Chemical Signals in Interactions Between Hylobius Abietis and Associated Bacteria

Chemical signals in interactions between Hylobius abietis and associated bacteria

Karolin Axelsson

Doctoral Thesis

Stockholm 2016

Akademisk avhandling som med tillstånd av Kungl Tekniska Högskolan i Stockholm framlägges till offentlig granskning för avläggande av doktorsexamen i kemi med inriktning mot ekologisk kemi onsdagen den 15 juni kl 13 i sal F3, KTH, Lindstedtsvägen 26, Stockholm. Avhandlingen försvaras på engelska. Opponent är Doc. Benedicte Albrectsen, Umeå Univerisitet.

Cover: Pine weevil (Hylobius abietis L.) walking on a pine twig. (Photo collage by Karolin Axelsson)

ISBN 978-91-7595-972-6 ISSN 1654-1081 TRITA-CHE-Report 2016:21

“But the smell of a forest is infinitely changeful; it varies with the hour of the day, not in strength merely, but in character; and the different sorts of trees, as you go from one zone of the wood to another, seem to live among different kinds of atmosphere. Usually the resin of the fir predominates. But some woods are more coquettish in their habits; and the breath of the forest of Mormal, as it came aboard upon us that showery afternoon, was perfumed with nothing less delicate than sweetbrier. /.../

A whole forest, healthy and beautiful, giving colour to the light, giving perfume to the air: what is this but the most imposing piece in nature’s repertory?”

From An Inland Voyage by Robert Louis Stevenson, 1878

~ To my beloved family ~

Feeding pine weevil as pictured by Linnea Axelsson, 5 years

Karolin Axelsson, 2016:”Chemical signals in interactions between Hylobius abietis and associated bacteria”, KTH Chemical Science and Engineering, Royal Institute of Technology, SE-100 44 Stockholm, Sweden.

Abstract The pine weevil (Hylobius abietis L.) is one of the two topmost economically important insect pests in Swedish conifer forests. The damage increase in areas were the silvicultural practice is to use clear cuttings were the insects gather and breed. During egglaying the female protects her offspring by creating a cave in roots and stumps were she puts her egg and covers it with frass, a mixture of weevil feces and chewed bark. Adult pine weevils have been observed to feed on the other side of the egg laying site and antifeedant substance has been discovered in the feces of the pine weevil. We think it is possible that microorganisms present in the frass contribute with antifeedant/repellent substances. Little is known about the pine weevils associated bacteria community and their symbiotic functions. In this thesis the bacterial community is characterized in gut and frass both from pine weevils in different populations across Europe as well as after a 28 day long diet regime on Scots pine, silver birch or bilberry. Volatile substances produced by isolated bacteria as well as from a consortium of microorganisms were collected with solid phase micro extraction (SPME) and analyzed with GC-MS. The main volatiles were tested against pine weevils using a two-choice test. Wolbachia, Rahnella aquatilis, Serratia and Pseudomonas syringae was commonly associated with the pine weevil. 2-Methoxyphenol, 2-phenylethanol, 3-methyl- 1-butanol were found in the headspace from Rahnella aquatilis when grown in substrate containing pine bark. 2-Methoxyphenol and 3-methyl-1-butanol, phenol and methyl salicylate were found in pine feces. Birch and bilberry feces emitted mainly linalool oxides and bilberry emitted also small amounts of 2- phenylethanol.

A second part of the thesis discusses the role of fungi in forest insect interactions and the production of oxygenated monoterpenes as possible antifeedants. Spruce bark beetles (Ips typhographus L.) aggregate with the help of pheromones and with collected forces they kill weakened adult trees as a result of associated fungi growth and larval development. A fungi associated with the bark beetle, Grosmannia europhoides, was shown to produce de novo 2-methyl-3-buten-2-ol, the major component of the spruce bark beetle aggregation pheromone. Chemical defense responses against Endoconidiophora polonica and Heterobasidion parviporum were investigated using four clones of Norway spruce with different susceptibility to Heterobasidion sp. Clone specific differences were found in induced mono-, sesqui and diterpenes. A number of oxygenated monoterpenes which are known antifeedants for the pine weevil were produced in the infested areas.

Keywords: Hylobius, Rahnella, Pseudomonas, bacteria, fungi, metabolites, 2- methoxyphenol, 2-phenylethanol

Abbreviations and definitions

GC-MS Gas Chromatography-Mass spectrometry SPME Solid phase microextraction OTU Operational taxonomic unit 16S rRNA 16S ribosomal ribonucleic acid PAL Phenylalanine ammonia lyase IPP Isopentenyl diphosphate DMAPP Dimethylallyl diphosphate MEP Methyl-D-erythritol phosphate MVA Mevalonate NA Nutrient agar NB Nutrient broth PCR Polymerase chain reaction DNA Deoxyribonucleic acid PDMS/DVB Polydimethylsiloxane/Divinylbenzene HS Headspace NMDS Non-metric multidimensional scaling

Semiochemicals: Chemical signals that transmit information between individual organisms

Infochemicals: A chemical that conveys information in an interaction between two individuals, evoking in the receiver a behavioural or physiological response that is adaptive to either one of the interactants or to both.

Antifeedant Any substance that reduces consumption by an insect

Deterrent Any substance that prevents consumption by an insect

Repellent Insects orient away, repelled from feeding without contact with plant material

List of Publications This thesis is based on the following papers, referred to in the text by their Roman numerals I-III: I. Antifeedants produced by bacteria associated to the gut of the pine weevil (Hylobius abietis) Karolin Axelsson, Vera Konstanzer , Gunaratna Kuttuva Rajarao, Olle Terenius, Lisa Seriot, Henrik Nordenhem, Göran Nordlander and Anna- Karin Borg-Karlson Microbial ecology in review, 2016 II. The gut microbiota of the pine weevil (Hylobius abietis, Coleoptera: Curculionidae) is similar accross Europe and resembles that of other conifer-feeding beetles Aileen Berasategui, Karolin Axelsson, Göran Nordlander, Anna-Karin Borg-Karlson, Axel Schmidt, Jonathan Gershenzon. Olle Terenius and Martin Kaltenpoth Molecular Ecology. 2016, in press III. Do pine weevil microbiota and corresponding volatiles change due to selective feeding? Karolin Axelsson, Louise Nilsson, Göran Nordlander, Anna-Karin Borg-Karlson and Olle Terenius Manuscript.

Appendix A Fungal symbionts of the spruce bark beetle synthesize the beetle aggregation pheromone 2-methyl-3-buten-2-ol Tao Zhao, Karolin Axelsson, Paal Krokene, Anna-Karin Borg-Karlson Journal of Chemical Ecology. 2015, 41, 848-852 Appendix B Clone specific chemical defense responses in Norway spruce to infestations by two pathogenic fungi. Karolin Axelsson, Amene Zendegi-Shiraz, Gunilla Swedjemark, Anna- Karin Borg-Karlson and Tao Zhao. Manuscript for Forest pathology

Paper II and Appendix A are reprinted by the kind permission from the publishers.

Table of Contents Abstract Abbreviations List of publications 1. Introduction 1 1.1. AIMS 2 1.2. CONIFERS 3 1.2.1. AROMATIC COMPOUNDS IN CONIFERS 3 1.3. PINE WEEVILS 4 1.3.1. LIFE CYCLE OF THE LARGE PINE WEEVIL 5 1.3.2. SEMIOCHEMICALS 6 1.4. MICROORGANISMS 8 1.4.1. BACTERIA 8 1.4.2. YEASTS AND FUNGI 9 1.4.3. MICROBIAL DERIVED METABOLITES 10 2. Materials and methods 12 2.1. BIOLOGICAL MATERIAL AND PROCEDURES 12 2.1.1. COLLECTION OF PINE WEEVILS 12 2.1.2. HOST PLANT MATERIALS FOR THE PINE WEEVIL 12 2.1.3. DISSECTION OF PINE WEEVILS 12 2.1.4. ISOLATION OF BACTERIA 13 2.1.5. MICROBIAL CULTURING MEDIA 14 2.1.6. IDENTIFICATION OF MICROORGANISMS 15 2.1.7. COLLECTION OF HEADSPACE VOLATILES RELEASED BY BACTERIA 16 2.1.8. SEPARATION AND IDENTIFICATION OF VOLATILES 17 2.1.9. BIOASSAY 18 3. Culturable bacteria associated with the pine weevil 19 4. Horizontal transfer of microorganisms 22 5. Dietary effects on microflora 24 6. Microbial volatiles affecting pine weevil behavior 28 7. Summary 31 8. Future prospects 32

Acknowledgements References Author’s contributions Appendix A Appendix B Appendix C

1. Introduction

The pine weevil (Hylobius abietis L.) has received much attention from both the forest industry and from researchers as it comprises one of the two topmost insect pests in Swedish conifer forests (Petersson et al. 2003, Wermelinger 2004). The other insect is the spruce bark beetle (Ips typhographus L.) which increases tremendously the years after heavy wind falls when there are plenty of weakened or killed trees were they can reproduce. Pine weevils, on the other hand, increase in areas were the silvicultural practice is to use clear cuttings, these are used by the insects to gather and breed. The clear cutting is followed by replantation of new seedlings which provides as easily available food for the new emerging generation. The seedlings are easy to girdle for an adult pine weevil and the plants dry and die. The newly planted seedlings has mainly been protected by insecticides which have adverse effects on the ecosystem as well as on the forestry workers applying them (Kolmodin- Hedman et al. 1995). The use of insecticides is now being phased out in Sweden. Other, more environmentally friendly methods are being developed where the pine weevil’s life cycle and behavior has to be considered and to further explore their part of the ecosystem. To the pine weevil ecosystem belongs a microbial community associated with the pine weevil itself as well as with its hosts. These microorganisms may provide the pine weevil with nutrients, detoxification of the chemical defense of the host as well as information of the health status of the tree.

