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
© Joakim Bång, 2011
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
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.
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.
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.
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.
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
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
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.
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).
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
H3C
H3C
CH3 OH
OH
H3C OH
H3C O
CH3 H3C
CH3
H3C CH3 CH3
Hexan-1-ol Butane
A. Positional isomers: B. Skeletal isomers: C. Functional group isomers:
Hexan-2-ol Isobutane
Ethanol
Methoxymethane
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).
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).
Figure 3. (R)- and (S)-configuration.
Cl
CH3 H3C
H
CH3
Cl H3C
H E
Z COOH
HO
CH3 H
HOOC OH
CH3 H
H COOH
CH3 HO
H2N H
HO COOH
CH3 H NH2 H
C. E/Z isomers (diastereomers)
A. Enantiomers B. Diastereomers
(High)
(High)
(High) (High)
(Low)
(Low)
(Low) (Low)
COOH HO
CH
3H HOOC OH
CH
3H
(R)-2-hydroxypropanoic acid (S)-2-hydroxypropanoic acid
1 2
3 4 2 1
3 4
(R) (S)
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).
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).
Figure 4. The different groups and subgroups of semiochemicals.
Semiochemicals
Chemical substances carrying messages
Pheromones
Between individuals of the same species
Alarm
Repellent
Aggregation
Trail
Sex
Allelochemicals
Between individuals of different species
Allomones
Kairomones
Synomones
Apneumones
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).
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
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).
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.
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,
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).
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).
CH3
OH
CH3 CH3
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-‐‑
CH3 OH
CH3 CH3
CH3 OH
CH3 CH3
13 7
7
3 2
15 3
2
* *
*
* *
*
CH3 OH
CH3
15 3
2
* CH3 *
OH
CH3 CH3
3 2
* 14 *
7*
CH3 OH
CH3 CH3
CH3
13 7 3
2
* *
*
9* CH3
OH
CH3 CH3
CH3
13 7 3
2
* *
* 11*
CH3 OH
CH3 CH3
3 2
*
* 9*
11
Diprion spp (2S,3R,7R)
Diprion spp, Gilpinia spp and Neodiprion spp (2S,3S,7S) and (2S,3R,7R)
Gilpinia pallida (2S,3R,7R) Macrodiprion nemoralis
(2S,3R,7R,9S)
Microdiprion pallipes (2S,3S,7S,11S) and (2S,3S,7S,11R) Diprion nipponica
(2S,3R,7S)
Gilpinia frutetorum and Gilpinia socia (2S,3R)
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.
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).
CH3 O
CH3 CH3
CH3 O
CH3 O
CH3 CH3
O
CH3
Propionate of (2S,3R,7R)-3,7-dimethyltridecan-2-ol Acetate of (2S,3R,7R)-3,7-dimethyltridecan-2-ol
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-‐‑dimethyl-‐‑
pentadecan-‐‑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).
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).
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).
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).
CH3 O
CH3 CH3
CH3 O
CH3 O
CH3 CH3
O CH3
Propionate of (2S,3S,7S)-3,7-dimethylpentadecan-2-ol Acetate of (2S,3S,7S)-3,7-dimethylpentadecan-2-ol
