lowest priority. If the order is clockwise the centre is named R (rectus) and S (sinister) if it is counted clockwise (Figure 3).

4 4 H H 1 2 2 1 HO COOH HOOC OH (R) (S) 3 3 CH CH3 3

(R)-2-hydroxypropanoic acid (S)-2-hydroxypropanoic acid

Figure 3. (R)- and (S)-configuration.

2 Stereochemistry, and the ability to separate and detect stereoisomers, is of great importance for several reasons. The molecules that build up life (amino acids, nucleotides, sugars) are chiral and only exist in nature in enatiomerically pure forms. Semiochemicals (see Section 1.2.), used by organisms for communication, are often stereoisomers (Mori 2007). Many of the active components in drugs are chiral, and it is often crucial to produce them in enatiomerically pure forms and test both enantiomers for toxicity (Gübitz and Schmid 2001; Anslyn and Dougherty 2005).

3 1.2. Semiochemicals

Chemicals that mediate interactions between organisms are defined as semiochemicals (Gk. semeon = mark or signal). They are subdivided in two groups, pheromones and allelochemicals (Nordlund and Lewis 1976; Nordlund 1981). Pheromones mediate chemical communication between individuals of the same species and allelochemicals between individuals belonging to different species. They are further divided in subgroups based on the purpose or benefits (Figure 4).

Semiochemicals Chemical substances carrying messages

Pheromones Allelochemicals Between individuals of the Between individuals of same species different species

Alarm Allomones

Repellent Kairomones

Aggregation Synomones

Trail Apneumones

Sex

Figure 4. The different groups and subgroups of semiochemicals.

4 1.2.1. Pheromones

In 1959 Karlsson and Lüscher suggested the term pheromone (Gk. phereum = to carry, horman = to excite) to define a substance released by an animal that trigger a specific reaction in a member or members of the same specie (Karlson and Lüscher 1959; Nordlund and Lewis 1976). The existence of pheromones was, however, known long before that. The fact that male dogs are attracted to secretions from female dogs in heat were known already to the ancient Greeks, and in 1609 Charles Butler described how a single bee sting attracts other bees to attack (Wyatt 2009). Jacentkovski discovered in 1932 that a trap containing a female gypsy moth attracted a large amount of males (Nandagopal et al. 2008).

The word pheromone was launched at the right time. That same year (1959) Butenandt et al. reported the first isolation and identification of a pheromone, (10E,12Z)-hexadeca-10,12-dien-1-ol (bombykol), the sex pheromone of the silkworm moth Bombyx mori (Wyatt 2009). Since then hundreds of pheromones have been discovered, ranging from small molecules as formic acid to polypeptides. The early beliefs that pheromones were single compounds have been revised. In many cases, they are mixtures of different compounds in very exact ratios. Also, the stereochemistry of the pheromone can be of crucial importance (see Section 1.4.1.).

Examples of some pheromone subgroups:

• Alarm: Dispersion or aggressive behaviour as a response to predators. The workers of the honeybee Aplis mellifera release a mixture of isopentyl acetate and more than 20 other substances to coordinate an attack when they feel threatened (Slessor et al. 2005). • Repellent: A warning signal to avoid sources unsuitable for food or colonisation. Used by bark beetles to warn other individuals from attacking healthy trees with high amounts of poisonous substances (Francke et al. 1995). • Aggregation: Congregation for feeding or mating. Bark beetles use aggregation pheromone to coordinate feeding and mating when a suitable tree has been found (Seybold et al. 2006). • Trail: Path marking, common among social insects. Ants use trail pheromone to guide other workers to a food source (Morgan 2009). • Sex: Usually emitted from females to attract males for mating. Females of the Asian elephant Elephas maximus uses (Z)-7-dodecen-1-yl acetate to attract males. The same substance is also used as one of the components in the pheromone blend of some 140 moth species (Rasmussen et al. 1997).

5 1.2.2. Allelochemicals

Substances that mediate interaction between individuals of different species are defined as allelochemicals. They are divided into four groups (Nordlund 1981):

• Allomones (Gk. allo = different): beneficial to the emitter. Plants release allomones as defence against herbivores by attracting their enemies. • Kairomones (Gk. kairos = opportunistic): beneficial to the receiver. Parasites use kairomones to detect hosts. • Synomones (Gk. syn = with or jointly): beneficial to both the emitter and receiver. Flowers attract pollinators, which receive nectar as reward. • Apneumones (Gk. a-pneum = breathless or lifeless): chemicals from a non- living source that are favourable to a receiving organism but unfavourable to another species that are found on the non-living material.

In combination with plant volatiles from the host tree, the aggregation pheromone of bark beetles is used by predators to locate them. In this case the bark beetle pheromone is a kairomone and the plant volatiles a synomone to the predator (Mumm and Hilker 2006).

1.2.3. Semiochemicals in pest management

An increased environmental awareness, resulting in the ban of many synthetic pesticides, has made it important to develop alternative and more environmental- friendly methods to fight pests. A growing world population, resulting in an increased demand for food, and an on-going change in climate has made this even more important and challenging.

A problem with pesticides is the development of resistance after long-term use, and many times the natural enemies to the pest are more affected than the target species (Witzgall et al. 2010). The optimal solution would be methods that only affect the pest and leaves the rest of the ecosystem unaltered. This must, of course, also be economically viable.

Both allelochemicals and pheromones can be used for control of pest insects. Especially sex pheromones have several advantages: they are species-specific, very small amounts are needed, and they are almost all non-toxic to other animals (Witzgall et al. 2010). Although sex pheromones often consist of a mixture of different substances (or stereoisomers) in very exact ratios, it is sometimes effective even in incomplete blends. This reduces the costs to produce synthetic

6 pheromones. Sometimes host volatiles, used for aggregation of many insects, are combined with pheromones for control of pests, e.g. the apple fruit moth Argyresthia conjugella (Norin 2007). The use of semiochemicals instead of pesticides does not affect predators, which thus can reduce secondary pests.

Monitoring The most common use of semiochemicals in pest management is for monitoring. Traps with synthetic sex pheromone are used to detect the presence of a certain pest or if a larger outbreak is imminent. This is often used in combination with pesticides, which thereby reduces the amount of chemicals needed. Otherwise pesticides are often used “just in case” (Witzgall et al. 2010).

Mass trapping Mass trapping is mainly used for species that use aggregation pheromones and thereby captures both males and females. This method has been used sucessfully against the bark beetle Ips duplicatus in China, where a synthetic pheromone blend of ipsdienol and E-myrcenol strongly reduced tree mortality by bark beetle attacks in a spruce forest (Schlyter et al. 2001).

Attract and kill This technique combines an attractive semiochemical with an insecticide. It reduces the need of chemicals to a minimum. The house fly Musca domestica can effectively be caught with commercial traps containing the female sex pheromone (Z)-9-tricosene and the insecticide imidacloprid (Butler et al. 2007).

Mating disruption Sex pheromones are normally released from females to attract males for mating. By saturating an area with synthetic sex pheromone, the male will not be able to find the female and thus mating is prevented. This method is more efficient in large areas, where the movement of females in and out of the treated area (border effect) is insignificant (Östrand et al. 1999; Witzgall et al. 2010), and/or with females that show limited movement after mating (Martini et al. 2002). Mating disruption has now become the most common method to control pests with semiochemicals, and is used in vineyards, orchards, forests, and can also be used for indoor pests (Ryne et al. 2006; Witzgall et al. 2010).

Repelling Compounds from non-host-plants that a pest avoids can be used to prevent feeding. The pine weevil Hylobius abietis, although polyphagous, avoids feeding on certain plants even when no choice is given. An example of such a compound is nonanoic acid from linden bark (Månsson et al. 2005).

7 Push and pull “Push and pull” is a method that uses an attractive stimuli in combination with a repellent. An example of a successful application is the control of the cereal stem borers Chilo partellus and Busseola fusca on cereal crops in Africa (Khan et al. 2008). A repellent plant is planted inside the fields and an attractive trap plant at the borders. For this method to work, the attractive plant has to be more appealing than the crop.

1.3. Olfactory system of insects

The olfactory system of insects is very selective and can discriminate between a pheromone and other molecules with minimal structural difference, even between different stereoisomers. The discrimination is made by odorant-binding proteins (OBP), that transport the pheromone across an aqueous barrier, and odorant receptors (Leal 2005). Many male insect species have large and strongly branched antennas to be able to detect low amounts of sex pheromone emitted by the female, sometimes hundreds of meters away. The chemical signal is picked up by hair- liked sensilla on the surface of the antenna, transported by OBP to the olfactory receptor neurons (ORN), which are in contact with the antennal lobes in the brain by their axons. The signal is processed in the brain and instructions are given to the motor system. This results in the male navigating towards the female in a zigzag pattern to pinpoint the source of the pheromone (Leal 2005).

The number of ORN in most sensilla are normally between two and five, but can be as many as 140 in wasps (Leal 2005). In the pine sawfly specie Neodiprion sertifer all but one of its 8 to 12 ORN are specialized for reception of the chiral sex pheromone, and the last one is tuned to an inhibiting stereoisomer (Hansson et al. 1991). For the closely related Diprion pini, all of the 8 to 9 ORN are specialized for the attractive stereoisomer of the sex pheromone (Anderbrant et al. 1995). Co- localization of the ORN for the pheromone and a behavioural antagonist is common among male moths (Baker et al. 1998). The moth detects the time difference (milliseconds) in the arrival of the different odour molecules. This makes it possible for them to discriminate between plumes of a mixture of the pheromone and antagonist from the same source, or plumes of each from different sources that have been mixed in the air (Hansson 2002). The sensilla of Japanese beetle Popillia japonica have two ORN, one tuned to the pheromone (R)-japonilure and the other to the antagonist (S)-japonilure, which is the pheromone of another beetle, living in the same habitat (Nikonov and Leal 2002). This shows the importance of the ORN to discriminate between different stereoisomers.

8 1.4. Pine sawflies

The family of pine or conifer sawflies (Diprionidae) belongs to the order Hymenoptera, with two suborders, Symphyta (sawflies and horntails) and Apocrita (wasps, ants and bees). Diprionidae is one of 14 families of Symphyta. The name pine sawflies comes from its main host, Pinus spp. and the sawlike ovipositor of the females (Smith 1993; Anderbrant 1999).

Although a small family with about 130 species in 11 genera, it has received great attention from scientists. This is due to its harmful defoliation of conifers, causing damages with great economical consequences. Pine sawflies are widespread over the Holarctic region, with a southern limit in Central America, Thailand, northern India, and northern Africa. The species considered as the most severe pests are those that have been introduced from Europe to North America, such as Diprion similis and Neodiprion sertifer (Smith 1993). Two of the worst outbreak species (and also most studied) in Europe are D. pini, and N. sertifer. An outbreak species is defined as having a population eruption two or more times per 100 years, a host defoliation of >50%, lasting for at least two years per eruption, and affecting an area of more than 1000 ha (Larsson et al. 1993).