The pine weevil has been thoroughly studied over the years with the beginning of the description of Carl von Linné in his tenth edition of Systema Naturæ published in 1758, a few studies in the beginning of 1920-30 and then more intensively from 1960 and onwards. A part of the research body covers their egg laying behavior. Under dry conditions pine weevil females creates a hole in the root bark of a conifer tree. She places an egg inside the hole and covers it with feces and nibbles of gnawed bark as a plug which protects her offspring. Other adult pine weevils has been observed to feed on the other side of the root, opposite to the egg laying site and antifeedant substances has been discovered in their feces. The microbial community may continuously contribute with the production of volatile substances. This thesis will hopefully contribute with a piece to this immense research puzzle by adding knowledge of how bacteria possibly acts as vectors for communication between adult weevils by producing antifeedant and deterrent substances at the oviposition site.

1.1. Aims The main objectives of the studies underlying this thesis were to understand the role of microbes, with main focus on bacteria, in the chemical interactions between host trees and the large pine weevil (Hylobius abietis).

The specific aims of the studies were:

 To characterize the microflora associated with the pine weevil

 To find microbial derived metabolites with potential antifeedant and repellent functions to pine weevils which could be explored in development of pine weevil control methods

The hypothesis that forms the base for this thesis is, that microorganisms present in the plug constructed by the female pine weevil consisting of bark and feces with which she covers the oviposition site, continuously produce chemical substances that deter conspecifics from feeding at the said site.

A consortium of microorganisms consisting of yeasts, fungi and bacteria may all contribute to the production of deterrent substances. The yeast and fungal flora has been investigated and summarized in a previous thesis (Azeem 2013). This thesis aims to cover the bacterial flora which has ability to contribute with odors during the first important days at the oviposition site.

To test the hypothesis five studies were performed. Three studies are presented in this thesis that focus on the pine weevil bacteria consortium while two additional studies, presented as Appendix A and B, focus on the role of fungi in related forest insect interactions.

Paper I and III Investigation of the production of volatile substances emitted by selected bacterial isolates and from a consortium of bacteria and their effect on pine weevil behavior.

Paper II and III Investigation of the consistency of the microbial community associated with the pine weevil gut from different populations across Europe and from diet changes.

Appendix A Investigation of the production and role of major fungal volatiles from four bark beetle associated fungi, Grossmannia europhioides, G. penicillata (Grosmann) Goid., Endoconidiopthora polonica (Münch) Syd. & P. Syd and Ophiostoma piceae (Münch) Syd. & P. Syd

Appendix B Investigation of tree defense reactions in response to Endoconidiophora polonica and the economically important root rot fungus Heterobasidion parviporum Niemelä & Korhonen.

1.2. Conifers The boreal forests thrives in the dry and cold climate of the northern hemisphere and is estimated to comprise from a range between 9-17 million km2 (Evans 2001). The coniferous plants contribute to the major part of the woody plant diversity with patches of deciduous trees as e.g. birch, alder and aspen. In Sweden 80 % of the forest area consists of conifers of which Norway spruce (Picea abies L.) and Scots pine (Pinus sylvestris L.) contribute with around 40 % each and the lodgepole pine (Pinus contorta Dougl. ex. Loud.) with around 1 % (Nilsson et al. 2015). The understory mainly consists of mosses, lichens and shrubs as bilberries (Vaccinium myrtillus L.).

Forest living insects use different niches of the trees for breeding and feeding (Nordenhem et al. 1994, Turgeon et al. 1994, Långström et al. 2004). The trees has adapted both to the biotic pressure from insects and pathogens and to the abiotic stresses from the environment e.g. drought and heat with different physiological and chemical defense mechanisms. The chemical defense rely mainly of a large structural variety of stored and de novo production of phenolics and terpenoids and alkaloid compounds (Franceschi et al. 2005). Feeding insects and pathogenic fungi can induce the biosynthesis of these defense chemicals and thereby the tree can avoid major damage from attacking pest during their long life span.

1.2.1. Aromatic compounds in conifers The metabolic pathways of aromatic compounds are well described among plants and include ca 4000 different compounds (Ralph et al. 2006). The compounds in conifers have different functions, some are together with terpenes proposed to be a part of the conifer defense against pathogens and insects, others such as lignin is important in the formation

3 of cell walls and support tissues. The major part of the aromatic substances in conifers has its start point in the shikimate acid pathway. It is initialized by the condensation of erythrose-4-phospate and phosphoenol pyruvate which produce chorismate after a series of seven steps. From chorismate the three aromatic amino acids L-tryptophan, L- tyrosine and L-phenylalanine are synthesized. Phenylalanine is enzymatically transformed to trans-cinnamic acid with the enzyme phenylalanine ammonia-lyase (PAL) and is the precursor for the volatile phenylpropanoids and benzenoids. (Pichersky et al. 2006, Maeda et al. 2012, Dudareva et al. 2013)

1.3. Pine weevils The true weevil family, the Curculionidae, is a large insect family which comprises more than 40 000 species divided into 18 subfamilies. In general they are plant feeding insects, more or less specialized. Some are attacking crops and therefor they are often considered as pests. The large pine weevil is widely distributed in the coniferous areas throughout Europe, North America and Asia. (Leather et al. 1999) Apart from the large pine weevil, three other species of the genus Hylobius are distributed in Europe. H. pinastri Gyll. and H. excavates Laicharting, generally prefer moist areas and tends to feed on Norway spruce. The forth species, H. transversovittatus (Goeze) is not associated with forest trees but feed on the roots of Purple-loosestrife (Lythrum salicaria L.) (Långström et al. 2004).

In North America there are seven species; pine root collar weevil H. radicis (Buchanan), H. warreni, Pales weevil H. pales (Herbst), H. congener, H. aliradicis, H. pinicola, H. assimilis (=H. rhizophagus) (Warner 1966, Day et al. 2004). H. pales and H. congener share similar niches as H. abietis. They breed in roots of dying or recently dead trees and the adult weevils feed on coniferous seedlings.(Långström et al. 2004) H. pales feeds on several pine species with white pine (Pinus strobus L.) as the preferred host (Warner 1966) and are regarded as a severe pest in Christmas tree plantations throughout the eastern parts of North America (Lynch 1984, Rieske 2000). H. congener is reported to cause high mortality in new pine plantations in Nova Scotia (Day et al. 2004).

1.3.1. Life cycle of the large pine weevil Adults of the large pine weevil are 8-14 mm long without their snout. The brown elytrum is covered with patches of yellow hairs. They feed on phloem on a range of plants e.g. berry bushes and shrubs as well as deciduous trees such as birch and ash, however pine is their preferred host (Månsson et al. 2004). They favor the actively growing parts of conifer trees but are able to utilize bark from both living and recently dead trees. Feeding on mature trees do not cause any major harm to the trees but young seedlings, under 3 years of age, needs to be protected from feeding weevils as they are easily girdled. Girdling destroys the capillary transportation system in the plants which means that sugars and nutrients can no longer be transported from the needles and the transport of water from the root system is hindered. The seedlings die from drought and malnutrition.

Pine weevils breed in the roots of dying or freshly killed conifer trees. The strong odors of ethanol and α-pinene emitted from the freshly killed tree Fig. 1 Feeding pine stumps in clear cut areas attracts large numbers weevil on girdled of pine weevils of both sexes during the spring migration in May (Nordlander 1991, Örlander et seedling. . al. 2000). After migration the flight muscles in Photo: Claes Hellqvist the weevils degenerate and the weevils walks to find food and suitable oviposition sites. The overwintering adult females are ready to lay mature eggs as soon as they reach the clear cut area. During the summer period the weevils eat and mate. About two eggs are laid per day per female and during the summer season a female lays 70- 80 eggs. She oviposit in the soil close to stump roots or in the root bark of stumps depending on the moisture level (Nordlander et al. 1997). In the oviposition process she creates a cavity in the bark root with the help of her snout where she place one egg together with feces. She creates a bark plug consisting of nibbles of bark and uses it to close the cavity. This trait is shared among the Curculionidae family and several Hylobius species also do this. This inherited trait may be a way of protecting the eggs from predation or a way of signaling to other females to lay their eggs elsewhere to reduce the competition between the offspring and thereby increase fecundity of the species. Adult weevils avoid feeding close to the egg cavities (Bylund et al. 2004) were the feces are and methanol extracts of feces has shown antifeedant effects on pine weevils which lasted for up to 24 hours (Borg-Karlson et al. 2006).

1.3.2. Semiochemicals Semiochemicals are substances produced by one organism that elicits a response in another. For insects a semiochemical may mediate interactions between insects and plants, or other animals or microorganisms. Host and non-host plants emit substances which can guide insects to make decisions about suitable places for oviposition, host selection or feeding and give information about the resistance capability and toxin levels of the plant. The cues can act as attractants which draw the insect to move towards the source, repellents which elicit a movement away from the source, feeding deterrents which inhibit feeding by the insect and antifeedants which reduce the feeding activity. Communication within a species is mediated by pheromones, a term coined in 1959 by Karlson and Lüscher defined as any substance secreted by an organism to the outside that causes specific reactions in the receiving organism of the same species. Several types of pheromones exists which are categorized by their behavioral functions. Two examples are sex pheromones and aggregation pheromones. Sex pheromones affect interactions between the sexes where the first studied system is bombykol in silk worm moths (Bombyx mori L.). Bombykol is released by calling females which attracts males. Aggregation pheromones draw the animal closer to an odour source and increase the density of the animals. This is exemplified by many bark beetle species that produce blends of 2-3 oxygenated monoterpenes or other compounds. In spruce bark beetle (Ips typographus) the main aggregation pheromone components are 2- methyl-3-buten-2-ol and (S)-cis-verbenol (Schlyter et al. 1987).

No long-distance pheromone has been described for H. abietis even though pentane extracts of females and of juvenile males have been found to elicit a copulatory response in males at close range (Kalo et al. 1986, Tilles et al. 1986, Tilles et al. 1986). Long-range attractive compounds have been described to be emitted by the conifer hosts. (Tilles et al. 1986). Pine weevils are able to migrate more than 10 km towards clear cut areas guided by odors emitted from the stumps (Solbreck 1980, Tilles et al. 1986). The monoterpene α-pinene (unknown mix of enantiomers) and ethanol are among these attractive compounds and these volatile compounds have also been found to be attractive in laboratory bioassays and in field studies (Mustaparta 1975, Nordlander 1990, Schlyter 2007). Recently it was revealed that (-)-α-pinene do not have an attractive effect on the pine weevil (Lina Lundborg et al submitted).

