Mid Sweden University

Four-eyed bark beetles of the genus Polygraphus have been involved in large bark beetle outbreaks in different parts of the world, resulting in major economic losses. A striking example is the invasive species Polygraphus proximus which is a pest on Abies sibirica in Russia. In Sweden, Polygraphus poligraphus has been involved in bark beetle outbreaks on Norway spruce, Picea abies, together with the European spruce bark beetle Ips typographus. Two related species, Polygraphus punctifrons and Polygraphus subopacus are also present in Sweden. Recently, aggregation pheromones or pheromone components have been identified for these four Polygraphus species. However, questions remain regarding the complete composition of their pheromones, particularly for P. subopacus and P. proximus, whose aggregation pheromones appear to be very similar. In an attempt to better understand the chemical communication of these species, additional studies were conducted on P. poligraphus, P. punctifrons and P. subopacus using solid phase microextraction coupled with gas chromatography and mass spectrometry (SPME–GC–MS), electroantennography (EAG) as well as SPME–GC–MS and GC–MS with electroantennographic detection (EAD). Field experiments were also conducted. In P. punctifrons, some male-specific compounds were found in addition to those previously identified. In EAG and SPME–GC–MS/EAD studies, all three Polygraphus species responded strongly to grandisol. Using a chiral column, GC–MS/EAD revealed that they were able to detect both enantiomers of grandisol. In summary, this work presents our current understanding of the aggregation pheromones in four Polygraphus species and the challenges we have met in identifying species-specific pheromone blends for some of these species.

Extracts of Diprion similis females contained about 15 ng of the sex pheromone precursor 3,7‐dimethylpentadecan‐2‐ol per female. After derivatisation with (S)‐2‐acetoxypropanoyl chloride, we found that the major stereoisomer in the extract was (2S,3R,7R)‐3,7‐dimethylpentadecan‐2‐ol. Small amounts of other stereoisomers of 3,7‐dimethylpentadecan‐2‐ol were also identified in the extract, namely 1% of (2R,3S,7S), 0.3% (2R,3R,7R) and 0.4% of (2R,3R,7S). An unknown fifth substance showed a very similar spectrum to 3,7‐dimethylpentadecan‐2‐ol, both in SIM and full scan mode. None of the earlier suggested behavioural synergistic isomers ((2S,3S,7S), (2S,3S,7R) and (2S,3R,7S)) were detected in the extracts. In field tests in Ontario, Canada, the earlier identified main pheromone component, viz. the propanoate of (2S,3R,7R)‐3,7‐dimethylpentadecan‐2‐ol, was tested alone and in combination with other stereoisomers, earlier reported to be synergistic. No synergistic effects were detected and the threo four‐isomer blend was as attractive as the pure main compound. Thus, one of the few examples of a diprionid sawfly using more than one substance in its sex pheromone could not be confirmed. The results also suggest that monitoring programs can use the more easily synthesized threo‐blend without losing efficiency. Furthermore, the study suggests that other diprionid pheromones may benefit from a reinvestigation, to clarify possible synergistic effects of stereoisomers. 

Bark beetles of the genus Polygraphus have recently been involved in large bark beetle outbreaks in central Sweden, together with the European spruce bark beetle Ips typographus. Three species of Polygraphus can be found in this region; Polygraphus poligraphus, Polygraphus punctifrons and Polygraphus subopacus. Efficient pheromone traps would facilitate further investigations of these species and their role in bark beetle outbreaks. Pheromone compounds have previously been identified in P. poligraphus and P. punctifrons, but not in P. subopacus. Thus, we allowed males and females of P. subopacus to bore in the bark of stem sections of Norway spruce (Picea abies) in the laboratory. Volatile organic compounds from boring insects were sampled with SPME and analysed with GC–MS and several male-specific compounds were observed. The male specific compounds were 3-methyl-3-buten-1-ol, 3-methyl-2-buten-1-ol, 3-methyl-2-butenal, grandisol, fragranol, (Z)-2-(3,3-dimethylcyclohexylidene)-ethanol, (E)-2-(3,3-dimethylcyclohexylidene)-ethanol, (Z)-2-(3,3-dimethylcyclohexylidene)-acetaldehyde, (E)-2-(3,3-dimethylcyclohexylidene)-acetaldehyde, geranial and γ-isogeraniol. (Z)-2-(3,3-dimethylcyclohexylidene)-ethanol, [(Z)-DMCHE], was identified from GC–MS analysis to be the major male-specific compound while the (E)-isomer, [(E)-DMCHE], was found as a minor compound. These two compounds gave positive responses in EAG analyses with antennae from males and females of P. subopacus. Thus, (Z)- and (E)-DMCHE were used in a field experiment in central Sweden but only (Z)-DMCHE was found to be attractive to males and females of P. subopacus. Consequently, (Z)-DMCHE was established to be a component of P. subopacus aggregation pheromone. 