Pinus sylvestris can release volatiles that attract egg parasitoids when they are attacked by the two common pine sawfly species D. pini and N. sertifer, but does not do so against the less common Gilpinia pallida (Mumm and Hilker 2006).

The female uses the saw-like ovipositor to slice a slit in the needles of conifers, where she lays one or several eggs, depending on species. Many pine sawflies oviposit on the needles from the previous year, although the second generation can be laid on needles of the current year (Géri et al. 1993). Depending on species, the sawfly passes the winter either as an egg or as a prepupal cocoon on the ground. Depending on the climate and species, one or more generations can occur each year. Normally, only fertilized eggs evolve into females, but for some species (e.g. Gilpinia hercyniae) females are produced from unfertilized eggs and males are very rare (Knerer 1993; Anderbrant 1999). Larvae of Diprionidae has developed a defence mechanism where they store resin from the tree in foregut pouches and regurgitate it when they are disturbed (Mumm and Hilker 2006).

1.4.1. Pine sawfly pheromone

Coppel et al. (1960) were the first to study the female pine sawfly pheromone. They discovered that a caged female of D. similis could attract thousands of males,

9 although they were not able to isolate and identify the attractant (Coppel et al. 1960). It was not until 1976 that the sex pheromone of a pine sawfly species was identified, when Jewett et al. identified 3,7-dimethylpentadecan-2-ol (Figure 5), later named diprionol, as an inactive pheromone precursor of N. lecontei, N. sertifer and D. similis (Jewett et al. 1976). They also found that diprionol is stored in the female body and just prior to release it is esterified to the active pheromone. N. lecontei and N. sertifer are attracted to the acetate and D. similis to the propionate. No production site for the pheromone precursor has been found in female pine sawflies. The alcohol is distributed equally over the whole body, indicating that the biosynthesis is not concentrated to a specific part of the body (Anderbrant 1993). Diprionol has three stereogenic centres and thus can exist as eight different stereoisomers. Each of these have been synthesised in isomerically pure forms, and this, in combination with improved analytical methods, have allowed for more reliable pheromone identification as well as field-testing of the different stereoisomers (Högberg et al. 1990).

CH3

CH3 CH3

Figure 5. 3,7-Dimethylpentadecan-2-ol (diprionol).

Since the first sex pheromone precursor was identified, several more have been found in other pine sawfly species. They all have a very similar structure: a secondary alcohol with a chain of 11, 13, 14 or 15 carbons, substituted with one to three methyl groups (Figure 6). The alcohols can exist as 4 to 16 stereoisomers, with normally one of the isomers (as esters) as the major pheromone component and sometimes other isomers with synergistic or antagonistic effect (Anderbrant 1999; Hedenström and Andersson 2002; Keeling et al. 2004). Almost every alcohol precursor have the (2S)-configuration, although Hedenström et al. identified several alcohols with (2R)-configuration in extracts of Gilpinia pallida (Hedenström et al. 2006). In one field study D. similis was trapped with the propionate of (2R,3R,7R)-diprionol, being significantly more attractive than the (2S,3S,7S)-isomer, and with increasing catches with increasing dose. (Longhurst et al. 1980). These results have not been able to reproduce in later studies (Kikukawa et al. 1982; Olaifa et al. 1988; Anderbrant et al. 2011).

10 OH OH 2 * 2 * CH3 CH3 11 9 * 3 * 13 7 * 3 *

CH3 CH3 CH3 CH3

Diprion nipponica Diprion spp (2S,3R,7S) (2S,3R,7R)

OH OH 2 * 2 * CH3 CH 13 9 * 7 * 3 * 13 11 * 7 * 3 * 3

CH3 CH3 CH3 CH3 CH3 CH3

Macrodiprion nemoralis Microdiprion pallipes (2S,3R,7R,9S) (2S,3S,7S,11S) and (2S,3S,7S,11R)

OH OH 14 2 * 2 * CH3 CH3 7 * 3 * 15 3 * CH3 CH3 CH3 Gilpinia pallida Gilpinia frutetorum and Gilpinia socia (2S,3R,7R) (2S,3R)

OH 2 * CH3 15 7 * 3 *

CH3 CH3

Diprion spp, Gilpinia spp and Neodiprion spp (2S,3S,7S) and (2S,3R,7R)

Figure 6. Sex pheromone precursors of pine sawflies that have been identified in female extracts (*stereogenic centre).

The following list includes all pine sawfly species that have been studied by field-testing, EAG/EAD-experiments, or analysis of pheromone precursor content in female extract.

Diprion jingyuanensis The propionate of (2S,3R,7R)-3,7-dimethyltridecan-2-ol is the main component of the sex pheromone (Zhang et al. 2005). Anderbrant et al. (unpublished results) found that a mixture of (2S,3R,7R) and (2S,3R,7S) caught significantly more than the isomers alone, and these two isomers gave also the strongest response in EAG. In extracts they found both threo [(2S,3R,7R) or (2S,3R,7S)] and erythro [(2S,3S,7S)/(2S,3S,7R) or (2R,3R,7R)/(2R,3R,7S)] at a ratio of 3:1.

Diprion nipponica The propionate of (2S,3R,9S)-3,7-dimethylundecan-2-ol is the main component of the sex pheromone, confirmed by field studies and analysis of female body extracts (Tai et al. 2002). Propionates of (2S,3R,7S)- (2S,3R,8S)- and (2S,3R,9R)-3,7-

11 dimethylundecan-2-ol were also attractive in field studies (Tai et al. 1998; Tai et al. 2002).

Diprion pini The acetate or the propionate of (2S,3R,7R)-3,7-dimethyltridecan-2-ol is the main component of the sex pheromone (Figure 7), with an amount of the precursor alcohol of about 8 ng/female (Bergström et al. 1995; Bång et al. 2011). Bergström et al. also found minor amounts (0.5-4%) of the alcohol analogues with a carbon chain length of 12, 14, 15 and 16, with the first three giving EAG response. None of the minor component resulted in any effect in field test. Anderbrant et al. discovered that 3,7-dimethyltridecan-2-ol, acetic, propionic, butyric, and, isobutyric acid, together with the acetate, propionate and butyrate esters of 3,7-dimethyltridecan-2- ol are released from the female (Anderbrant et al. 2005). Both EAG and field tests showed a reaction and attraction to different esters (acetate, propionate, butyrate, isobutyrate) of the alcohol.

O CH3

CH3

CH3 CH3 Acetate of (2S,3R,7R)-3,7-dimethyltridecan-2-ol O

CH3 O

CH3

CH3 CH3 Propionate of (2S,3R,7R)-3,7-dimethyltridecan-2-ol

Figure 7. The pheromone of Diprion pini.

Diprion similis The propionate of (2S,3R,7R)-3,7-dimethylpentadecan-2-ol is the main component of the sex pheromone, and Anderbrant et al. showed that it alone caught most males, with no synergistic effect by the other isomers or any attraction at all by the acetate (Kikukawa et al. 1982; Olaifa et al. 1988; Anderbrant et al. 2011). Female body extract contained about 15 ng of (2S,3R,7R)-3,7- dimethylpentadecan-2-ol, with minor amounts of (2R,3S,7S), (2R,3R,7S) and (2R,3R,7R), 1%, 0.4% and 0.3%, respectively, of the main component (Anderbrant et al. 2011).

12 Gilpinia frutetorum The acetate of (2S,3R,7(R/S))-3,7-dimethylpentadecan-2-ol was most attractive in field test (Kikukawa 1982). EAG recordings gave the strongest response for the acetates of (2S,3R,7R)- and (2S,3R,7S)-3,7-dimethylpentadecan-2-ol, and the following alcohols were found in female body extracts: (2S,3R,7R)-3,7- dimethylpentadecan-2-ol (1-2 ng/female), (2S,3R)-3-methylpentadecan-2-ol (1-2 ng/female), and (2R*,3R*)-3-methylpentadecan-2-ol (0.1-0.2 ng/female) (Hedenström et al. 2009).

Gilpinia pallida The propionate of (2S,3R,7R)-3,7-dimethyltetradecan-2-ol was most attractive in field test, followed by the propionate of (2S,3R,7R)-3,7-dimethyltridecan-2-ol (Hedenström et al. 2006). Several isomers of 3,7-dimethyltridecan-2-ol, 3,7- dimethyltetradecan-2-ol and 3,7-dimethylpentadecan-2-ol were found in female body extracts in amounts from analytical trace levels to 750 pg, some of them with 2R configuration, the first observation of this in a pine sawfly species.

Gilpinia socia The acetate of (2S,3R)-3-methylpentadecan-2-ol gave the strongest response in EAG recordings, followed by the acetate of (2S,3R,7R)-3,7-dimethylpentadecan-2- ol, the propionate of (2S,3R)-3-methylpentadecan-2-ol, and the acetate of (2S,3R,7S)-3,7-dimethylpentadecan-2-ol (Hedenström et al. 2009). The following alcohols were found in female body extracts: (2R*,3S*,7R/S)-3,7-dimethylpentadecan-2-ol (1-8 ng/female), (2R*,3R*,7R/S)-3,7-dimethyl-pentadecan-2-ol (2 ng/female) (2R*,3S*)-3-methylpentadecan-2-ol (0.7-4 ng/female), and (2R*,3R*)-3- methylpentadecan-2-ol (1 ng/female).

Macrodiprion nemoralis The acetate of (2S,3R,7R,9S)-3,7,9-trimethyltridecan-2-ol is the main component of the sex pheromone (Wassgren et al. 2000). About 0.8 ng per female of the alcohol precursor was found in body extract. A mixture of all 16 stereoisomers of the acetate caught a large amount of males, indicating no antagonistic effect of any isomer.

Microdiprion pallipes Field tests and EAG recordings indicate that the propionate of (2S,3S,7S,11R)- 3,7,11-trimethyltridecan-2-ol is the main sex pheromone component (Bergström et al. 1998; Östrand et al. 2003). About 1.5 ng/female of the isomer has been found in female body extract (Bergström et al. 1998).

13 Neodiprion abbotti The propionate of 3,7-dimethylpentadecan-2-ol obtained from an extract of N. sertifer gave the strongest EAG response, when compared with the acetylated extract from of N. sertifer, the acetate and propionate of D. similis extract, and synthetic acetate and propionate of 3,7-dimethylpentadecan-2-ol (Jewett et al. 1976).

Neodiprion abietis The propionate of 3,7-dimethylpentadecan-2-ol gave a stronger EAG response than the acetate (Jewett et al. 1976).