Host plants, non-host plants, pine weevil feces and fungi isolated from pine weevil feces are all known sources for antifeedant substances. The terpene substance limonene can inhibit the attraction of pine weevils to α-pinene as well as to other host volatiles (Nordlander 1990, Nordlander 1991). Verbenone is another monoterpene pine weevil antifeedant which is found in various plants. This substance is also produced by fungi and bacteria associated with the bark beetle by conversion of verbenol to verbenone (Lindgren et al. 1996, Agrawal et al. 2000, Xu et al. 2015). Fungi are capable to oxidize monoterpene hydrocarbons to verbenol and other oxygenated monoterpenes as pinocarveol and -terpineol (Fig 4, Appendix A).

Many aromatic compounds act as antifeedants. For instance ethyl trans- cinnamate and ethyl 2,3-dibromo-3-phenylpropanoate isolated from the lodgepole pine significantly reduce feeding in laboratory tests. Structure activity studies of derivatives from synthetic hydroxy, methoxy benzoic methyl esters, phenyl propanoic-, cinnamic- and benzoic acids (Bratt et al. 2001, Månsson et al. 2004, Unelius et al. 2006, Sunnerheim et al. 2007, Bohman et al. 2008, Eriksson et al. 2008).

Several aromatic antifeedants were found in methanol extracts of pine weevil feces, among others phenol, 2-methoxyphenol and 3-methylanisol (Borg-Karlson et al. 2006, Azeem et al. 2015). These were later found also in the volatile production of fungi associated with the pine weevil (Azeem et al. 2013, Azeem et al. 2015). The volatile nature of these compounds make them very short lived in bioassays and the antifeedant effect of these compounds was highly reduced after 24 hours (Borg- Karlson et al. 2006). However, a de novo production of antifeedants by microorganisms in the feces may be a way to protect the eggs from feeding pine weevils during the 1-4 weeks before the eggs hatch.

1.4. Microorganisms The microorganisms are grouped together based on the fact that they are not visible for the naked eye. The group is very diverse and includes organisms like virus, all prokaryotes (archeas and bacteria), protozoa as well as some eukaryotes, nematodes and some members of the fungi kingdom such as yeasts and molds. In the ecosystem they have different functions, for instance as primary producers which produce biomass. The photosynthetically active cyanobacteria which produce oxygen belong to this group. Another group is the detrivores which utilize decomposing plants, animals and feces as energy sources and by that recycles the nutrients. The microorganisms are also important for the different biogeochemical cycles of phosphor, sulfur, carbon, nitrogen and oxygen. Microorganisms are also important in more direct interactions with other living organisms including insects. These symbiotic relationships can be divided into five types. 1) The interactions may be mutualistic were both organisms benefit from each other, 2) commencialistic were one organism benefit without affecting the other, 3) parasitic were one organism is harmed by the benefit of the other, 4) amensalistic were one organism is harmed without affecting the other, and lastly 5) synnecrotic were the symbiosis is detrimental to both organisms involved which is rare. (Klepzig et al. 2009). These interactions are not only two dimensional, the importance of multitrophic intractions has gained increasing interest in research lately. When an insect feed on a plant, it does not only come in contact with the plant and the plant produced metabolic substances but also with the extensive microbiota on the surface of the host which also produce a variety of substances. The insect itself will in turn, together with its associated microorganisms expose the host for metabolic products in the feces and in the saliva.

This thesis focuses on the association of bacteria and their chemical interactions with the pine weevil.

1.4.1. Bacteria Many studies describes different symbiotic relationships between bacteria and insects ranging from antagonistic relations with pathogenic bacteria such as Bacillus thuringiensis (Ruiu 2015) to more beneficial relationships. One interesting field is the endosymbiotic relationships between gut microbes and their hosts. Insects are unable to synthesize sterols, 9 amino acids and B-vitamin. These nutrients has to be provided through their diet, and gut bacteria are known to provide different types of nutrients to insects feeding on plants with low or unbalanced nutrient content (Douglas 2013). Phloem, bark and wood feeding insects feed on

8 a carbon rich nutrient source with polysaccharides, cellulose, hemicelluloses and lignin as main components. Many insects have the intrinsic ability to digest cellulose but still utilize cellulolytic microorganisms (Behmer et al. 2003, Douglas 2009). Cellulose- degrading Actinobacteria associated with the invasive woodwasp Sirex noctilio Fabricius compensates for the insects inability to produce cellulases (Adams et al. 2011). Some wood feeding insects are known to harbor bacteria capable of nitrogen fixation and/or hydrolyzes of urea which compensates for a low abundance of nitrogen (Morales-Jiménez et al. 2013, Ayayee et al. 2014). The wood feeding insects encounter the chemical defense from their hosts and many insects are associated with terpene metabolizing bacteria (Adams et al. 2013, Boone et al. 2013) and aromatic degrading bacteria (Adams et al. 2013, Raffa 2014) which helps to detoxify these substances. Bacteria emit a wide range of volatiles which can act as signals in interactions with insects (Davis et al. 2013). For instance microorganisms in feces in German cockroach (Blatella germanica L.) produce volatile carboxylic acids which acts as aggregations agents to the host (Wada-Katsumata et al. 2015).

Only a few studies covers the microbial community of H. abietis and only one bacteria, Ewingella spp., has been isolated from the frass/feces and identified (Azeem et al. 2015). In his doctoral thesis, Azeem reports that feces from pine weevils feeding on Scots pine contain more bacteria and yeasts, and frass contains more fungi species (Azeem 2013).

1.4.2. Yeasts and fungi Yeasts and fungi occupy similar niches as bacteria. In antagonistic relationships with insects pathogenic fungi such as Beuveria and Metarhizium are well known examples and are often used as biopesticides (Scheepmaker et al. 2010). Pine weevils has been shown to be susceptible against these fungi in laboratory conditions (Ansari et al. 2012). Like bacteria fungi are known in many mutualistic relationships involving food processing, nutrient contribution and food detoxification (Christiaens et al. 1987, Douglas 2009). Wood feeding insects such as the Asian longhorned beetle and several termites are known to be associated with lignin and cellulose degrading fungi (Geib et al. 2008, Long et al. 2010). Fungi are able to produce sterols as do plants, an ability lacked by insects and most bacteria. In some mutualistic relationships fungal symbionts contribute with sterol synthesis to insects (Douglas 2009). In bark beetles (Dendroctonus) fungal symbionts may provide the host with ergosterol (Bentz et al. 2006) and also help in detoxification of monoterpenes (DiGuistini et al. 2011). Bark beetles are vectors for

9 phytopathogenic fungi which are introduced to trees when constructing mating chambers. These fungi produce toxins which may help the bark beetles in colonizing the trees (Paine et al. 1997, Klepzig et al. 2009, Lieutier et al. 2009).

Several fungi have been isolated and identified in association with pine weevil e.g. Ophiostoma piceae, O. pluriannulatum, Penicillium solitum, P. expansum, Mucor racemosus, Candida sequanensis. Some of them produce aromatic compounds with antifeedant or deterrent effects on the pine weevil (Azeem et al. 2013, Azeem et al. 2015, Azeem et al. 2015).

1.4.3. Microbial derived metabolites Microorganisms produce a wide variety of aromatic, terpene and aliphatic substances as a part of their metabolic processes. Their functions are not fully understood but many of them are essential for growth and development as well as being used in interactions with other organisms (Schulz et al. 2007, Citron et al. 2012).

Aromatic compounds are derived mainly from the shikimate pathway or by degradation of L-phenylalanine, L-tyrosine and lignin. Aromatic volatiles are produced by many types of bacteria (e.g. Pseudomonas, Enterobacteriacea and Klebsiella), yeasts and fungi (e.g. Candida tropicalis, Ophiostoma sp. and Heterobasidion occidentale) (Schulz et al. 2007, Gosset 2009, Hansson et al. 2012, Krastanov et al. 2013). In pine weevils one bacteria Ewingella sp. and several yeasts and fungi (e.g. Candida sequanensis, Penicillum expansum and Ophiostoma piceae) produce styrene, benzylalcohol, 2-phenylethanol, 2-methoxyphenol etc. which have antifeedant effect on the pine weevil (Azeem et al. 2013, Azeem et al. 2015).

Terpene compounds in microorganisms are derived from the condensation of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). The production of these precursors are in bacteria mainly served by the methyl-D-erythritol phosphate (MEP) pathway and in eukaryotes and archaea mainly from the mevalonate (MVA) pathway (Lombard et al. 2011, Yamada et al. 2015). Microorganisms are also able to transform terpenes they encounter in their environment to use them as a source of carbons or for detoxification in high concentrations. Transformation often involve oxidation or hydroxylation of the original terpenes (Marmulla et al. 2014). Several bacteria associated to bark beetles (Dentroctonus valens) are capable to

10 produce verbenone, an anti-aggregation pheromone in some bark beetles feeding on fresh dead wood and a feeding deterrent in pine weevils (Lindgren et al. 1996, Staffan Lindgren et al. 2002, Xu et al. 2015). Verbenone has also been found in the volatile production by fungi associated with other bark beetles (Brand et al. 1975, Hunt et al. 1989).

Aliphatic compounds are derived from carbohydrates, fatty acids or amino acids (Schwab et al. 2008). Saturated and unsaturated hydrocarbons, alcohols, aldehydes, ketones etc. are all common parts of the volatile profile from bacteria and fungi (Kai et al. 2009).

2. Materials and methods

2.1. Biological material and procedures

2.1.1. Collection of pine weevils Paper I and III Field collected female weevils were first brought to the lab were they were kept in darkness at 10°C until the start of the experiments. The weevils were kept in large boxes and provided with fresh branches of Scots pine (Pinus sylvestris), and water. The box had two bottoms with a grid were the insects were kept. All frass could fall down through the grid to the second bottom, this way the box could be kept clean.

Paper II Pine weevils were collected at fresh clear cuts from three countries in Europe; Sweden (Umeå, Uppsala, Asa and Gotland), Germany (Hannover) and Spain (Galicia). The weevils were captured in pit-fall traps baited with α-pinene and ethanol. The weevils were placed in boxes with holes for ventilation and sent alive to the laboratory. The weevils were kept at 10°C no longer than two days before dissection.