Bark beetles have caused extensive damage to forests in central Sweden during the past decade, and the four-eyed spruce bark beetle, Polygraphus poligraphus, seems to be involved. However, its role in these bark beetle outbreaks is still not clear. The purpose of this study was to develop an efficient pheromone bait for P. poligraphus, which would make it possible to study the species more carefully and thereby contribute to protect exposed forests in an environmentally friendly way. Three field studies were conducted in 2015, 2016 and 2018 in Medelpad, county of Västernorrland, Sweden. The pheromone of P. poligraphus, (−)-terpinen-4-ol, was tested at different release rates and in different enantiomeric purities, to find the most attractive formulation for the beetles. It was also tested in combination with racemic frontalin, a compound which has previously been shown to produce a synergistic effect together with (−)-terpinen-4-ol of low enantiomeric purity; 52% ee. Other compounds, chosen based on responses from electroantennographic studies, were also tested in an attempt to find additional attractants and repellents for P. poligraphus. The most attractive treatment tested was enantiomerically pure (−)-terpinen-4-ol (99% ee). When the enantiomeric purity was lower (50% ee), the trap catches was lowered to levels comparable to the catches for unbaited control traps. A strong synergistic effect with frontalin was observed for (−)-terpinen-4-ol of low enantiomeric purity (50% ee) but not for the enantiomerically pure compound (99% ee). The release rate of (−)-terpinen-4-ol (99% ee) was shown to be an important factor. For the combination of frontalin and (−)-terpinen-4-ol (50% ee), the attraction seemed strongest when (−)-terpinen-4-ol was released at a higher rate than frontalin. An interesting and novel result was that a repellent compound, α-terpineol, was identified in our studies. Our results from field studies and electroantennography recordings also indicate that (+)-terpinen-4-ol is a repellent for P. poligraphus.

Gas chromatography (GC) and mass spectrometry (MS) were used to determine the stereoisomeric compositions of 6,10,14-trimethylpentadecan-2-ol and 6,10,14-trimethylpentadecan-2-one in wing extracts from 17 Bicyclus butterfly species from different regions of Africa. All samples were purified using solid phase extraction (SPE). Since some species contained both alcohol and ketone, these were separated and the ketone was reduced to the alcohol before analysis as either (R)-trans-chrysanthemoyl or (S)-2-acetoxypropionyl esters. A novel asymmetric synthesis was developed for a reference mixture of (2R/S,6S,10R)-6,10,14-trimethylpentadecan-2-ol with known composition of the eight stereoisomers. The mixture then was used as the (R)-trans-chrysanthemoyl esters to correlate each of the eight gas chromatographic peaks to a specific stereoisomer of the extracted wing compounds. Seven butterfly species showed (2R,6R,10R)-configuration of the alcohol, four species contained minute amounts of alcohol too small to determine the stereochemistry, nine species showed (6R,10R)-configuration of the ketone, and one species contained minute amounts of ketone too small to determine the stereochemistry. No other stereoisomers of alcohol or ketone could be detected in the extracts, and the quantities of the compounds in the wing extracts varied from 5 to 900 ng per sample for each species.

Male orchid bees (Euglossini, Apidae, Hymenoptera) expose species-specific blends of volatile chemicals (perfume bouquets) during their courtship display. The perfumes are acquired by collecting fragrant substances from environmental sources, which are then accumulated in specialised hind leg pouches. To balance the perfume composition, the males need to find and collect the required substances in specific relative amounts while facing seasonal, local or habitat-dependent differences in compound availability. Experience-dependent choice of odours, i.e. 'learned avoidance' of recently collected components, has been proposed as the mechanism that mediates the accumulation of the stereotypical compound ratios. In the present study, we used the presence of certain compounds in male hind leg pouches as proxy for the respective local compound availability, and investigated whether differences in content are correlated with differences in chemical choice assays. Our results suggest that volatile availability differs between localities (n = 16) as well as habitats (n = 2; coastal vs. inland) across the Yucatán peninsula, Mexico, for both studied species. Male Euglossa dilemma showed a pronounced preference for benzyl benzoate and eugenol at locations where those compounds were rare in hind leg extracts, as predicted by the learned avoidance model. No equivalent correlations were found for Euglossa viridissima. This is the first study to combine chemical analyses of perfumes with bioassays of odour choice. It strengthens the view that negative feedback from collected odours modifies future chemical choice and helps males to acquire specific perfume blends. © 2013 Springer-Verlag Berlin Heidelberg.