Neodiprion dailingensis Both the acetate and propionate of (2S,3R,7R)-3,7-dimethylpentadecan-2-ol were highly attractive to males, the propionate caught a little more, but with no significant difference (Anderbrant et al. 1997).

Neodiprion dubiosus A 1:1 mixture of the propionates of (2S,3R,7R)- and (2S,3R,7S)-3,7- dimethylpentadecan-2-ol caught most males, pure (2S,3R,7R) was also attractive but pure (2S,3R,7S) showed very little attraction (Kraemer et al. 1984). Propionates containing (2S,3R,7S) gave the strongest EAG response.

Neodiprion lecontei The acetate of (2S,3S,7S)-3,7-dimethylpentadecan-2-ol is the main component of the sex pheromone (Kraemer et al. 1981). In field test by Matsumura et al. the acetate of (2S,3S,7S/R)-3,7-dimethylpentadecan-2-ol was the only active isomer blend, and was shown to be equally effective as females in trap experiments by Wilkinson et al. (Matsumura et al. 1979; Wilkinson et al. 1982). About 7 ng/female of (2S,3S,7S)-3,7-dimethylpentadecan-2-ol was found in extract (Paper VI).

Neodiprion nanulus nanulus The acetate of (2S,3S,7S)-3,7-dimethylpentadecan-2-ol is the main component of the sex pheromone, with about 2 ng/female of the alcohol precursor found in extract (Olaifa 1987). In field test Kraemer et al. found that the propionate of (2S,3S,7S) and acetate of (2S,3S,7R) were just as attractive as the acetate of (2S,3S,7S) (Kraemer et al. 1983).

Neodiprion nigroscutum Samples containing the propionate of (2S,3R,7S)-3,7-dimethylpentadecan-2-ol gave the strongest response in EAG-recordings (Kraemer et al. 1984).

14 Neodiprion pinetum The acetate of (2S,3S,7S)-3,7-dimethylpentadecan-2-ol is the main component of the sex pheromone (Kraemer et al. 1979; Kraemer et al. 1981), with (2S,3R,7R) acting as a synergist (Olaifa et al. 1988). A 1:2 ratio of the isomers was the most attractive blend i field test. About 10 ng/female was found of the alcohol precursor in extract, with a major peak of (2S,3S,7S) and a minor of (2S,3R,7R/S) (Olaifa et al. 1988).

Neodiprion pratti banksianae A mixture of the acetates of (2S,3S,7S)- and (2S,3R,7R)-3,7-dimethylpentadecan- 2-ol in an ratio of 5:1 attracted most males in field test (Olaifa et al. 1984). According to Kraemer et al. it was instead a 1:1 mixture of the propionates of (2S,3R,7R) and (2S,3R,7S) that was most attractive in field test (Kraemer et al. 1983).

Neodiprion pratti paradoxicus A 1:1 mixture of the propionates of (2S,3R,7R)- and (2S,3R,7S)-3,7- dimethylpentadecan-2-ol attracted most males in field test (Kraemer et al. 1983).

Neodiprion pratti pratti The propionate of (2S,3R,7R)-3,7-dimethylpentadecan-2-ol attracted most males in field test (Kraemer et al. 1983). A 1:1 mixture of the propionates of (2S,3R,7R) and (2S,3R,7S) gave the strongest respons in EAG-recording.

Neodiprion rugifrons A 1:1 mixture of the propionates of (2S,3R,7R)- and (2S,3R,7S)-3,7- dimethylpentadecan-2-ol caught most males (Kraemer et al. 1984). Propionates containing (2S,3R,7S) gave the strongest EAG response.

Neodiprion sertifer The acetate or the propionate of (2S,3S,7S)-3,7-dimethylpentadecan-2-ol is the main component of the sex pheromone (Figure 8) (Kikukawa et al. 1983; Olaifa et al. 1987; Tai et al. 1992). Both field tests and EAG-recordings showed equal attractiveness and response for the two esters (Kraemer et al. 1983). Field tests showed an antagonistic effect of (2S,3R,7R) (Anderbrant et al. 1992), increasing from Japan to Europe and Canada, but in Siberia it was synergistic (Anderbrant et al. 2000; Anderbrant et al. 2010). Tests with (2S,3R,7S) gave results with a similar pattern. Female body extracts contained 5-20 ng/female of (2S,3S,7S)-3,7- dimethylpentadecan-2-ol (Wassgren et al. 1992; Bång et al. 2011). Wassgren et al. also found minor amounts (2-5%) of the alcohol analogues with a carbon chain length of 13, 14, and 16, with the first two giving EAG response. None of the minor component showed any effect in field test (Wassgren et al. 1992).

15 O

O CH3

CH3

CH3 CH3 Acetate of (2S,3S,7S)-3,7-dimethylpentadecan-2-ol O

CH3 O

CH3

CH3 CH3 Propionate of (2S,3S,7S)-3,7-dimethylpentadecan-2-ol

Figure 8. The pheromone of Neodiprion sertifer.

Neodiprion swainei The propionate of (2S,3S,7S)-3,7-dimethylpentadecan-2-ol attracted most males in field test (Kraemer et al. 1984). EAG-recordings showed the strongest response to the propionate of (2S,3S,7S) and propionates containing (2S,3R,7S).

Neodiprion taedae linearis The acetate and the propionate of (2S,3S,7S)-3,7-dimethylpentadecan-2-ol attracted most males in field test (Kraemer et al. 1983). These gave also the strongest response in EAG-recordings.

1.5. Bicyclus anynana (Squinting Bush Brown)

The genus Bicyclus (“bush-browns”) belongs to the order Lepidoptera, family Nymphalidae and subfamily Satyrinae. It is an endemic African (sub-Sahara) genus with about 80 species (Nieberding et al. 2008). The Bicyclus species are known for the seasonal polyphenism of their wing pattern, with large eyespots during the rain-season and small or absent spots during the dry season. The size of the eyespots is determined by the temperature during the larvae period. Bicyclus anynana has been a popular model for the study of life history evolution, morphology, mating choice and genetics. It has a suitable size and is readily reared in captivity (Robertson and Monteiro 2005; Costanzo and Monteiro 2007; Brakefield et al. 2009).

16 Not only the eyespots differ between the dry and wet seasons, the mating behaviour also changes. During the dry period it is mostly the female courting the male, but in the wet season it is the opposite (Prudic et al. 2011).

In Bicyclus anynana, the sex pheromone is produced by the male in its fore- and hind wing androconia. The pheromone is a mixture of (Z)-9-tetradecenol, hexadecanal and (2R,6R,10R)-6,10,14-trimethylpentadecan-2-ol, see more in Section 4.2.3. (Nieberding et al. 2008). The tetradecenol is mainly produced in the fore wings, hexadecenal in the hind wings, and pentadecan-2-ol in both the fore- and hind wings.

1.6. Euglossa (Orchid bees)

The genus Euglossa belongs to the group Euglossini, orchid bees (order Hymenoptera, family Apidae). It is a group of more than 200 brightly coloured species occurring in lowland forests of the Neotropics. They are important pollinators, responsible for 10% of the pollination of the Neotropical orchid flora (Eltz et al. 2005; Ramírez 2009).

The male bees are unique in that they collect fragrances from orchid flowers and other sources, like decaying wood and fungi. The fragrances, mostly sesquiterpenes, monoterpenes and aromatics, are stored in hair-filled pouches in their enlarged hind tibiae. The pouches will over time become large and contain a complex mixture of volatile substances (Eltz et al. 2003; Ramírez 2009; Ramírez et al. 2010). The mixture can be stored for a long time and is finally released during courtship at special display territories. Although there is no proof, it is believed that this blend of fragrances is used for mate recognition and choice (Eltz et al. 2005; Zimmermann et al. 2009). These blends are species-specific and surprisingly independent of the type of habitat. Different populations of a species in different environments will contain the same major compounds (Zimmermann et al. 2006; Ramírez et al. 2010). One of the largest fragrance molecules found in the tibiae of orchid bees is (6R,10R)-6,10,14-trimethylpentadecan-2-one (Eltz et al. 2010). It is believed that this kind of low-volatile compounds functions as “base-notes” in the complex mixture of fragrances.

17 2. ANALYTICAL METHODS 2.1. Antennal response

Electroantennogram (EAG) is a method for measuring the response of an insect antenna to a chemical substance. A stream of air, containing a compound, is passed over an antenna, which is connected to a pair of electrodes. Response to a pheromone generates a difference in potential between the electrodes, which is amplified and recorded (Jones and Oldham 1999). A drawback with this method is that it requires pure chemicals.

A development of EAG is gas chromatography coupled with electroantennographic detection (GC-EAD). The sample is first passed through a GC column, and then split between the EAD (with the antenna) and the GC detector (for example FID or MS). This enables one to analyse a mixture of substances and identify those that generates a response from the insect antenna (Jones and Oldham 1999; Leal 2005).

It is also possible to measure the response of a single sensillum with SSR (single sensillum recording) or a single cell (neuron) with SCR (single cell recording). The latter gives more detailed information because the receptor cells are specialised for different pheromone components (Jones and Oldham 1999; Leal 2005). The different cells can even discriminate between stereoisomers of the same substance, where one can be the sex pheromone and another an antagonist (see Section 1.3.).

An antennal response does not guarantee that a substance is a pheromone. To confirm pheromone activity field trials must always be used. Many times, a substance generating an antennal response can either be inactive or even inhibit catches in pheromone traps (Jones and Oldham 1999).

2.2. Collection of volatiles

One way to collect the pheromone released from insects is to keep some insects in a chamber with a flow of clean air passing through. The volatiles are collected from the air stream on an adsorbent, for example activated charcoal or a porous polymer phase, and then removed from the adsorbent by solvent extraction (Jones and Oldham 1999; Millar 2005). In an early method, the air stream was collected in a condensing coil, kept at a temperature of -60 °C (Jones et al. 1965).

18 In another method Casida et al. kept the insects in a jar together with a filter paper for 5-7 days. Then they rinsed the filter paper and the inside walls with diethyl ether (Casida et al. 1963).

Another method is to use solid-phase microextraction (SPME). A fused silica fibre with a solid phase coating (or uncoated) adsorbs the analyte from e.g. the air. The collected analyte is then desorbed thermically or with a solvent (Arthur and Pawliszyn 1990; Jones and Oldham 1999). To collect the volatiles the SPME fibre can either be placed in a cage with an insect or wiped directly on the insects (Millar 2005). The fibre is then placed directly in the heated injector port of the GC for analysis. Although this method doesn’t isolate the active compounds or collects material for bioassays, it is an excellent method for detecting emitted volatiles from insects. SPME is also a useful technique to follow continuous changes in the production and release of semiochemicals and how the changes depend on different parameters (Francke and Dettner 2005). SPME is a fast, simple, solvent- free method that causes minimal interference with the samples, which is important when samples are collected from living organisms (Musteata and Pawliszyn 2007). A drawback is that the collected substances do not always give the whole picture of what is emitted from the source. The properties of the solid phase on the fibre determine what is collected.