2.1.2. Host plant materials for the pine weevil Paper III The weevils were divided into three groups and assigned to eat Scots pine, silver birch or bilberry. All plant material was collected in the forest in Hemmesta, Värmdö, Sweden. Pine and birch twigs were collected from one tree respectively, and bilberry branches were collected from shrubs growing in close vicinity from each other. All weevils were kept in Falcon tubes with airholes. The weevils were provided fresh food and water and their cages were cleaned every day. The weevils were reared on the diet for 28 days.

2.1.3. Dissection of pine weevils Before dissection all weevils were cleaned to remove outside contaminants. This procedure differed between studies.

Paper I The weevils were surface sterilized by dipping them individually in 70% ethanol followed by rinsing them in phosphate buffer saline.

Fig. 2 Dissection of the pine weevil gut Paper II and III The procedure started with submerging them in 70% ethanol for 15 seconds, then 1 time in 10% hypochlorite with 1% tween 20 for 15 seconds, and last 2 times in PBS-buffer for 15 seconds. Dissection was performed under sterile conditions with the weevils submerged under sterile water. All instruments were dipped in ethanol and flamed between the treatments of each individual. The hind guts were cut out from the weevils and placed in sterile Eppendorf tubes containing sterile saline with 20% glycerol was added. All samples were frozen in - 80°C.

2.1.4. Isolation of bacteria Isolation of bacteria was done for paper I by homogenizing guts from five female weevils in saline solution. This mix was diluted 1:10, 1:100, and 1:1000 times in sterile saline solution. These diluted samples and the original solution were spread (100 µl) on 1.5% Nutrient agar (NA)-plates and the plates were incubated for 24 hours at 20°C (room temperature), 30°C or 37°C. Single colonies growing on the NA-plates were isolated and pure cultures of the bacterial species were maintained on plates and also kept in glycerol stocks.

2.1.5. Microbial culturing media Since the volatile metabolites produced by bacteria may vary depending on the nutrients available a set of different culturing media were used (paper I)

These were: a) NB-medium b) NB medium diluted in autoclaved millipore water to 10% c) 1% autoclaved grated Scots pine phloem from twigs (natural nutrient). in 10% diluted NB-medium d) 1% autoclaved grated phloem in autoclaved millipore water e) NB-medium diluted to 10% and supplemented with ring-13C6 L-phenylalanine (99 %, Larodan)

The different media were used for different purposes a) to screen for interesting volatiles; b) to follow up the ability to produce volatiles under less nutrition; c) to identify host specific degradation products; d) to study the ability of the bacteria to utilize host plant for production of volatiles under more realistic conditions; e) to confirm and characterize the different ability of the bacterial isolates to utilize L-phenylalanine for the production of aromatic compounds. The incorporation of the carbon isotope in a microbial produced compound was measured by calculating the isotope ratio with the following formula [(M+6)/M x 100%], were M is molecular ion and M+6 is the molecular weight of the isotope containing molecular ion.

The volatile emissions of all isolates were screened for substances with possible antifeedant effect. The isolates were grown over night, i.e. 16 hour growth, in 50 ml of Nutrient Broth (NB) medium in a 250 mL Erlenmeyer flask which was closed first with aluminum foil and then with Parafilm. The isolates identified as Rahnella aquatilis and Brevundimonas nasdae were selected for additional investigations based on their differences in volatile profiles.

All isolates were pre grown in NB-medium for 7 hours at their optimal temperature. Isolates Rahnella aquatilis isolate Ha1 and Brevimundimonas nasdae isolate Ha2 were incubated at 30°C, and R. aquatilis isolate Ha3 was incubated at 25°C. After incubation the pre culture was added to 20 ml of one of the four media. Media of type b), c) and d) were incubated at the optimal incubation temperature for 16h. Medium of type e) were grown for 24 h at 25°C.

2.1.6. Identification of microorganisms

Bacterial DNA extraction and PCR

Paper I Guts from five individually dissected pine weevils were used. For identification of bacterial isolates, PCR of 16S was performed on pure colonies from each strain using Illustra PuRe Taq Ready-To-Go PCR Beads (GE Healthcare, Uppsala, Sweden). The 16S rRNA was amplified using primers 341f (5’-CCTACGGGNGGCWGCAG-3’) and 1401r (5’- CGGTGTGTACAAGACCC-3’). Amplification products were sequenced at Macrogen (South Korea).

Paper II and III Guts from dissected pine weevils collected from the same trap were pooled with an average of five individuals in paper II and from five pooled individuals from the same treatment in paper III. The guts were flash frozen in liquid nitrogen and homogenized. The samples were pretreated with an enzymatic lysis buffer for Gram-positive bacteria and the DNA was extracted by a Quiacube automated extraction robot (Qiagen) using the QIAamp DNA mini kit (Quiagen) (Paper II and III).

In paper II, the identification of bacteria was performed using 454 sequencing at SciLife Lab, Uppsala. The 16S rDNA genes were targeted using general eubacterial primers 8f (5’-AGAGTTTGATIITGGCTCAG- 3’) and 1501r (5’-CGGITACCTTGTTACGAC-3’) (Lindh et al. 2005). For the identification of bacteria, only the V1-V2 regions of 16S were used. Analysis of 454 sequences was done at the Max Planck Institute for Chemical Ecology in Jena (Paper II).

In paper III the identification of bacteria was performed using MiSeq sequencing at SciLife Lab, Uppsala. The 16S rDNA (Escherichia coli position 341-805) was targeted using general bacterial primers 341F (CCTACGGGNGGCWGCAG) and 805R (GACTACIIVGGGTATCTAATCC). This primer pair matches around 90% of all good quality bacterial sequences and covers all phyla in the Ribosomal Database Project release 10.25 (Herlemann et al. 2011).

2.1.7. Collection of headspace volatiles released by bacteria Solid Phase Micro Extraction (SPME) is a solvent free extraction method used to sample and concentrate minute amounts of volatiles (Zhang et al. 1993, Fäldt et al. 2000, Vas et al. 2004). It is extremely sensitive especially when used in static analysis, e.g. the head space (HS) above a sample in an open or closed container without moving air (Fäldt et al. 2000). The method is non-destructive and is therefore well suited for detection of the small amounts of the volatiles continuously produced by living organisms. Insects, plants, microorganisms as well as non-living objects as drugs, trace amounts of insecticides in food etc. has all been studied with the help of SPME (Borg-Karlson et al. 1996, Fäldt et al. 1999, Dickschat et al. 2005, Lindh et al. 2008, Sreekumar et al. 2009, Levi-Zada et al. 2011, Zhao 2011, Cordero et al. 2012, Risticevic et al. 2012, Azeem et al. 2013, Kännaste et al. 2013). A wide range of commercially available fiber coatings exists for the detection and analysis of various compounds. In this thesis 65 µm polydimethylsiloxane/ divinylbenzene (PDMS/DVB Supelco, USA) SPME fiber was used to detect low and medium weight molecules such as monoterpenes, sesquiterpenes, alcohols, ketones, aromatic compounds etc. which are known to be released by growing bacteria. In paper I volatiles from isolated bacteria inoculated to different media, e.g. 1% grated bark in water, as well as uninoculated media (control) were collected by SPME. The volatile extractions were done at room temperature and the extraction time was 3 hours. The isolates were grown in 50 ml of medium in 250 mL Erlenmeyer flasks which were closed with aluminum foil and then with Parafilm wrapped around the rim of the opening to seal the aluminum close to the glass. The heat sterilized SPME needle was inserted through a hole made in the aluminum foil.

In paper III volatiles from feces of pine weevils reared on pine, birch or bilberry were collected. Feces and water were placed in SPME vials. The naturally occurring microorganisms were allowed to grow with the feces as substrate in water. Volatiles were collected at two time points, the first after 3-5 hours, and the second after 24 hours. The volatiles were collected with an SPME autosampler for 40 minutes.

In this thesis we have focused on a qualitative analysis of the volatiles emitted from microorganisms. The relative proportions of the emitted substances are important for the pine weevil behavior as they come in close contact with the substrate. Furthermore the continuous production of volatiles from the bacteria creates a system which never reach an equilibrium between the concentration of substances in the HS and the

16 adsorption on the fiber. This system has been termed biodynamic SPME instead of static (Azeem 2013). A quantification approach in such a system is possible but maybe not completely reliable.

2.1.8. Separation and identification of volatiles In paper I a Varian 3400 GC was used to separate the volatiles produced by the isolated bacteria. The GC was equipped with a DB-wax (Agilent, USA) capillary column. The temperature program of the GC oven was set to 40°C for 1 min followed by an increase of 5°C/min up to 225°C, then isothermal for 12 min. Injector temperature was 220°C and the split was closed for 30 sec (splitless injection). The volatiles were transferred to a Finnigan SSQ7000 MS instrument were they were fragmented with Electron Ionization (EI at 70eV). The mass spectra and retention times of the compounds were compared with those of authentic standards and mass spectra obtained from NIST 2008 reference library and Mass finder version 3.

In paper III volatiles were analyzed using an Agilent HP7890A GC equipped with a HP-5MS capillary column (30 m, ID 0.25 mm, film thickness 0.25 µm, Agilent Technologies, CA, USA). The temperature program was set to 40ºC for 1 min, increasing to 160ºC at a rate of 4ºC/min followed by a rise to 230ºC at a rate of 10ºC/min, and then remaining constant for 6 min. The GC was connected to a 5975C inert MSD with triple axis detector. The volatiles were identified using the retention times and mass spectra which were compared to the spectra in the NIST Mass Spectral Library using the G1701EA MSD ChemStation software (Agilent Technologies, CA, USA).

2.1.9. Bioassay

Fig. 3 Two-choice laboratory bioassay

The response of both sexes of pine weevils towards selected bacterial volatiles was tested by means of a two-choice laboratory bioassay (Paper I). This test has been used in several previous studies (Bratt et al. 2001, Legrand et al. 2004, Borg-Karlson et al. 2006, Unelius et al. 2006, Sunnerheim et al. 2007). Twigs of Scots pine wrapped in aluminum foil with two equally sized holes were applied with pure substances dissolved in methanol at one end and pure solvent at the other according to a standardized method. The solvent was allowed to evaporate before the bioassay started which lasted for 6 or 24 hours. After the stipulated time an antifeedant index (AFIa) was calculated based on the proportions of the treated bark area and control bark area that had been consumed by the pine weevils in each test twig. AFIa = 100x(C-T)/(C+T); C represents the mean area of control surfaces consumed and T the mean area of treated surfaces consumed. Positive values (up to a maximum of 100) reflect an antifeedant effect, whereas negative values (down to a minimum of -100) indicate a stimulant effect on feeding.