Elater ferrugineus is a saproxylic click beetle inhabiting old deciduous trees in Europe. It is threatened throughout its area of distribution due to habitat loss. No efficient monitoring method has been available for this species, but observed attraction of females to (R)-(+)-γ-decalactone, which is a male-produced sex pheromone of its prey, the scarab beetle Osmoderma eremita, has led to the development of an odour lure for monitoring. In addition, four esters have recently been identified from the pheromone-producing gland in female E. ferrugineus, and a blend of these esters is highly attractive to conspecific males in the field, revealing an alternative odour-based method for monitoring this species. However, no rigorous analysis has been performed to check whether all four esters show biological activity in male E. ferrugineus, and whether its own sex pheromone is a more potent lure than the prey kairomone for monitoring of E. ferrugineus. In this study, we reinvestigated the E. ferrugineus sex pheromone, using electrophysiological and behavioural analyses, and found that only one of the esters, 7-methyloctyl (Z)-4-decenoate, is active. In addition, trapping experiments revealed that 7-methyloctyl (Z)-4-decenoate is a much more efficient attractant for male E. ferrugineus than the prey pheromone is for conspecific females, or any sex of O. eremita. With a very efficient odour lure at hand, novel information about current distribution, local population sizes, and dispersal ranges in E. ferrugineus can now be obtained, which can aid in conservation efforts to protect this threatened insect and its habitat. © 2011 Springer Science+Business Media B.V.

Stereoisomers of long-chain secondary alcohols are used as sex pheromone precursors among pine sawflies, which can be severe pest to conifers. To use the pheromone in environmentally friendly pest managements, methods are needed that can determine the exact stereochemistry of the precursor alcohols. Combinations of 11 acid chloride derivatives and 10 GC columns were evaluated for separation of the stereoisomers of 3,7-dimethylundecan-2-ol, 3,7-dimethyldodecan-2-ol, 3,7-dimethyltridecan-2-ol, 3,7-dimethyltetradecan-2-ol, and 3,7-dimethylpentadecan-2-ol. Derivatisation with (2S)-2-acetoxypropionyl chloride in combination with the Chiraldex B-PA column separated all eight stereoisomers of 3,7-dimethylundecan-2-ol, 3,7-dimethyldodecan-2-ol and 3,7-dimethyltridecan-2-ol. A combination of two different derivatisation methods was needed to separate all stereoisomers of the longer chained alcohols, 3,7-dimethyltetradecan-2-ol and 3,7-dimethylpentadecan-2-ol. An extract of the pine sawfly Neodiprion lecontei was also analysed, and the stereochemistry of the sex pheromone alcohol prcursor was determined to be (2S,3S,7S)-3,7-dimethylpentadecan-2-ol, at an amount of about 7 ng/female. This paper presents the first GC-MS-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 analytical run, and also the first determination of the stereochemistry of the sex pheromone precursor of N. lecontei.

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.

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.

A GC-MS method to analyze the stereoisomeric composition of chiral secondary alcohols found in whole body extracts of pine sawfly females was developed. The tested alcohols were derivatized with optically pure (S)-2-acetoxypropionyl chloride prior to GC-MS analysis. Baseline separation was obtained for all sixteen stereoisomers of 3,7,9-trimethyltridecan-2-ol and for the four 3-methylpentadecan-2-ol stereoisomers. For 3,7-dimethyltridecan-2-ol, 3,7-dimethyltetradecan-2-ol and 3,7-dimethylpentadecan-2-ol baseline separation was obtained for 6 of the possible 8 stereoisomers. When a mixture of 16 stereoisomers of 3,7,11-trimethyltridecan-2-ol was tested, baseline separation of 7 peaks out of 16 possible was obtained. The investigated alcohols are pheromone precursors for some pine sawfly species that are severe defoliators of pine. Females from several Diprion, Neodiprion, Macrodiprion, Microdiprion, and Gilpinia species emit esters of such secondary alcohols as sex pheromones that attract males for mating. To quantify the small amounts of the precursor alcohol and its stereoisomeric composition found in whole body extracts from female pine sawflies, a purification method was optimized. An extract of 20 females of D. pini contained about 8 ng of (2 S,3 R,7 R)-3,7-dimethyltridecan-2-ol per female, and three extracts of 18, 20, and 90 females of N. sertifer contained between 5 and 13 ng of (2 S,3 S,7 S)-3,7-dimethylpentadecan-2-ol per female.