2.3. Extraction and purification

The extraction of pheromone content in insects is normally done with an organic solvent. To extract the wings of the butterfly Bicyclus anynana 15 min in hexane was sufficient, without any need of further purification prior to analysis (Nieberding et al. 2008). A longer time is normally needed for extraction of larger parts, or whole insect bodies. Casida et al. soaked body parts with sodium sulphate in diethyl ether (Casida et al. 1963). For extracting whole bodies of pine sawflies Kikukawa et al. used methanol with overnight reflux, or overnight immersal in diethyl ether, (Kikukawa et al. 1982; Kikukawa et al. 1983). Wassgren et al. did an experiment with different solvents: ethyl acetate, dichloromethane, hexane, diethyl ether:hexane (1:1), and ethyl acetate:hexane (1:1). The conclusion was that ethyl acetate is the most efficient extraction medium (Wassgren et al. 1992). Extraction with ethyl acetate in 72 h at room temperature has become a common method for extracting the sex pheromone precursor alcohols present in whole pine sawfly bodies (Wassgren et al. 1992; Bergström et al. 1995; Hedenström et al. 2006; Hedenström et al. 2009). The similarity in polarity of ethyl acetate and the analyte (secondary alcohols) makes it a suitable extraction solvent. One must, however, consider that co-eluted contaminants can be extracted simultaneously. A less polar

19 solvent may result in lower recovery, but due to a cleaner extract it may result in an increased detection limit.

An internal standard is normally added after the extraction as a control of the method and for quantification. It should have similar properties to those of the bioactive compound.

For the analysis of extracts from whole body insects, a purification step is normally needed. An important reason is to remove large amounts of body lipids, which can cause problems in later separation and analysis steps (Francke and Dettner 2005). The extracts also contain large amounts of acids that can reduce the performance and shorten the lifetime of the analytical columns. One can remove most of the acids by washing the extract with a basic alkaline solution and is normally combined with gradient elution on a silica column. Often, the pheromone or pheromone precursor are present in very low amounts in a complex matrix. Therefore, to be able to detect them, the sample should be purified as much as is possible.

The silica column can be self-packed with activated silica (Wassgren et al. 1992) or a pre-packed solid phase (SPE) silica column (Bergström et al. 1995; Bång et al. 2011). A step-wise gradient elution with an increasing polarity eluent is then used. Pentane with 0-10% of ethyl acetate in steps of 1% is common, but higher amount of the polar solvent are sometimes needed. To determine which fractions to collect, a reference sample of the analyte (or a reference compound with the same polarity) in the extract is chromatographed and the fractions analysed.

When the fractions containing the desired analyte have been collected and combined, they are normally washed with an alkaline solution to remove co-eluted acids. The organic phase is dried and concentrated before analysis. Concentration of the sample is often important because the pheromone analyte can be in the pg to ng scale (Wassgren et al. 1992; Bergström et al. 1995; Bång et al. 2011).

A normal procedure for analysing a pine sawfly female whole body extract is:

• Extraction of whole body insects in ethyl acetate for 72 h. • Addition of internal standard. • Purification by gradient elution on a silica column. • Washing with KOH solution and drying with sodium sulphate. • Concentration and analysis on GC-MS.

See Section 4.1.1. for more details on purification methods.

20 2.4. Separation of stereoisomers using GC

Because pheromones are volatile compounds they are well suited for gas chromatography (GC). Many of them are diastereomers, like E/Z isomers, and the isomers can often be separated on an ordinary GC column (achiral stationary phase) with a polarity equal to the analyte (Jones and Oldham 1999). For enantiomers and some diastereomers separation on an ordinary GC column may not be possible and other methods need to be applied.

Normally, one column is enough to solve a separation problem, but sometimes the resolution power of two different columns is needed. This can be done by connecting two different column in a series, or splitting the sample between two columns in parallel (Jones and Oldham 1999). Another method is to use two- dimensional GC (2D-GC), where a peak that is not resolved on the first column is cut out and sent to a second column in a separate oven, so called heart cutting (Jones and Oldham 1999; McNair and Miller 2009).

2.4.1. GC columns with a chiral stationary phase

The easiest way to separate enantiomers is to use a column coated with a chiral stationary phase (CSP). Using (L)-N-trifluoroacetyl isoleucine lauryl ester as the stationary phase, Gil-Av et al. were the first to perform a direct separation of enantiomers using chiral capillary GC (Gil-Av et al. 1966b; Schurig 2005).

The CSP for GC can be divided into three groups:

• Chiral amino acid derivatives • Chiral metal complexes • Cyclodextrin derivatives

The chiral analyte interacts via hydrogen bonding to amino acid derivatives or by coordination to metal complexes in the stationary phase (He and Beesley 2005). Chiral metal complex phases are limited by a low temperature range and are rarely used nowadays. Depending on the substituents, the cyclodextrin derivatives form either an inclusion complex or interacts with the analyte via other mechanisms.

The chiral amino acid phases are mainly used for specific applications, for example analysis of amino acids in natural products (He and Beesley 2005). They have also been used for pheromone analysis, where König et al. developed a method for chiral alcohols using a XE-60-(S)-valine-(S)-α-phenylethylamide

21 column. The separation was enhanced by first turning the alcohols into isopropyl urethanes (König et al. 1982). This method has also been used for the separation of the chiral precursor alcohols of the pine sawflies pheromone. Separation of all the stereoisomers was, however, not possible (Högberg et al. 1990; Wassgren et al. 1992; Bergström et al. 1995; Wassgren and Bergström 1995; Wassgren et al. 2000).

Cyclodextrin derivatives have become the dominating chiral phases, accounting for 90% of the separations (Schurig 2001; He and Beesley 2005). Cyclodextrin are cyclic oligosaccharides, with six, seven or eight glucose units, named with the Greek letters α, β and γ (Juvancz and Szejtli 2002). They have a truncated cone- shaped structure, with three hydroxyl groups on each glucose unit that can be substituted with different groups (Figure 9). Chiral recognition occurs not only with the whole or a part of the molecule on the inside of the cyclodextrin (inclusion), but also on the outside (Krupcík et al. 1994). The selectivity depends on the percentage and size of the cyclodextrin (α, β or γ), the degree of substitution, and the type of substituents (He and Beesley 2005). β-Cyclodextrin is the most commonly used, as it separates a large amount of stereoisomers.

O HO O O OH HO OH O OH HO HO O OH O O

HO HO O OH OH O HO OH HO O OH O OH O O HO

HO O OH OH 3

HO 2

6 O OH

Figure 9. β-Cyclodextrin.

22 Substituents at the 3 position (Figure 9) are expected to have the most influence on selectivity. They orient in parallel to the cyclodextrin axis and with their steric features have a strong impact on the interactions with the analyte (Juvancz and Petersson 1996). For most enantiomeric mixtures, acetylation of the 3 position generally gives good selectivity, but acetylation at the 6 position has a negative effect. Substituents at the 6 position do not directly interact with the analytes, but affects the shape of the cyclodextrin. Because substituents at the 2 position are orientated outside the wide opening of the cyclodextrin cone, it does not have any major influence on chiral selectivity (Juvancz and Petersson 1996). Some common substituent groups on modified cyclodextrins are methyl, trifluoroacetyl, propionyl butyryl and tert-butyldimetylsilyl (Jones and Oldham 1999).

Pure cyclodextrin decomposes instead of melting upon heating. Therefore, to lower the melting point and allow for a liquid state, and to increase its efficiency, the cyclodextrin is normally modified and dissolved in a polydimethylsiloxane phase. By covalent bonding of these components it is possible to use up to 50% (w/w) cyclodextrin (Juvancz and Petersson 1996; Schurig 2001; Juvancz and Szejtli 2002). This chemical bonding also improves efficiency and selectivity, and gives the column a longer lifetime.

In chiral separation, method development normally starts with a permethylated β-cyclodextrin column (methyl groups on position 2, 3 and 6) because this has the widest separation range. Enantiomeric separation of chiral alkanes is difficult due to absence of functional groups. This is, however, sometimes possible to achieve with modified cyclodextrin columns (Sicoli et al. 2009).

2.4.2. Pre-column derivatisation

Achiral derivatisation of the analyte can be used to make the analyte less polar and more volatile. Elution at lower temperatures often results in better selectivity, and introduction of additional structure and new functional groups can also improve separation (Juvancz and Petersson 1996).

The main purpose of pre-column chiral derivatisation of chiral compounds is to turn them into diastereomers, which should separate on a column with an ASP. Already in 1966 Gil-Av et al. demonstrated that this method worked for secondary alcohols (Gil-Av et al. 1966a). A chiral reagent is often tens of times cheaper than a column with a CSP, and ASPs are also more robust and long-lived. The small differences in physical properties of the diastereomers are often enough to separate

23 them on a readily available and cheap column with an ASP. Derivatisation of the analyte with rigid and bulky groups can also enhance separation.

Functional groups on the analyte, like OH, NH2, COOH, SH, NH and C=O, are necessary for derivatisation, and additional groups on the chiral reagent can also increase detector response and add sites for hydrogen bonding (Srinivas 2004; Stalcup 2010). Chiral alcohols can be derivatised with carboxylic chlorides, sulfonyl chlorides and anhydrides. The reaction requires anhydrous conditions and a basic catalyst, such as pyridine. The purpose of the pyridine is also to neutralise the acid formed by the reaction. Sterically hindered secondary alcohols are often derivatised with acyl chlorides because of their high reactivity (Zhou et al. 1994).

Examples of acyl chlorides that have been used to derivatise chiral alcohols in pheromone analysis are (2S)-2-acetoxypropionyl chloride (Doolittle and Heath 1984; Slessor et al. 1985; Jones and Oldham 1999; Bång et al. 2011), (R)-trans- chrysanthemoyl chloride (Murano 1972; Brooks et al. 1973; Brand 1985; Jones and Oldham 1999; Nieberding et al. 2008), Mosher’s acid chloride (Dale et al. 1969; Jones and Oldham 1999), pentafluorobenzoyl chloride (Wassgren and Bergström 1995), and 2-naphthoyl chloride (Bång et al. 2011) (Figure 10).

O H3C Cl H3C O Cl CH3

O CH3 H3C CH3

(2S)-2-Acetoxypropionyl chloride (R)-trans-Chrysanthemoyl chloride

CF OCH O H3CO 3 F3C 3

Cl Cl Cl

O O

(R)- and (S)-MTPA chloride (Mosher's acid chloride) 2-Naphthoyl chloride

Figure 10. Some of the acyl chlorides used for derivatisation of alcohols.