3. Culturable bacteria associated with the pine weevil Paper I During egg laying the pine weevil females plugs the hole where she has put her egg with feces and chewed bark as a protection from other feeding pine weevils. Our hypothesis is that microorganisms present in the feces and in the bark are able to produce antifeedants which acts on pine weevils and will prevent them from feeding on the egg laying site. To test this hypothesis we isolated bacteria that exist in close association with the pine weevil female and examined the volatile compounds emitted from cultures of those bacteria. To mimic the conditions at the egg laying site the isolates were cultured in aerobic conditions at room temperature. Different culture media were chosen to be able to examine the variations in the volatile profiles of the bacteria depending on nutrient availability. A medium with only grated bark as a nutrient source was utilized as a substitute for the natural medium for the bacteria. Since this media were closest to the natural situation we expected to detect the most relevant antifeedant compounds from bacteria growing on this media.

We focused on the following questions: 1. Which bacteria can be isolated from the gut of pine weevil females? 2. Which volatiles are emitted by the isolated bacteria? 3. Do the volatiles correspond with the substances detected from pine weevil feces?

From five female guts a total of 16 isolates were isolated and out of those 13 belonged to Rahnella aquatilis. The tree others were identified as Brevundimonas nasdae, Kocuria kristinae and Micrococcus terreus. Furthermore, R. aquatilis was found in at least 50% of all samples across Europe (Paper II). R. aquatilis has been found in various environments and in associations with both plants and animals, for example in all life stages of the wood feeding cerambycid beetles (Rhagium inquisitor L.) (Gruenwald et al. 2010), in the bark beetles such as the red turpentine beetle (Dendroctonus valens LeConte) and in a bark beetle endemic to Mexico (D. rhizophagus Thomas&Bright),(Morales-Jimenez et al. 2012). R. aquatilis is known to be able to decrease the concentrations of monoterpenes and worth to mention is its ability to reduce the amount of α-pinene (Boone et al. 2013, Raffa 2014) as well as fix nitrogen with urea as a nitrogen source (Morales-Jiménez et al. 2013). The ability to degrade terpene detoxifies the food from and provides the weevil with available nitrogen as well as recycling the nitrogen. Another Brevundimonas sp, Brevundimonas vesicularis. has been found in mountain pine beetle (D.

19 ponderosae Hopkins) where it reduces diterpenes (Boone et al. 2013). The two gram positive and closely related bacterial species K. kristinae and M. terreus. were both found in the gut of the tobacco horn worm (Manduca sexta) and in addition the Kocuria species were detected in the wood boring beetle (Anoplophora chinensis Forster, (van der Hoeven et al. 2008, Rizzi et al. 2013).

Volatiles from bacterial isolates

The headspace volatiles of all isolates grown in NB-medium were screened and R. aquatilis and B. nasdae were chosen for further analysis based on their differences in volatile profiles. Emissions of aromatic compounds were especially interesting as they have previously been found in feces of pine weevils and some with have an antifeedant function for pine weevils (Borg-Karlson et al. 2006). R. aquatilis produced mainly 3-methyl-1-butanol and 2-phenylethanol in NB- medium and developed a greater variety of substances in grated bark phloem in water (the natural medium), than in NB-medium. The compound detected in highest amount was 2-methoxyphenol while 2- phenylethanol, phenylacetaldehyde, phenylmethanol, benzyl methyl ether and 2,3-dihydrobenzofuran were also detected. Besides the aromatic compounds we found oxygenated terpenoids, 3-octanone and structurally similar aliphatic compounds (paper I). When growing R. aquatilis in diluted NB-medium supplemented with 13C-ring-labeled L-phenylalanine its ability for production of aromatic compounds was confirmed, 2-phenylethanol, phenylacetaldehyde, phenylmethanol, benzyl methyl ether and benzaldehyde where all labelled which were expected from known metabolic pathways. 2-methoxyphenol on the other hand was not labelled suggesting that it is not produced by the same pathway, R. aquatilis may degrade lignin or polyphenols in the grated phloem as a precursor for this compound.

B. nasdae produced small amounts of the aromatic compounds phenylmethanol and 2-phenylethanol in NB-medium together with oligosulfides and an alkylpyrazine. B. nasdae was found to have a low ability to utilize grated bark as a substrate and L-phenylalanine as a precursor for aromatic compounds.

In the work of Azeem (2013) Ewingella sp. was isolated and grown in a medium made of pine weevil feces. In this medium the Ewingella sp. produced 2-methoxyphenol and 2-methoxy-4-vinylphenol as the main compounds (detected in highest amount) followed by 3-methyl-1- butanol, methyl salicylate and phenol (Azeem 2013). In that study

20 microorganisms were isolated on sterile agar plates supplemented with pine weevil frass and feces. Ewingella and Rahnella are closely related and taken together the results presented in this thesis and by Azeem 2013 indicate that they share the metabolic pathways involved in production of aromatic compounds.

The volatile emissions from the Rahnella and Ewingella species corresponds in part with the aromatic substances found in solvent extractions of pine weevil feces. Among other compounds were phenol, 2-methoxyphenol and 2,3-dihydrobenzofurane together with many other compounds related to lignin degradation detected in the extracts. Bacteria and fungi present in association with the pine weevil and its host, seems to be able to contribute to the degradation of lignin. Bacteria and yeasts starts their production of volatile compounds within 24 hours after inoculation on culture media whereas fungi starts their volatile production after 3-4 days which thereafter continues for weeks (Azeem 2013), as long as there are nutrients available. Taken together this suggests that if the microbial consortium associated with the pine weevil contribute to protection of the pine weevil egg at the egg laying site several species of both fungi and bacteria are probably important since the egg have to be protected from egg laying to egg eclosion (up to a month in field conditions)(Nordlander et al. 1997).

4. Horizontal transfer of microorganisms

In paper II we studied the gut bacterial community in adult pineweevils across Europe. Weevils were collected from four sites in Sweden (Uppsala, Umeå, Asa and Gotland), as well as one site each in Germany and in Spain.

We focused on the following questions:

1. Which bacteria are present in pine weevils from all countries? 2. What is the potential role of the bacteria associated with the pine weevils?

The bacterial sequences were taxonomically assigned into operational taxonomic units (OTUs) using 97% similarity as a threshold. The core microbiota was defined as OTUs present in more than 50% of the samples in this study. Irrespective of the geographic region the most dominant bacteria in the gut microflora was one strain of Wolbachia. To be able to get a more detailed insight into the microbial community this species had to be removed from the data analysis. This revealed 16 OTUs that were found in the gut from all three countries. Thirteen belonged to the Enterobacteriacea family and the remaining three to the Firmicutes phylumn. Only four OTUs were found in more than 50% of the samples, all of them belonged to the Enterobacteriaceae family. The closest relatives were Erwinia typographi, Rahnella and Serratia. E. typographi was mainly found in the German and Spanish samples, but were detected in the Swedish samples in low amounts. The Ewingella sp. which has previously been isolated from pine weevil frass and feces, (Azeem et al. 2015) was also detected in pine weevils collected in most locations in this study. Ewingella was present in 13 of 28 samples and did not reach the 50% limit to be core bacteria.

Germany and Sweden (Gotland and some samples in Uppsala) shared a common abundance of Firmicutes which was absent from the Spanish samples. A high abundance of Firmicutes was correlated with a low abundance of Proteobacteria and vice versa, perhaps by a mutual competitive exclusion in the weevil’s gut.

The microbial community of the pine weevil was compared with the communities of other wood feeding insects. It was found that the bacterial community in pine weevils resemble the gut communities in bark beetle species feeding on conifers but not to weevils feeding on non-

22 conifer food sources. Several bacteria belonging to the Enterobacteriacea family in the pine weevil, including all of the core bacteria, were closely related phylogenetically with microbiota from Dendroctonus ponderosae Hopkins, D. rhizophagus, D. frontalis Zimmerman D. valens and Ips pini Say.

In the metagenome of the pine weevil bacterial flora genes of symbiotic importance and genes corresponding with diterpene degradation as well as genes corresponding with metabolism of amino acids, carbohydrates and vitamins were found to be likely present. In other wood feeding insects bacteria has been found to contribute with terpene and diterpene degradation. In the bark beetle Dendroctonus ponderosae, the bacteria Rahnella aquatilis, Serratia marcescens, Pseudomonas mandelii, P. migulae and Brevundimonas vesicularis degrade terpenes (Boone et al. 2013, Raffa 2014). Other bacteria has been found to degrade diterpenes in vitro such as Pseudomonas rhodesiae, P. fluorescens and Burkholderiales spp. (Bicas et al. 2008, Adams et al. 2013) and aromatic compounds Pseudomonas sp. and R. aquatilis (Jensen et al. 2001, Vasudevan et al. 2011).

The pine weevil feed on a substrate with low nitrogen content and this may be overcome by nitrogen fixing bacteria. Several Rahnella strains are known to have nitrogen fixing capacity or urolytic capacity and by that make the nitrogen available for the insect host (Morales-Jiménez et al. 2013). Wolbachia are commonly infecting insect`s reproductive organs and guts. Up to two-thirds of all insects examined has been found infected (Hilgenboecker et al. 2008). In their associations with insects they are known to be involved in both parasitic and mutualistic relationships with their host insects. For instance, they have been described to effect the host reproduction, increase the host resistance towards virus infections and to contribute with nutrition. Due to the high abundance in the pine weevils examined in this study and the location in the guts we speculate that Wolbachia may have a nutritional function in this system (Douglas 2009).

In conclusion, the bacteria associated with the gut of the pine weevils appear to have the capacity to provide the host with nutrition as well as to be able to detoxify the food from pine tree host chemical defenses.

5. Dietary effects on microflora

In paper III we studied the dietary effects on the microflora in the gut and frass of the pine weevil. The weevils were reared on Scots pine, silver birch or bilberry which are all part of their natural diets.

We focused on the following questions: 1. What effect does diet have on the gut and frass microflora? 2. What volatiles are emitted from the feces and how do they correspond to the different diets? 3. Do the volatiles from the microbial consortium correspond with volatiles known to be emitted from bacteria previously isolated from the feces?

No significant difference in gut microflora was observed between the weevils feed on different diets for 28 days. The two dominant species in the gut was a Wolbachia sp. with a mean relative abundance of 48% (range: 13-94%), and R. aquatilis with a mean relative abundance of 48% (range 3-84%). The microbial community in the frass differed from that in the guts (Fig. 4).

Fig. 4 NMDS plots of bacteria OTUs in pine weevil guts and in their frass colored according to food source. A. with all bacteria OTUs, stress value: 0.098 B. Without Wolbachia OTUs stress value 0.88

Bacterial species belonging to the Proteobacteria phylum dominated the frass microflora and of those species in the gammaproteobacteria class was most abundant. In contrast in one sample a high number of species belonging to the Firmicutes phylum were detected. This samples also had a low abundance of proteobacteria. A similar pattern was observed in paper II were the gut samples with high abundance of proteobacteria had low numbers of Firmicutes. R. aquatilis and two Pseudomonas species were the most abundant bacterial species in the frass. R. aquatilis had a mean relative abundance of 39% in pine, 12% in birch and 47% in bilberry.

An interesting pattern was found for the two Pseudomonas species. P. syringae was mainly found in birch and P. rhizospherae was found in bilberry. The pine samples had an intermediate ratio of the two species (Fig. 5). P. syringae is known as a pathogen of many plants and birch is a host tree for one of the strains (Stead et al. 2003, Morris et al. 2008).

Fig. 5 Proportions of Pseudomonas syringae and P. rhizospherae in the three different diets. An indicator value was calculated to find bacteria that were strongly associated with the diet. An indicator value of 1 suggests that the bacterium is only present in that diet. In pine four alphaproteobacteria and in bilberry a Stenotropononas spp. were found with an indicator value of 1, while birch was strongly associated with Pseudomonas syringae.

(Table 1. Indicator species in frass from pine weevils on different diets. aSpecies/other: lowest classification based on SILVAmod-database. bIndicator value calculated by indicator species analysis in R.

a Indicator Diet OTU Phylum Class Species/other b P-value value

568 Acetobacteraceae 1 0.009 Rhizobiales;1174-901- 387 1 0.015 Proteo- Alphaproteo- 12 bacteria bacteria Pine 771 Methylocella spp. 1 0.01 48 Caulobacteraceae 1 0.015

Acido- 777 Acidobacteria Acidobacteriaceae 0.91 0.031 bacteria

Proteo- Gammaproteo- Birch 26 Pseudomonas syringae. 0.81 0.003 bacteria bacteria

34 Gammaproteo- Stenotrophomonas spp. 1 0.008 339 Proteo- bacteria Enterobacteriaceae 0.53 0.043 Bilberry bacteria Betaproteo- 5 Comamonas spp. 0.95 0.011 bacteria

The volatile profiles from feces originating from pine weevils fed on the different diets differed in their composition. Feces from pine feeding pine weevils emitted 2-methoxyphenol, 2-methoxy-4-methylphenol and methyl salicylate as the three main aromatic compounds and 4-terpineol as major oxygenated monoterpene. In addition 3-methyl-1-butanol and phenol were present in the volatile blend from the consortium. Feces from birch contained mainly cis- and trans- linalool oxides of furan type and 2-methoxyphenol. Small amounts of linalool, linalool oxides of pyrane type and eugenol were also detected. Bilberry feces contained among other compounds small amounts of benzylalcohol, 2- phenylethanol, 2-methoxyphenol and cis- and trans- linalool oxides.

The volatiles were most likely produced by a microbial consortium present in the feces that probably included species of bacterial, yeast and fungal origin. Bacteria and yeasts present in the feces are probably the producers of the volatiles during the first days. The reason for this is that fungi are not as commonly present in feces as in frass and when present they start their volatile production after 4-5 days (Azeem 2013) whereas bacteria and yeasts are able to start their metabolism within 24 hours.

Volatiles detected above R. aquatilis cultures in paper I corresponds with the volatiles emitted from the feces in this study. R. aquatilis grown on

26 pine bark emitted for instance 2-phenylethanol, 2-methoxyphenol, phenol and 3-methyl-1-butanol (Paper I). All but 2-phenylethanol were detected in emissions from pine feces. Pseudomonas sp. are also known to be able to degrade aromatic compounds and may contribute to the volatile bouquet (Gosset 2009). P. rhizospherae and P. syringae were identified in paper III from pine weevil frass and their abundance were coupled to the food source. The two different strains may have slightly different metabolism something that is also likely to affect their volatile profile. Other bacteria that were less abundant in paper III may contribute to the volatile as well, but there influence should be less pronounced. Yeasts and fungi were not identified in this study but pine weevils reared on pine has been found associated with the yeasts species Debaryomyces hansenii and Candida sequanencis previously (Azeem 2013). D. hansenii is capable of methyl salicylate production while C. sequanencis is capable of production of styrene, 2-phenylethanol and 2- methoxyphenol when grown in medium with pine extracts (Azeem 2013, Azeem et al. 2015). Linalool oxide is produced in a numbers of plants for instance bilberry (Du et al. 2014) and birch (Hakola et al. 2001) and is known as a mosquito attractant (Nyasembe et al. 2015). One linalool oxides of furan type have been reported from volatile emissions of Pseudomonas sp. (Madyastha et al. 1977).

6. Microbial volatiles affecting pine weevil behavior

Paper I, III, Appendix A and B Under natural conditions the pine weevil encounters a mixture of volatiles which together forms the chemical information used. The feces contain various microorganisms with the capability to produce a large number of substances. The chemical defense induced in a host tree varies depending on its susceptibility to the invader and the type of infestation.

In paper I and III we searched for volatiles with a presumed antifeedant effect produced by individual isolates of bacteria as well as microbial consortiums. We wanted to examine the effect different growth substrates have on the production of volatile compounds and how pine weevils respond to the presence of selected microbially produced volatiles.

In paper I we examined how the pine weevil respond to 2-phenylethanol, 2-methoxyphenol and phenol in a two-choice laboratory bioassay. The compounds were tested in concentrations of 50 mM, 25 mM or 5 mM dilutions in methanol. An antifeedant effect (AFIa) was calculated based on the feeding area in the test site compared to the control site treated with pure methanol. 2-Phenylethanol and 2-methoxyphenol showed a strong dose dependent antifeedant effect that lasted for the entire bioassay period of 24 h except for the lowest dose tested for 2- methoxyphenol. Phenol was only tested at the highest concentration and the effect were short lasted with an AFIa of 36 after 24 h. The high concentrations used may give a deterrent effect initially but the highly volatile samples evaporate quickly. The lowest concentration are closer to natural emission rates during a 24 hour period based on the styrene productions of yeasts (10 µg/day) in weevil frass broth.

Yeast and fungi associated with pine weevils produce volatiles with deterrent effect when grown on pine bark extracts. Pure compounds were tested together with a pine twig in a multi choice arena. Methyl salicylate, styrene, 2-methoxyphenol and 2-methoxy-4-vinylphenol were all shown to have deterrent effect also in the presence of their natural food (Azeem 2013).

In Appendix A and B we also searched for volatiles with a presumed antifeedant effect produced by associated symbiotic fungi. Bark beetles are closely associated with fungi which facilitate the colonization of their host trees Picea abies. In Appendix B we showed that Endoconidiophora polonica and Heterobasidion parviporum produce a number of

28 oxygenated monoterpenes, and induce mono-, sesqui-, and diterpenes after inoculation in spruce clones. The clones have different capabilities to produce defense chemicals for the two fungi. H. parviporum inoculation resulted in a larger number of oxygenated monoterpenes than inoculation with E. polonica. Among others, terpinen-4-ol, pinocarveol, α-terpineol and myrtenal were found in the Heterobasidion infested tissues. Out of these, α-terpineol was a component in all clones of the Heterobasidion infested tissues especially pronounced in clone 393 and 457, two clones with high resistance towards Hetereobasidion infection, of the E. polonica infested tissues. The oxygenated monoterpenes are known semiochemicals (Schlyter et al. 2004) to the pine weevil and many have antifeedant properties.

In Appendix A we present evidence that the fungus Grosmannia europhioides is capable of de novo production of the major component in the bark beetle aggregation pheromone, 2-methyl-3-buten-2-ol (Zhao et al. 2015) but is also, as E. polonica (Appendix B), capable of producing a number of oxygenated monoterpenes which acts as antifeedants for the pine weevil.

Taken together an increase of oxygenated monoterpenes and aromatic compounds may serve as a cue of a host that is unsuitable for food (Schlyter et al. 2004).

Fig. 6 Pine weevil infochemicals

7. Summary

 Bacteria with the capability of producing pine weevil deterrent substances were isolated from adult pine weevil frass/feces.  These substances were produced from the first days of the period that is needed for protection of the egg.  The core gut bacteria were found to be closely related to Rahnella, Serratia, Pantoea and Erwinia spp.. A Wolbachia sp. was found in all guts.  Diet did not have any effect on the bacteria present in the gut in the period tested. However microorganisms from the host made a contribution to the difference found in bacterial flora between feces samples from pine weevils in the different diet regimes.  The bacterium Rahnella aquatilis was found in high abundance in all feces and gut samples from pine weevils.  The major volatiles found in the emissons from R. aquatilis isolates were 2-methoxyphenol, phenol and 2-phenylethanol. These compounds were also detected above the microbial consortium that were active in pine weevil feces.  Grosmannia europhioides is capable of de novo production of the major component in the bark beetle aggregation pheromone, 2-methyl-3-buten-2-ol.  We showed the different ability of Endoconidiophora polonica and Heterobasidion parviporum to produce oxygenated monoterpenes, and induce mono-, sesqui-, and diterpenes after inoculation in a clonal spruce trees of wich many act as antifeedants for the pine weevil

8. Future prospects To further investigate the function and generality in forest insect community of the chemical signals released from the dynamic complex mixture of bacteria, yeast and fungi, that produce semiochemicals.