24 Isopropyl isocyanate has also been used for long-chained and cyclic secondary alcohols (König et al. 1982; Högberg et al. 1990), and 1-phenylethyl isocyanate has shown to be an excellent derivatisation agent for chiral secondary alcohols with remote stereogenic centers (Pereira et al. 1970; Habel et al. 2007). For diastereomeric ester formation of chiral alcohols Westley et al. used mentyl chloroformate (Westley and Halpern 1968) (Figure 11).

CH3 CH3

CH 3 NCO NCO

H3C NCO

Isopropyl isocyanate (R)- and (S)-1-Phenylethyl isocyanate

H3C CH3 H3C CH3

O O O O

Cl Cl

CH3 CH3

(1R)- and (1S)-Menthyl chloroformate

Figure 11. Isocyanates and chloroformates used for derivatisation of alcohols.

2.5. Detection and identification

Flame ionization detection (FID) is a common detector in GC analysis of pheromone compounds because it is universal and has a linear response over a large concentration range (Jones and Oldham 1999). Compared with FID, electron capture detection (ECD) and nitrogen phosphorus detection (NPD) are more sensitive but also more specific and requires certain functionalities in the analyte. ECD detectors are very sensitive to compounds containing electronegative groups, such as halogens and nitro groups, while NPD detectors have similar specificity

25 and sensitivity towards analytes with phosphorus and nitro groups (Srinivas et al. 1995; Jones and Oldham 1999).

FID has been used for the esters and underivatised secondary alcohol precursors of pine sawfly pheromone, and in GC-EAD experiments (Olaifa et al. 1987; Olaifa et al. 1988; Högberg et al. 1990; Wassgren et al. 1992; Bergström et al. 1995). For the analysis of secondary alcohol derivatives, ECD was used for pentafluorobenzoates (Bergström et al. 1995; Wassgren and Bergström 1995), and NPD for isopropyl carbamates (Högberg et al. 1990; Wassgren et al. 1992).

The major drawback with FID, ECD and NPD detectors is that they give no identification of the analytes, they only show a peak (or absence of a peak) at a certain retention time. In addition to the retention time of a peak, GC coupled with mass spectrometry (GC-MS) also gives a mass spectrum, which gives a fingerprint of the compound that can be used for comparison with a standard or in library searches (Jones and Oldham 1999). Furthermore GC-MS has a very high sensitivity. Hence, this is the method of choice in pheromone analysis.

Ionization of the sample in the MS detector is normally made with electron impact (EI) or chemical ionization (CI). EI is a “hard” ionization that gives a lot of fragments, suitable for structure elucidation and library searches. CI is softer, with few fragments and often a distinct molecular ion. A combination of EI and CI is often necessary for identifying new and unknown compounds.

When higher sensitivity is needed (low amounts of analyte in complex samples), selected ion monitoring (SIM) is often used. Instead of scanning a large area of ions (full scan), only a few ions are selected. Thereby the detection time for these ions are increased, so does the signal to noise ratio (Jones and Oldham 1999). Generally, compared with a full scan, SIM gives an improvement of the detection limit by 10 to 100 times (van Asten 2002). The selected ions shall be typical for the analyte and enough for identification, and they should preferably be not present in the background (van Asten 2002; Bång et al. 2011). The working range of the different GC detectors are shown in Figure 12 (McNair and Miller 2009).

26 FID

ECD

NPD (N)

NPD (P)

MS SIM Full scan

fg pg ng µg mg

Figure 12. Working range of different GC detectors.

GC coupled to Fourier transform infrared spectroscopy (GC-FTIR) has not been widely used, but can give important information about functional groups and double bond geometry (Jones and Oldham 1999; Eltz et al. 2010). When a light-pipe interface is used, the detection limit is down to 10-50 ng (Heaps and Griffiths 2005).

Nuclear magnetic resonance (NMR) is rarely used in the analysis of pheromones because it requires pure samples and is not as sensitive as MS (nanogram amounts at the best, normally microgram amounts required). The advantage of NMR compared to MS is that it gives a much more predictable spectra and thereby a more secure identification (Francke 2010).

The final step in the identification of an unknown bioactive compound is its synthesis with the proposed stereostructure, and comparison of its analytical data with that of the natural compound.

27 3. OBJECTIVES

The goal with this work has been to develop methods for the analysis of pheromones and pheromone precursors in extracts of different insect species.

The focus has been on:

• Optimisation of the purification method. • Separation of stereoisomers of secondary alcohols. • Application of the methods on extracts.

28 4. RESULTS 4.1. Method development

4.1.1. Purification (Paper IV)

During workup of natural extracts the combination of organic solvents and plastic equipment often leads to contamination of the samples with plasticizers. When one works with analytes in low concentrations and with sensitive analytical instruments, such as GC-MS, plasticizers can cause problems. The plasticizers appear in the chromatogram as one or several large peaks with characteristic mass spectra. Commonly used plasticizers are various phthalates and with electron impact (EI) ionization they always give a dominant fragment of m/z 149 (Shen 2005). To avoid this problem, solid phase extraction (SPE) columns of glass or with Teflon coating were tested. Compared with ordinary SPE columns both showed significantly lower amounts of contamination peaks, and with pre-conditioning of the columns with solvent (pentane and ethyl acetate), these peaks were reduced even further. The Teflon coated SPE columns were chosen for further use, but the glass columns worked just as well.

For the purification of extracts, the selected SPE column was used with a gradient elution method developed by Bergström et al. (Bergström et al. 1995; Wassgren and Bergström 1995; Östrand et al. 2003). The method was optimised to elute all of the analytes of interest, with a minimum of co-eluted contaminants. The SPE column was preconditioned with three column volumes each of ethyl acetate, 10% ethyl acetate in pentane, and pentane. This reduced the contamination from the column itself to a minimum. A standard solution of 3,7-dimethyltridecan-2-ol and 2-tridecanol was then applied on the column and eluted with a gradient elution of 0 to 15% of ethyl acetate in pentane, in steps of 1%. The elution volume for each fraction was 150 µl for 100 mg columns and 750 µl for 500 mg columns. The alcohol standards eluted in fraction 9-11 (9-11% ethyl acetate in pentane added). Twice the elution volume was chosen for these three fractions to make sure that all of the alcohol eluted. Because many pheromone components occur in very low amounts close to the detection limit, the collected and merged fractions needs to be concentrated (from 0.9-4.5 ml to 50 µl). This means that co-eluting contaminants will also be concentrated, which can interfere with the analyte.

The collected fractions were combined, evaporated to dryness under a stream of argon and dissolved in 1 ml pentane. The solution was washed by adding 200 µl of 0.2 M KOH and then mixing on a vortex mixer. Care must be taken in this step because, with dirty extracts, the analyte can sometimes be trapped in the water phase inside micelles formed by anions of fatty acid salts. The organic layer was

29 dried by passing it through a Pasteur pipette containing sodium sulphate and a piece of glass wool, and additional pentane (1 ml) was added to elute all of the sample. The glass wool was pre-cleaned with pentane and dried a few minutes in an oven before use. The collected pentane solution was evaporated to dryness under a stream of argon and dissolved in 50 µl cyclohexane for further analysis.

The general principle of the method can be seen in Figure 13. It has also been tested with other alcohols which all eluted in the same fractions as the standard alcohols, and with ketones and esters which eluted in earlier fractions. Adequate reference samples should always be analysed first to determine the right fractions to collect. When purifying whole body extracts, a 100 mg column was chosen for 1- 10 insects and a 500 mg column for larger samples. For extracts containing more than 50 insects, the extract was either purified twice or divided into smaller parts, which were purified separately (Paper IV). All solvents used were of spectrophotometric grade or higher. Due to its higher boiling point (compared with pentane or similar) cyclohexane was found suitable for long-time storing and was usedboth as the final solvent after purification or derivatisation, and for diluting and storing reference samples.

Pre-cleaning of SPE column with EtOAc and pentane

Application of sample on column

Gradient elution with 0-11% EtOAc in pentane

Collection of fraction 9-11 and evaporation to dryness

Dissolving in pentane and washing with 0.2 M KOH

Drying of the organic layer with Na2SO4

Evaporation to dryness and dissolving in cyclohexane

Figure 13. Purification method for pheromone precursor alcohols in extracts.

4.1.2. Derivatisation (Paper IV and VI)

Different derivatisation methods were optimised with solutions of synthetic alcohol standards to be used in separation experiments with different GC columns (Section 4.1.3.).

(2S)-2-Acetoxypropionyl chloride The first approach was to use (2S)-2-acetoxypropionyl chloride on a standard mixture of (2R*,3S*,7R/S)-3,7-dimethyltridecan-2-ol (Figure 14). The derivatisation procedure was based on a method by Doolitle et al., but optimised and down- scaled to suit forthcoming analysis of extracts (Doolittle and Heath 1984).

OH O

O O + Cl CH3

H CH3 CH CH3 CH3 3

Pyridine (dry) O

O O O

H CH3 CH3 CH3

CH3 CH3

Figure 14. Derivatisation of chiral alcohols with (2S)-2-acetoxypropionyl chloride

Optimisation was done by varying the following parameters:

• molar ratio between the alcohol and chloride • reaction temperature • reaction time

The results are shown in Table 1 and 2. A molar ratio of 1:10, a temperature of 80 °C and a reaction time of 30 min were chosen for further analysis. These experiments were done with a solution of 3,7-dimethyltridecan-2-ol. In a pine sawfly extract, the amount of pheromone precursor alcohol and internal standard together rarely exceeds 1 µg. But because the amount of alcohol is unknown before

31 analysis, and there could be other substances in the extract reacting with the chloride, the alcohol:chloride ratio was kept much larger than 1:10 to ensure that all of the alcohol was esterified. This is important as different diastereomers react at different rates.

Table 1. Amount of underivatised alcohol left after derivatisation with different molar ratios of alcohol:chloride (80 °C, 45 min).

1:1 1:3 1:10 65% 19% 0.3%

Table 2. Amount of underivatised alcohol left after derivatisation at different temperatures and reaction times (molar ratio alcohol:chloride 1:10).

10 min 30 min 45 min 60 min 40 °C - 39% - 35% 80 °C 1.2% 0.3% 0.3% 0.2% - not investigated

The optimised method used for subsequent separation experiments and analysis of extracts is as follows:

• 65 ul of pyridine solution (1% (v/v) pyridine in cyclohexane) is added to the sample dissolved in 50 ul in cyclohexane in a GC vial. • 100 µl of chloride solution (1% (v/v) (2S)-2-acetoxypropionyl chloride in dichloromethane) is added under argon, and the sample vial is heated in an oil bath at 80 °C for 30 min. • The solution is evaporated to dryness by passing argon through the sample vial in the oil bath. • The residue is dissolved in 1 ml pentane and 200 µl of 1 M HCl is added. • After mixing, the organic layer is separated and dried by passing it through a column of sodium sulphate. • The solution is evaporated to dryness and dissolved in 50 µl cyclohexane.