Among others:  Identify and determine the function of the enantiomers of the chiral substances we found, e.g. linalool oxides,  Identify larger molecules, non-volatile substances, with deterrent effects with the use of LC-MS/HPLC  Test mixtures of volatiles emitted from a known consortia consisting of Rahnella aquatilis together with yeasts and fungi towards pine weevils in multiple choice arena tests.  Test the effect a microbial consortium has on pine weevils in lab situation on long term basis, a month, to see if the behavior is consistent and if an antifeedant effect would cover the period before egg eclosion.  Develop a matrix supplemented with nutrients which could harbor a microbial consortium. The matrix should be applied to the stem of conifer seedlings as a protection, both physically and by semiochemicals.  Investigate the competition among bacteria and fungi in the pine weevil gut to possibly explain the absence or presence of certain bacteria.  Investigate how bacteria are being transferred between the life stages of the pine weevil

Acknowledgements

This thesis could not have been finished without the help of my supervisors, colleagues, friends and family. I’m sincerely grateful and touched for all support, all help and all love I have received from you. My heartfelt thank you to all of you!

I would like to express my sincere gratitude to my supervisors for accepting me as a PhD-student. Prof. Anna-Karin Borg-Karlson you create a warm atmosphere which makes it easy to thrive and grow as a curious person in all aspects of life. My deepest thanks to Dr. Jenny Lindh for accepting the role as my main supervisor during the last shivering months of my Phd. You made this possible in so many ways by giving me constructive critizism in writing, finding good references and ways to solve problems of all sorts. I’m truly looking forward to visit your garden in July. My heartfelt appreciation goes to Assoc. Prof. Guna Kuttuva Rajarao. Your support and warm encouragement during my time in the microbiology lab is invaluable. Dr Tao Zhao, my warmest thank you for all your support during these years. I truly appreciate our trips to do field work and all time we spend together. You made me go to work with a smile and come home with an even bigger one!

My sincere gratitude for invaluable comments and suggestions during the writing process Prof. Torbjörn Norin, Prof. Anna-Karin Borg-Karlson, Dr. Jenny Lindh, Dr Tao Zhao, Prof. Göran Nordlander and Dr. Olle Terenius.

I want to thank all my collaborators in Sweden and abroad for the nice atmosphere and fruitful cooperation we have had. Especially I would like to thank Prof. Göran Nordlander, Research engineer Henrik Nordenhem, Dr. Olle Terenius, Louise Nilsson, Aileen Berasategui, Prof. Dr. Jonathan Gershenzon, Dr. Paal Krokene, Dr. Gunilla Swedjemark, Dr. Tao Zhao and Dr. Muhammad Azeem. Thank you all for your contributions to this thesis and publications.

I have had the opportunity to work in a group were my colleagues also are my friends. I will remember all interesting group meetings with presentations pitching all sorts of interesting topics, how we baked Lussekatter in the lunch room during the Christmas party, all birthday parties, the beach party in Grisslinge and all fun we have had together in and outside the lab. I want to thank you all for these fantastic years: Anna- Karin Borg-Karlson, Tao Zhao, Jenny Lindh, Lynda Kirie Eneh, Lina

33 Lundborg, Astrid Kännaste, Raimondas Mozuraitis, Marie Danielsson, Katinka Pålsson, Nélida González, Amene Zendegi, Peter Baeckstrom, Nancy Cabezas, Torbjörn Norin, Savina Asiimwe, Norihisa Kusumoto, Anders Molin, Rushana Murtazina, Muhammad Azeem, Maia Lescano, Xosé López, Madina Adia Mohamed, Francis Ocheng, Hesham El-Seedi, Alexey Kravchenko, Hiromo Saijo, Fredrik Schubert, Hylobius abietis and all former members of the Ecological Chemistry group.

A special thanks to all students and pupils I’ve met during the years. It was fun teaching you!

My sincere gratitude to all of you working in the administration. A special thanks to Lena Skowron, Henry Challis, Ulla Jacobsson and Vera Jovanovic for all help in practical questions.

FORMAS, SSF (Paretree), KTH (Department of chemistry), KTH and ÅF are gratefully acknowledged for financial support and travel stipends.

Thank you mum and dad, all my friends and relatives for your unconditional love and support during these years.

To Michael, thank you for being here by my side when I needed you the most, for better and worse. I love you!

To Linnea and Alve, my small giggles, I love you! Let’s go play in the garden!

References

Adams, A. S., F. O. Aylward, S. M. Adams, N. Erbilgin, B. H. Aukema, C. R. Currie, G. Suen and K. F. Raffa (2013). "Mountain Pine Beetles Colonizing Historical and Naive Host Trees Are Associated with a Bacterial Community Highly Enriched in Genes Contributing to Terpene Metabolism." Applied and Environmental Microbiology 79(11): 3468-3475. Adams, A. S., M. S. Jordan, S. M. Adams, G. Suen, L. A. Goodwin, K. W. Davenport, C. R. Currie and K. F. Raffa (2011). "Cellulose-degrading bacteria associated with the invasive woodwasp Sirex noctilio." ISME J 5(8): 1323-1331. Agrawal, R. and R. Joseph (2000). "Bioconversion of alpha pinene to verbenone by resting cells of Aspergillus niger." Applied Microbiology and Biotechnology 53(3): 335-337. Ansari, M. A. and T. M. Butt (2012). "Susceptibility of different developmental stages of large pine weevil Hylobius abietis (Coleoptera: Curculionidae) to entomopathogenic fungi and effect of fungal infection to adult weevils by formulation and application methods." Journal of Invertebrate Pathology 111(1): 33-40. Ayayee, P., C. Rosa, J. G. Ferry, G. Felton, M. Saunders and K. Hoover (2014). "Gut Microbes Contribute to Nitrogen Provisioning in a Wood-Feeding Cerambycid." Environmental Entomology 43(4): 903-912. Azeem, M. (2013). Microbes associated with Hylobius abietis: A chemical and behavioural study. PhD thesis, KTH. Azeem, M., G. Kuttuva Rajarao, O. Terenius, G. Nordlander, H. Nordenhem, K. Nagahama, E. Norin and A. Karin Borg-Karlson (2015). "A fungal metabolite masks the host plant odor for the pine weevil (Hylobius abietis)." Fungal Ecology 13: 103-111. Azeem, M., G. K. Rajarao, H. Nordenhem, G. Nordlander and A. K. Borg-Karlson (2013). "Penicillium expansum Volatiles Reduce Pine Weevil Attraction to Host Plants." Journal of Chemical Ecology 39(1): 120-128. Azeem, M., O. Terenius, G. K. Rajarao, K. Nagahama, H. Nordenhem, G. Nordlander and A.-K. Borg-Karlson (2015). "Chemodiversity and biodiversity of fungi associated with the pine weevil Hylobius abietis." Fungal Biology 119(8): 738-746. Behmer, S. T. and W. D. Nes (2003). "Insect sterol nutrition and physiology: A global overview." Advances in Insect Physiology, Vol 31 31: 1-72. Bentz, B. J. and D. L. Six (2006). "Ergosterol Content of Fungi Associated with Dendroctonus ponderosae and Dendroctonus rufipennis (Coleoptera: Curculionidae, Scolytinae)." Annals of the Entomological Society of America 99(2): 189-194. Bicas, J. L., P. Fontanille, G. M. Pastore and C. Larroche (2008). "Characterization of monoterpene biotransformation in two pseudomonads." Journal of Applied Microbiology 105(6): 1991-2001. Bohman, B., G. Nordlander, H. Nordenhem, K. Sunnerheim, A. K. Borg-Karlson and C. R. Unelius (2008). "Structure-activity relationships of phenylpropanoids as antifeedants for the pine weevil Hylobius abietis." Journal of Chemical Ecology 34(3): 339-352.

35 Boone, C. K., K. Keefover-Ring, A. C. Mapes, A. S. Adams, J. Bohlmann and K. F. Raffa (2013). "Bacteria Associated with a Tree-Killing Insect Reduce Concentrations of Plant Defense Compounds." Journal of Chemical Ecology 39(7): 1003-1006. Borg-Karlson, A. K. and R. Mozuraitis (1996). "Solid phase micro extraction technique used for collecting semiochemicals. Identification of volatiles released by individual signalling Phyllonorycter sylvella moths." Zeitschrift Fur Naturforschung C-a Journal of Biosciences 51(7-8): 599-602. Borg-Karlson, A. K., G. Nordlander, A. Mudalige, H. Nordenhem and C. R. Unelius (2006). "Antifeedants in the feces of the pine weevil Hylobius abietis: Identification and biological activity." Journal of Chemical Ecology 32(5): 943-957. Borg-Karlson, A. K., R. Nordlander, A. Mudalige, H. Nordenhem and C. R. Unelius (2006). "Antifeedants in the feces of the pine weevil Hylobius abietis: Identification and biological activity." Journal of Chemical Ecology 32(5): 943-957. Brand, J. M., J. W. Bracke, A. J. Markovetz, D. L. Wood and L. E. Browne (1975). "Production of verbenol pheromone by a bacterium isolated from bark beetles." Nature 254(5496): 136- 137. Bratt, K., K. Sunnerheim, H. Nordenhem, G. Nordlander and B. Langstrom (2001). "Pine weevil (Hylobius abietis) antifeedants from lodgepole pine (Pinus contorta)." Journal of Chemical Ecology 27(11): 2253-2262. Bylund, H., G. Nordlander and H. Nordenhem (2004). "Feeding and oviposition rates in the pine weevil Hylobius abietis (Coleoptera : Curculionidae)." Bulletin of Entomological Research 94(4): 307-317. Christiaens, E., W. Hansen and J. Moinet (1987). "Rahnella aquatilis isolated from the sputum of a patient with chronic lymphatid-leukemia." Medecine Et Maladies Infectieuses 17(12): 732-734. Citron, C. A., P. Rabe and J. S. Dickschat (2012). "The Scent of Bacteria: Headspace Analysis for the Discovery of Natural Products." Journal of Natural Products 75(10): 1765-1776. Cordero, C., S. A. Zebelo, G. Gnavi, A. Griglione, C. Bicchi, M. E. Maffei and P. Rubiolo (2012). "HS-SPME-GCxGC-qMS volatile metabolite profiling of Chrysolina herbacea frass and Mentha spp. leaves." Analytical and Bioanalytical Chemistry 402(5): 1941-1952. Davis, T. S., T. L. Crippen, R. W. Hofstetter and J. K. Tomberlin (2013). "Microbial Volatile Emissions as Insect Semiochemicals." Journal of Chemical Ecology 39(7): 840-859. Day, K. R., G. Nordlander, M. Kenis and G. Halldorson (2004). General biology and life cycles of bark weevils. Dordrecht, Kluwer Academic Publishers. Dickschat, J. S., H. Reichenbach, I. Wagner-Döbler and S. Schulz (2005). "Novel Pyrazines from the Myxobacterium Chondromyces crocatus and Marine Bacteria." European Journal of Organic Chemistry 2005(19): 4141-4153. DiGuistini, S., Y. Wang, N. Y. Liao, G. Taylor, P. Tanguay, N. Feau, B. Henrissat, S. K. Chan, U. Hesse-Orce, S. M. Alamouti, C. K. M. Tsui, R. T. Docking, A. Levasseur, S. Haridas, G. Robertson, I. Birol, R. A. Holt, M. A. Marra, R. C. Hamelin, M. Hirst, S. J. M. Jones, J. Bohlmann and C. Breuil (2011). "Genome and transcriptome analyses of the mountain pine beetle-fungal symbiont Grosmannia clavigera, a lodgepole pine pathogen." Proceedings of the National Academy of Sciences 108(6): 2504-2509.