See Paper IV for further details. The separation experiments with different alcohols and GC columns, using this derivatisation method, are presented in Section 4.1.3.

32 (R)-trans-Chrysanthemoyl chloride Because (R)-trans-chrysanthemoyl chloride is not commercially available it has to be synthesized from the acid. The method of Brooks et al. was used, with minor modifications (Brooks et al. 1973). To ensure that all of the alcohol was esterified the original molar ratio between alcohol and chloride of 1:3 was increased to 1:10. An increased reaction temperature from 40 °C to 80 °C did not improve the result, and the same for a reaction time of 2 h instead of 1 h. Despite the use if pure standard solutions during derivatisation a lot of contamination peaks were generated. To remove these, the last steps from the previuos method [(2S)-2- acetoxypropionyl chloride] were added to the method. These steps removed most of the contaminations.

The final method used for further experiments:

• 2 µl of (R)-trans-chrysanthemic acid is added to 100 µl of dry toluene under argon. • 200 µl thionyl chloride is added and the solution is kept under argon on an oil bath at 60 °C for 1 h. • Excess of thionyl chloride is removed with argon and an increased temperature of the oil bath to 70-80 °C. • The formed (R)-trans-chrysanthemoyl chloride is dissolved in 1 ml of dry toluene for immediate use. • 200 µl of the freshly made chloride solution is added to the sample solution and the solution is kept under argon on an oil bath at 40 °C for 1 h. • The solution is evaporated to dryness with argon while heated 70-80 °C on the oil bath. • The sample is dissolved in 1 ml pentane, and 200 µl of 1 M HCl is added. • After mixing, the organic layer is separated and dried by passing through a column of sodium sulphate. • The solution is evaporated to dryness and dissolved in 50 µl cyclohexane.

33 Other acid chlorides For further derivatisation experiments with secondary alcohols the following acid chlorides were used:

• Benzoyl chloride • (R)-(–)- and (S)-(+)-MTPA-Cl (Mosher’s acid chloride) • 1- and 2-Naphthoyl chloride • 2,3,4,5,6-Pentafluorobenzoyl chloride • Phenylacetyl chloride

The method described for pentafluorobenzoyl chloride by Wassgren et al. was used, with some modifications (Wassgren and Bergström 1995). A reaction time of 5 min was sufficient for all of the chlorides, except for 2-naphthoyl chloride. Probably due to steric hindrance and/or to effects from the electron rich aromatic rings, this acid chloride reacted much slower, and thus, the reaction time was increased to 60 min. In all experiments the molar ratio between alcohol and chloride was 1:10 or higher.

The method was as follows:

• 2 µl of the chloride is dissolved in 20 µl pyridine. • 3 µl of the freshly made chloride solution is added to a GC vial containing the alcohol sample. • The solution is kept on an oil bath at 70 °C for 5 min (60 min for 2-naphthoyl chloride). • 10 µl MeOH is added to the vial, which is kept on the oil bath for an additional 5 min. • The solution is evaporated to dryness under a stream of argon on the oil bath. • The residue is extracted with 2 x 100 µl pentane and washed with 2 x 10 µl H2O (Milli-Q). • The organic layer is separated and dried with sodium sulphate. • The solution is evaporated to dryness and dissolved in 50 µl cyclohexane.

34 Menthyl chloroformate (1R)-(–)- and (1S)-(+)-Menthyl chloroformate were employed by using the same method as for (2S)-2-acetoxypropionyl chloride. The only difference was an increase of the 1% dichloromethane solution volume from 100 to 175 µl.

1-Phenylethyl isocyanate 100 µl of a 1% (v/v) solution in toluene of (R)- and (S)-1-phenylethyl isocyanate was added to an alcohol solution and kept in a vial in an oil bath at 120 °C for 1 h. The product was analysed without any further purification.

35 4.1.3 GC separation (Paper IV and VI)

For method development, and to identify the alcohol derivatives and to detect any remaining underivatised alcohol the samples were first analysed with a fast method (10 °C/min) and full scan. Then the separation was improved by using a less steep gradient (down to 0.01 °C/min or isocratic), and also by using SIM with 1-5 significant ions. These very long temperature programmes (10-12 h) is often necessary to get the best possible separation of the stereoisomers, and also to separate them from co-eluting contaminants that is present in extracts even after purification.

For SIM analysis of standard samples during method development, a single significant ion is often sufficient. But for secure identification, especially of more complex matrixes like extracts, 4-5 ions were selected: three arising from the derivatisation agent part and one derived from the alcohol part (two if an internal standard had been added). The presence of, and the relative size of the ions in the spectra, can be seen as a ”fingerprint” for identification. Examples of significant ions of 3,7-dimethyltridecan-2-ol derivatised with (2S)-2-acetoxypropionyl chloride and 2-naphthoyl chloride can be seen in Figure 15 and 17. The ion m/z 133 in Figure 15 is due to McLafferty + 1 rearrangement (double hydrogen rearrangement), characteristic for ester derivatives of alcohols with an aliphatic chain of at least three carbons (Smith 2004). The mechanism is shown in Figure 16.

O 115 87 O O O 133 210 H CH3 CH3 H3C CH3

CH3 CH3

Figure 15. Significant ions and their SIM spectra of (2S)-2-acetoxypropionyl chloride derivative of 3,7-dimethyltridecan-2-ol.

36 CH3 CH3 H H CH3 H δ δ R O R O R1 γ O 1 γ 1 γ a a +

β β β H3C O R2 H3C O R2 H3C O R2 α α α

H H3C H3C H H3C O b H O β β O b c γ β H C + H C + c O R2 3 γ 3 γ H3C α αO R2 H δ O R2 δ δ α H R R R 1 1 1 m/z 133

Figure 16. Mechanism of McLafferty + 1 rearrangement (path c).

The m/z 172 in the SIM spectrum shown in Figure 17 is due to McLafferty rearrangement (γ-hydrogen rearrangement) of the analyte, typical for carbonyl compounds with γ-hydrogen (McLafferty 1959; Smith 2004). The mechanism is shown in Figure 18. The reason that there is no McLafferty + 1 rearrangement for the 2-naphthoyl derivative is probably due to the electron rich aromatic rings affecting the stability of the cation radicals and/or due to steric hindrance, and thus disfavouring this rearrangement. The m/z 382 is the molecular ion.

155 127

O 172 210

H3C CH3

CH3 CH3

Figure 17. Significant ions and their SIM spectrum of 2-naphthoyl chloride derivative of 3,7- dimethyltridecan-2-ol.

37 CH3 CH3 R1 H R1 H R1 CH3 H γ O γ O γ O +

β β β H3C O R2 H3C O R2 H3C O R2 α α α

H O

O R2 α m/z 172

Figure 18. Mechanism of McLafferty rearrangement.

(2S)-2-Acetoxypropionyl chloride derivatives A standard mixture of (2R*,3S*,7R/S)-3,7-dimethyltridecan-2-ol derivatised with (2S)-2-acetoxypropionyl chloride was tested on different columns (all 30 m x 0.25 mm i.d., df = 0.25 µm): Agilent HP-1ms (non-polar), Agilent HP-5ms (non-polar), Varian FactorFour VF-23ms (polar), and Supelco β-DEX 120 (CSP). They all showed some separation of the four stereoisomers, but the FactorFour column gave the best separation and was chosen as the standard column for further method development.

The separation of the (2R*,3S*,7R/S)-3,7-dimethyltridecan-2-ol isomers could be optimised to give basline separation of the four isomers on the FactorFour column, but a mixture of all eight stereoisomers gave only partial separation of (2R,3R,7R) and (2R,3R,7S), and no separation at all of (2S,3S,7S) and (2S,3S,7R) (Figure 19).

Figure 19. Separation of derivatised 3,7-dimethyltridecan-2-ol isomers on the FactorFour VF-23ms column.

A mixture of all eight stereoisomers of 3,7-dimethylpentadecan-2-ol showed the same separation as 3,7-dimethyltridecan-2-ol, but with longer retention times (Figure 20). Diastereomers may show different detector response (respons factors) which can explain differences in peak size, as shown in Figure 19 and 20 (Srinivas 2004). However, some of the reference mixtures used in the experiments have been mixed manually from separate stereoisomer standards and may be of different concentrations, which can explain some of the differences.

Figure 20. Separation of derivatised 3,7-dimethylpentadecan-2-ol isomers on the FactorFour VF-23ms column.

39 The next approach was to try a 50 m Chrompack CP-Sil 88 (polar) column. The shapes of the peaks were improved, being more narrow and of equal size compared to the FactorFour column. The separation of (2R,3R,7R)- and (2R,3R,7S)- 3,7dimethylpentadecan-2-ol was slightly enhanced, but no baseline separation, and still no separation of (2S,3S,7S) and (2S,3S,7R) (Figure 21).

Figure 21. Separation of derivatised 3,7-dimethylpentadecan-2-ol isomers on the CP-Sil 88 column.

The CP-Sil 88 column was additionally connected in series with the FactorFour column, but this resulted in a separation and peak shape similar to Figure 20.

A 100 m long Agilent HP-88 column was then tested. It has a similar polarity and selectivity to the CP-Sil 88 column, but with a higher temperature limit. The longer column improved the separation even further, giving almost baseline separation of (2R,3R,7R)- and (2R,3R,7S)-3,7-dimethyltridecan-2-ol (Figure 22). Similar results were achieved with 3,7-dimethyltetradecan-2-ol and 3,7- dimethylpentadecan-2-ol. Neither of the two alcohol isomer mixtures could be separated to give resolution of the (2S,3S,7S) and (2S,3S,7R) isomers. See Paper IV for more details and chromatograms.

Figure 22. Separation of derivatised 3,7-dimethylpentadecan-2-ol isomers on the HP-88 column.

(2S)-2-acetoxypropionyl chloride derivatives of 3-methylpentadecan-2-ol, 3,7,9- trimethyltridecan-2-ol and 3,7,11-trimethyltridecan-2-ol were also analysed on the HP-88 column. 3-Methylpentadecan-2-ol was separated with baseline separation of all four stereoisomers without any problem, and so was all 16 isomers of 3,7,9- trimethyltridecan-2-ol. However, the 16 stereoisomers of 3,7,11-trimethyltridecan- 2-ol only separated into 7 peaks (Paper IV). This emphasizes the problem with remote stereogenic centres. 3-Methylpentadecan-2-ol and 3,7,9-trimethyltridecan-2- ol have no stereogenic centres more distant than, at the most, one carbon apart from its nearest stereogenic neighbour centre, while the other alcohols have at least one centre three carbons apart (Figure 6). Derivatisation with (2S)-2- acetoxypropionyl chloride was selected for the analysis of these sex pheromone precursor alcohols in pine sawfly extracts, because it was the one that separated most stereoisomers (Paper II, IV-VI).

Although having a similar structure to those of the previously mentioned alcohols 6,10,14-trimethylpentadecan-2-ol behaves different. Derivatisation with (2S)-2-acetoxypropionyl chloride gave separation of all eight stereoisomers. However, not all isomers gave baseline separation and a difference in peak area was observed (Figure 23). The separation of all of the stereoisomers, despite the presence of remote stereogenic centres, is probably due to the equal distribution of the centres and methyl groups along the carbon chain, in combination with a longer carbon chain (compared with 3,7,11-trimethyltridecan-2-ol). Obviously, this affects the possibility of the different stereoisomers to interact with the stationary phase of the column.

Figure 23. 6,10,14-Trimethylpentadecan-2-ol derivatised with (2S)-2-acetoxypropionyl chloride.

(R)-trans-Chrysanthemoyl chloride derivatives Derivatisation of 6,10,14-trimethylpentadecan-2-ol with (R)-trans- chrysanthemoyl chloride improved the separation and also showed a much smaller difference in area between the different peaks (Figure 24). This method was selected for the analysis of extract containing 6,10,14-trimethylpentadecan-2-ol and its ketone analogue (Paper I and III).

Figure 24. 6,10,14-Trimethylpentadecan-2-ol derivatised with (R)-trans-chrysanthemoyl chloride.

42 Derivatisation of 3,7-dimethyltridecan-2-ol with (R)-trans-chrysanthemoyl chloride did not improve the separation, compared with (2S)-2-acetoxypropionyl chloride. A mixture of all eight stereoisomers only separated into five peaks.

Other derivatives and columns One major problem to solve was the separation of the (2S,3S,7S)- and (2S,3S,7R)-isomers of 3,7-dimethyltridecan-2-ol, 3,7-dimethyltetradecan-2-ol and 3,7-dimethylpentadecan-2-ol. It is especially important for the latter alcohol, because (2S,3S,7S)-3,7-dimethylpentadecan-2-ol is the major sex pheromone precursor for several pine sawfly species (see Section 1.4.1.). A screening procedure was set up, using a mixture of (2S,3S,7S)- and (2S,3S,7R)-3,7-dimethyltridecan-2-ol in the ratio of 2:1 (for easy identification when separated). The tridecanol was chosen because it was available in lager amounts than the pentadecanol and we assumed the two alcohols to behave the same. The alcohol mixture was derivatised with different chlorides and tested on a large selection of GC columns, all but one with CSP. If any kind of separation for a certain combination of chloride and column was obtained, a mixture of all eight stereoisomers was tried (see Paper VI for details).

The only combination that could separate all eight stereoisomers of 3,7- dimethyltridecan-2-ol was (2S)-2-acetoxypropionyl chloride and the Chiraldex B- PA column. This method was also applied to the shorter and longer alcohol analogues, giving separation of all stereoisomers of 3,7-dimethylundecan-2-ol and 3,7-dimethyldodecan-2-ol, but no separation of the (2S,3S,7S)- and (2S,3S,7R)- isomers of 3,7-dimethyltetradecan-2-ol and 3,7-dimethylpentadecan-2-ol respectively.

Chiral recognition takes place by interactions, both on the inside and on the rims of the cyclodextrin, and can involve inclusion, hydrogen-bonding, dispersion forces, dipole-dipole interactions, electrostatic interactions or hydrophobic interactions (Juvancz and Petersson 1996; Schurig 2001). Sometimes one enantiomer is included in the cyclodextrin cavity while the other is excluded for steric reasons (Schurig 2001). The reason that there are no separation between the (2S,3S,7S)- and (2S,3S,7R)-isomers of 3,7-dimethyltetradecan-2-ol and 3,7- dimethylpentadecan-2-ol might be that their slightly longer carbon chains makes it equally more difficult for the two stereoisomers to fit inside the β-cyclodextrin cavity and/or connect properly to the binding sites, which is needed to discriminate between them.

43 This is (to our knowledge) the first separation of all eight stereoisomers of 3,7- dimethylundecan-2-ol, 3,7-dimethyldodecan-2-ol, and 3,7-dimethyltridecan-2-ol in a single run. Several combinations of columns and chlorides gave some degree of separation of the (2S,3S,7S)- and (2S,3S,7R)-isomers of 3,7-dimethyltridecan-2-ol, from a hint of to baseline separation. If the only goal is to separate these isomers, the best choice of those tested in this work is to use 2-naphthoyl chloride and the Varian FactorFour VF-23ms column. This combination worked just as well with (2S,3S,7S)- and (2S,3S,7R)-3,7-dimethylpentadecan-2-ol. The FactorFour column, being the only column tested with an ASP, has the advantage of being cheaper and more durable than the chiral ones. 1-Naphthoyl chloride is also an alternative, because it gives almost as good separation, but with a faster derivatisation method.

A mixture (2S,3S,7S)- and (2S,3S,7R)-3,7-dimethyltridecan-2-ol was also tested with (R)- and (S)-1-phenylethyl isocyanate. None of the two derivatives gave any separation between the (2S,3S,7S)- and (2S,3S,7R)-isomers on the FactorFour VF- 23ms column (to be published).

44 4.2. Application of methods

4.2.1. Purity of synthetic references (Paper II)

Field experiments with pheromone traps require pure substances, because low amounts of impurities can lead to erroneous conclusions. For the sex pheromone precursor alcohol of pine sawflies, both the chemical and stereochemical purity needs to be exactly known, as some stereoisomers and alcohol analogues can be either synergists or antagonists to the main component of the sex pheromone.

In the field studies presented in Paper II, the acetate of (2S,3S)-3- methylpentadecan-2-ol [(SS-)AcJ, synthesised in Japan] was an attractant for Neodiprion sertifer in Japan when tested in high amounts, but inactive in Sweden. The acetate of (2S,3R)-3-methylpentadecan-2-ol [(SR-)Ac, synthesised in Sweden] on the other hand, acted as an inhibitor in Sweden, but had very low effect in Japan. Because the purity of the standards used in these experiments was unclear, it had to be investigated.

The chemical purity of the acetate standards of 3-methylpentadecan-2-ol were estimated by analysis on GC-MS using full scan. Stereogenic purity were calculated by first reducing the esters to alcohols with lithium aluminium hydride and then converting them to diastereomeric esters with (2S)-2-acetoxypropionyl chloride. The standards were then analysed on GC-MS and the peaks identified by comparing the retention times and spectra with a known mixture of all four isomers. The column temperature was increased from 50 °C by 10 °C/min up to 100 °C, from 100 °C by 0.01 °C/min up to 106 °C, and from 106 °C by 10 °C/min up to 230 °C. SIM mode was used, with m/z = 87, 115, 133 and 224. The results are presented in Table 3 and Figure 25-27. The purities were equal or better, compared to other synthetic standards used in this and other field studies, and the minor component seemed not to influence the result.

Table 3. Purities of acetate standards of 3-methylpentadecan-2-ol.

Abbreviation Chemical purity Stereoisomericic purity Minor component (SS-)AcJ >99.9% 98.7% SS 1.3% RR (SR-)Ac >99.9% 98.6% SR 1.4% RS

Figure 25. Separation of derivatised 3-methylpentadecan-2-ol isomers.

Figure 26. GC-MS chromatogram of derivatised (2S,3S)-3-methylpentadecan-2-ol [(SS-)AcJ].

Figure 27. GC-MS chromatogram of derivatised (2S,3R)-3-methylpentadecan-2-ol [(SR-)Ac].

46 4.2.2. Pine sawflies (Paper IV-VI)

Analysis of pine sawfly extracts was made using the general method in Figure 28 (see Section 4.1.1. and 4.1.2. for further details). The gradient elution purification step after derivatisation was only added when necessary (for severely contaminated samples).

Addition of internal standard to the extract and evaporation to dryness

Gradient elution with 0-11% EtOAc in pentane and collection of fraction 9 to 11

Washing the collected fractions with KOH and drying with Na2SO4

Evaporation to dryness and dissolving in cyclohexane

Identification and quantification of alcohols with GC- MS

Addition of pyridine and (2S)-2-acetoxypropionyl chloride to the cyclohexane solution

Heating on oil bath at 80 oC for 30 min

Washing with HCl and drying with Na2SO4

Gradient elution with 0-6% EtOAc in pentane and collection of fraction 6

Evaporation to dryness and dissolving in cyclohexane

Identification of stereoisomers with GC-MS

Figure 28. General flowchart that presents the method for analysis of pine sawfly extracts.

47 The general problem associated with work on these insects is the lack of a certain gland for producing the pheromone precursor, which makes it necessary to extract the whole body, resulting in a large amount of different compounds being extracted. Although purification reduced the contamination, there were still some co-eluted impurities. Identification and quantification of the pheromone precursor alcohol was normally performed after purification but before derivatisation. However, sometimes neither the pheromone precursor nor the internal standard was visible with full scan. By selecting one ion from the spectra, preferably m/z 45 (CH3CHOH), the compounds could normally be visualised. But, because of co- eluted impurities during the GC analysis, and the intensity of the m/z 45 ion is different between the analyte and the internal standard, an exact quantification was, for the most, not possible. Derivatisation of the aclohol often made it easier to detect both the internal standard and the analyte; if not during full scan, at least with SIM and an optimised gradient for separation of the stereoisomers. If a correction factor was calculated, quantification could also be made at this stage (see Paper IV).

The results of the analysis of pine sawfly extracts are presented in Table 4. The amounts of the main component of the sex pheromone precursor alcohol for D. pini and N. sertifer are in correlation with previous publications (Wassgren et al. 1992; Bergström et al. 1995), although our analysis was made with improved separation and detection. In an earlier study of D. similis extract it was shown to contain about 10 ng/female of the pheromone precursor, with (2S,3R,7R)-3,7- dimethylpentadecan-2-ol as the major component and the (2S,3S,7S)-isomer in minor amounts (Olaifa et al. 1988). However, this analysis was made almost 25 years ago with FID as the detector, and hence only the retention time could be used as a means of identification. Our improved method resulted in about 15 ng of 3,7- dimethylpentadecan-2-ol per female, with (2S,3R,7R) as the major component, and minor amounts of (2R,3S,7S), (2R,3R,7S) and (2R,3R,7R). No previous analysis of N. lecontei extracts have been published, but our result with (2S,3S,7S)-3,7- dimethylpentadecan-2-ol as the pheromone precursor is in correspondence with earlier field trap experiments (Kraemer et al. 1981).

A total of 260 females of the Chinese pine sawfly Diprion jingyuanensis has been analysed at different occasions with the same method as for D. pini (Paper IV). None of the stereoisomers of 3,7-dimethyltridecan-2-ol were found in the extracts (unpublished results). According to previous studies, the propionate of (2S,3R,7R)- 3,7-dimethyltridecan-2-ol is the main component of its sex pheromone (Zhang et al. 2005). Anderbrant et al. (unpublished results) found several of the stereoisomers of 3,7-dimethyltridecan-2-ol in extracts, although no exact determination of the stereochemistry or quantification was made (see Section 1.4.1.). The reason that no

48 alcohol was found in these extracts could either be due to that the amounts were below the detection limit (< 0.1 ng/female), or that it is a (unknown) pine sawfly species. Furthermore, the extracts contained lots of contaminants, which made analysis difficult.

An extract of 68 females of the pine sawfly Gilpinia hercyniae was analysed with the same method as for D. pini (Paper IV). None of the alcohols used by other pine sawflies were detected in the extract (unpublished results). Nothing has been published regarding sex pheromone for this species, so it is unclear if it is even using pheromone for its mating behaviour (O. Anderbrant, personal communication).

Table 4. Results of the analysis of pine sawfly extracts.

Species Major stereoisomer Amount (ng/female) Reference D. jingyuanensis n.d. - Unpublished D. pini (2S,3R,7R)a 8 Paper IV D. similis (2S,3R,7R)b 15 Paper V G. hercyniae n.d. - Unpublished N. lecontei (2S,3S,7S)b 7 Paper VI N. sertifer (2S,3S,7S)b 5-13 Paper IV a3,7-dimethyltridecan-2-ol, b3,7-dimethylpentadecan-2-ol, n.d. not detected

49 4.2.3. Bicyclus (Paper I)

The extract of 200 male wings of B. anynana did not require any further purification, and contained large amounts (about 200 µg in total) of 6,10,14- trimethylpentadecan-2-ol (Figure 29).

Figure 29. GC-MS chromatogram of male wing extract of Bicyclus anynana, 6,10,14- trimethylpentadecan-2-ol gives the largest peak.

Three sex pheromone components were identified by using GC-EAD (see Section 2.5.): (Z)-9-tetradecenol, hexadecenal and 6,10,14-trimethylpentadecan-2-ol. However, the stereochemistry of the latter was unknown. In Paper I, the alcohol was identified as (2R,6R/S,10R/S)-6,10,14-trimethylpentadecan-2-ol. We have later confirmed it to be the (2R,6R,10R)-isomer. A reaction sequence involving lipase- catalysed reactions and starting materials from the chiral pool generated the different stereoisomers of 6,10,14-trimethylpentadecan-2-ol in certain proportions, which made it possible to identify all eight stereoisomers (Figure 30) and compare them with the peak in the extract (to be published). The smaller peaks seen in Figure 3C, Paper I, have retention times that correspond to (2S,6R,10R)- and (2S,6S,10S)-6,10,14-trimethylpentadecan-2-ol, but further analysis are needed to confirm them.

6,10,14-Trimethylpentadecan-2-ol is also used as the sex pheromone by some pest insects (moths). Thus the method described here can also be applied for extracts of these insects (Hall et al. 1987; Mori et al. 1991; Burger et al. 1993; Sasaerila et al. 2003).

Figure 30. GC-MS chromatogram of the identified stereoisomers of 6,10,14- trimethylpentadecan-2-ol [as their (2S)-2-acetoxypropionates].

In three male wing extracts (from Kenya, Malawi, and Uganda) of the African butterfly Bicyclus safitza, the stereochemistry of the 6,10,14-trimethylpentadecan-2- ol was determined to be (2R,6R,10R), the same as for the closely related specie B. anynana (Paper I). The extracts also contained 6,10,14-trimethylpentadecan-2-one, and there was a clear difference of the amounts and ratio of alcohol/ketone between the different locations (unpublished results).

Two wing extracts (from Malawi and Uganda) contained octadecenal, with an unknown position and stereochemistry of the double bond. By using dimethyl disulphide (Buser et al. 1983) it was identified as 11-octadecenal. Both (E)- and (Z)- octadecenal were then synthesised in our lab, and by comparing their retention times with the octadecenal in the extracts, it was determined to be (Z)-11- octadecenal (unpublished results).

At the moment we are analysing extracts from 15 other related African butterfly species and the results will be published in due time.

51 4.2.4. Euglossa (Paper III)

The purification method described in Section 4.1.1. is also applicable to compounds with other functionalities, although they might elute in other fractions. The less polar ketone (compared to alcohol) in the Euglossa hind-leg extracts eluted in fraction 4-6. Separation of the stereoisomers by GC-MS of chiral ketones can be achieved by reduction to alcohols followed by derivatisation with acid chlorides in the same way as earlier described for chiral alcohols. It is important to note that reduction of the ketone to alcohol creates a new stereogenic centre that must be disregarded when determining the stereochemistry of the original ketone. In this case, the identified single isomer (6R,10R)-6,10,14-trimethylpentadecan-2-one in the extracts generates two peaks when it is reduced to alcohol, due to a formation in equal amounts of the alcohols with (2R)- and (2S)-configuration (Figure 31).

CH3 CH3 CH3 O

10 6

H3C CH3

LiAlH4

CH3 CH3 CH3 OH

10 6 2 * H3C CH3

Figure 31. (6R,10R)-6,10,14-Trimethylpentadecan-2-one and (2R/S,6R,10R)-6,10,14- trimethylpentadecan-2-ol, *new stereogenic centre.

The amounts of ketone found in the extracts of the different Euglossa species were in the amount of hundreds of µg per pair of hind-legs (Table 5). This is in contrast to insects that produce their own sex pheromone, where amounts in the range of pg to ng are quite normal. Because the Euglossa species collect fragrances from flowers and other sources, and store them for a rather long time, there can be substantial amounts of each compound. This makes it easier to identify them, and allows one to use other analytical instruments than GC-MS, for example as in this case GC-FTIR (Figure 32), and sometimes even NMR.

52 Table 5. Amounts of (6R,10R)-6,10,14-trimethylpentadecan-2-one in different Euglossa species.

Species Amount (µg/pair of hindlegs) E. allosticta 152 E. crassipunctata 245 E. imperialis 192

Figure 32. GC/FTIR spectra of synthetic (6R,10R)-6,10,14-trimethylpentadecan-2-one and extract of Euglossa allosticta.

53 5. CONCLUDING REMARKS

The analysis of sex pheromones and other semiochemicals in insects is often a challenging task. To find very low amounts of substances in complex matrices is almost like looking for a needle in a haystack. This places high demands on the methods and analytical instruments. In other cases, the analytes occur in high amounts in rather pure extracts, and the “only” challenge is the separation of stereoisomers.

An example of both complex matrices and separation of stereoisomers is the analysis of sex pheromone precursor alcohols in extracts of pine sawflies. Because the alcohols are not produced in a special gland or part of the insect, the whole body of the insect needs to be extracted. The huge amounts of co-eluting substances, in combination with analyte levels in the pg-ng scale, make this one of the more difficult tasks in insect pheromone analysis.

The analysis of the Bicyclus and Euglossa extracts are examples of simpler cases, where the challenge is the separation of stereoisomers. This can sometimes be a difficult problem in itself, as this thesis has shown.

The separation experiments has shown that in some cases, as for 6,10,14- trimethylpentadecan-2-ol, 3-methylpentadecan-2-ol and 3,7,9-trimethyltridecan-2- ol, finding a right acid chloride for derivatisation is enough to separate all of the stereoisomers on a GC column with an achiral stationary phase. In other cases, a combination of acid chloride and a column with a chiral stationary phase is necessary (3,7-dimethylundecan-2-ol, 3,7-dimethyldodecan-2-ol, and 3,7- dimethyltridecan-2-ol), or even different acid chlorides depending on which of the stereoisomers needs to be separated (3,7-dimethyltetradecan-2-ol and 3,7- dimethylpentadecan-2-ol). Other chiral alcohols, e.g. 3,7,11-trimethyltridecan-2-ol, has not been successfully separated and only partly tested in this work, showing that there is still work to do.

The successful separation of all eight stereoisomers of 3,7-dimethylundecan-2- ol, 3,7-dimethyldodecan-2-ol, and 3,7-dimethyltridecan-2-ol in a single run has, to our knowledge, been performed for the first time.

The analysis of extracts of D. pini, D. similis and N. sertifer gave results in line with earlier publications. Although, most of them were done 15-30 years ago, and often with only FID as the detector for identification of analytes in the extracts. This can explain the somewhat different results regarding presence or absence of minor pheromone components. The result for N. lecontei (Paper VI) is the first ever published of extract analysis for this species.

54 For the extracts of B. anynana and Euglossa spp. the analysis was simplified by cleaner extracts and larger amounts of analytes. In both cases, this was the first determination of the stereochemistry for the respective semiochemical.

To conclude, this thesis presents improved methods for the separation of stereoisomers of different chiral secondary alcohols, and their related ketones and esters, in extracts of different species of insects. There are, however, still some obstacles to overcome. The separation of all of the stereoisomers of 3,7,11- trimethyltridecan-2-ol is a suitable task for future research, because this has not been tested with all acid chlorides and columns here. Also, to separate all stereoisomers of 3,7-dimethyltetradecan-2-ol and 3,7-dimethylpentadecan-2-ol in a single run is still a problem to be solved. More derivatives and columns needs to be tested to find a way to solve these problems.

55 ACKNOWLEDGEMENTS

I would like to thank the following people:

My supervisor Professor Erik Hedenström for all the help and support during this time.

My assistant supervisor Professor Emeritus Hans-Erik Högberg and my former assistant supervisor Dr Kristina Sjödin.

Erika and Natalia for all the interesting discussions (although some of it may be defined in other words). Erika, you will surely get your degree in a few years, and Natalia, hopefully you too one day. That is, if you stop having children all the time…

Present and former members of our group: Amelie, Ann (thanks for the proofreading!), Anna, Fredrik, Jimmy, Kerstin, Rebecka, Susanne and Yirgalem.

Håkan Norberg for all the help with ordering supplies and solving problems in the lab.

Professor Olle Anderbrant, Lund University, Professor Christer Löfstedt, Lund University, PD Dr Thomas Eltz, Ruhr-Universität Bochum, and Dr Caroline Nieberding, Université catholique de Louvain, for all the extracts and collaboration.

All other colleagues at NAT.

I would like to give a special thanks to my GC-MS that has worked nearly flawless during all this time. If it had behaved as one of our other instrument (who shall remain anonymous), I would have had several years left.

I am also grateful for the financial support from EU (Objective 2 the region of South Forest Counties) and Länsstyrelsen i Västernorrlands län.

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