Douglas, A. E. (2009). "The microbial dimension in insect nutritional ecology." Functional Ecology 23(1): 38-47. Douglas, A. E. (2013). "Microbial Brokers of Insect-Plant Interactions Revisited." Journal of Chemical Ecology 39(7): 952-961. Du, X. F. and R. Rouseff (2014). "Aroma Active Volatiles in Four Southern Highbush Blueberry Cultivars Determined by Gas Chromatography-Olfactometry (GC-O) and Gas Chromatography-Mass Spectrometry (GC-MS)." Journal of Agricultural and Food Chemistry 62(20): 4537-4543. Dudareva, N., A. Klempien, J. K. Muhlemann and I. Kaplan (2013). "Biosynthesis, function and metabolic engineering of plant volatile organic compounds." New Phytologist 198(1): 16- 32. Eriksson, C., P. E. Månsson, K. Sjödin and F. Schlyter (2008). "Antifeedants and Feeding Stimulants in Bark Extracts of Ten Woody Non-host Species of the Pine Weevil, Hylobius abietis." Journal of Chemical Ecology 34(10): 1290-1297. Evans, H. F. (2001). Forest Resources and Types, in The Forests Handbook, Volume 1: An Overview of Forest Science. The Forests Handbook, An Overview of Forest Science. J. Evans. Oxford, UK, Blackwell Science Ltd. 1. Franceschi, V. R., P. Krokene, E. Christiansen and T. Krekling (2005). "Anatomical and chemical defenses of conifer bark against bark beetles and other pests." New Phytologist 167(2): 353-375. Fäldt, J., M. Eriksson, I. Valterova and A. K. Borg-Karlson (2000). "Comparison of headspace techniques for sampling volatile natural products in a dynamic system." Zeitschrift Fur Naturforschung C-a Journal of Biosciences 55(3-4): 180-188. Fäldt, J., M. Jonsell, G. Nordlander and A. K. Borg-Karlson (1999). "Volatiles of bracket fungi Fomitopsis pinicola and Fomes fomentarius and their functions as insect attractants." Journal of Chemical Ecology 25(3): 567-590. Geib, S. M., T. R. Filley, P. G. Hatcher, K. Hoover, J. E. Carlson, M. Jimenez-Gasco Mdel, A. Nakagawa-Izumi, R. L. Sleighter and M. Tien (2008). "Lignin degradation in wood- feeding insects." Proc Natl Acad Sci U S A 105(35): 12932-12937. Gosset, G. (2009). "Production of aromatic compounds in bacteria." Current Opinion in Biotechnology 20(6): 651-658. Gruenwald, S., M. Pilhofer and W. Hoell (2010). "Microbial associations in gut systems of wood- and bark-inhabiting longhorned beetles Coleoptera: Cerambycidae." Systematic and Applied Microbiology 33(1): 25-34. Hakola, H., T. Laurila, V. Lindfors, H. Hellen, A. Gaman and J. Rinne (2001). "Variation of the VOC emission rates of birch species during the growing season." Boreal Environment Research 6(3): 237-249. Hansson, D., A. Menkis, A. Olson, J. Stenlid, A. Broberg and M. Karlsson (2012). "Biosynthesis of fomannoxin in the root rotting pathogen Heterobasidion occidentale." Phytochemistry 84: 31-39. Herlemann, D. P., M. Labrenz, K. Jurgens, S. Bertilsson, J. J. Waniek and A. F. Andersson (2011). "Transitions in bacterial communities along the 2000 km salinity gradient of the Baltic Sea." Isme j 5(10): 1571-1579.

37 Hilgenboecker, K., P. Hammerstein, P. Schlattmann, A. Telschow and J. H. Werren (2008). "How many species are infected with Wolbachia? – a statistical analysis of current data." Fems Microbiology Letters 281(2): 215-220. Hunt, D. W. A. and J. H. Borden (1989). "Terpene alcohol pheromone production by Dentroctonus ponderosae and Ips paraconfusus (Coleoptera, Scolytidae) in the absence of readily culturable microorganisms " Journal of Chemical Ecology 15(5): 1433-1463. Jensen, N., P. Varelis and F. B. Whitfield (2001). "Formation of guaiacol in chocolate milk by the psychrotrophic bacterium Rahnella aquatilis." Letters in Applied Microbiology 33(5): 339-343. Kai, M., M. Haustein, F. Molina, A. Petri, B. Scholz and B. Piechulla (2009). "Bacterial volatiles and their action potential." Applied Microbiology and Biotechnology 81(6): 1001-1012. Kalo, P. and A. Nederstrom (1986). "Female-specific compounds in the ovaries of the large pine weevil, Hylobius abietis (L) (Coleoptera, Curculionidae)." Annales Entomologici Fennici 52(3): 95-+. Karlson, P. and M. Lüscher (1959). "Pheromones: a New Term for a Class of Biologically Active Substances." Nature 183(4653): 55-56. Klepzig, K. D., A. S. Adams, J. Handelsman and K. F. Raffa (2009). "Symbioses: A Key Driver of Insect Physiological Processes, Ecological Interactions, Evolutionary Diversification, and Impacts on Humans." Environmental Entomology 38(1): 67-77. Kolmodin-Hedman, B., M. Åkerblom, S. Flato and G. Alex (1995). "Symptoms in forestry workers handling conifer plants treated with permethrin." Bulletin of Environmental Contamination and Toxicology 55(4): 487-493. Krastanov, A., Z. Alexieva and H. Yemendzhiev (2013). "Microbial degradation of phenol and phenolic derivatives." Engineering in Life Sciences 13(1): 76-87. Kännaste, A., T. Zhao, A. Lindstrom, E. Stattin, B. Langstrom and A. K. Borg-Karlson (2013). "Odors of Norway spruce (Picea abies L.) seedlings: differences due to age and chemotype." Trees-Structure and Function 27(1): 149-159. Leather, S. R., K. R. Day and A. N. Salisbury (1999). "The biology and ecology of the large pine weevil, Hylobius abietis (Coleoptera : Curculionidae): a problem of dispersal?" Bulletin of Entomological Research 89(1): 3-16. Legrand, S., G. Nordlander, H. Nordenhem, A. K. Borg-Karlson and C. R. Unelius (2004). "Hydroxy-methoxybenzoic methyl esters: Synthesis and antifeedant activity on the pine weevil, Hylobius abietis." Zeitschrift Fur Naturforschung Section B-a Journal of Chemical Sciences 59(7): 829-835. Levi-Zada, A., D. Fefer, L. Anshelevitch, A. Litovsky, M. Bengtsson, G. Gindin and V. Soroker (2011). "Identification of the sex pheromone of the lesser date moth, Batrachedra amydraula, using sequential SPME auto-sampling." Tetrahedron Letters 52(35): 4550- 4553. Lieutier, F., A. Yart and A. Salle (2009). "Stimulation of tree defenses by Ophiostomatoid fungi can explain attack success of bark beetles on conifers." Annals of Forest Science 66(8). Lindgren, B. S., G. Nordlander and G. Birgersson (1996). "Feeding deterrence of verbenone to the pine weevil, Hylobius abietis (L) (Col, Curculionidae)." Journal of Applied Entomology- Zeitschrift Fur Angewandte Entomologie 120(7): 397-403.

Lindh, J. M., A. K. Borg-Karlson and I. Faye (2008). "Transstadial and horizontal transfer of bacteria within a colony of Anopheles gambiae (Diptera: Culicidae) and oviposition response to bacteria-containing water." Acta Tropica 107(3): 242-250. Lindh, J. M., O. Terenius and I. Faye (2005). "16S rRNA gene-based identification of midgut bacteria from field-caught Anopheles gambiae sensu lato and A. funestus mosquitoes reveals new species related to known insect symbionts." Applied and Environmental Microbiology 71(11): 7217-7223. Lombard, J. and D. Moreira (2011). "Origins and early evolution of the mevalonate pathway of isoprenoid biosynthesis in the three domains of life." Mol Biol Evol 28(1): 87-99. Long, Y.-H., L. Xie, N. Liu, X. Yan, M.-H. Li, M.-Z. Fan and Q. Wang (2010). "Comparison of gut-associated and nest-associated microbial communities of a fungus-growing termite (Odontotermes yunnanensis)." Insect Science 17(3): 265-276. Lynch, A. M. (1984). "The pales weevil, Hylobius pales (Herbst) - A synthesis of the literature." Journal of the Georgia Entomological Society 19(3): 1-34. Långström, B. and K. R. Day (2004). Bark and wood boring insects in living trees in Europe, a Synthesis, Springer Netherlands. Madyastha, K., P. K. Bhattacharyya and C. S. Vaidyanathan (1977). "Metabolism of monoterpene alcohol, linalool, by a soil pseudomonad." Can J Microbiol 23(3): 230-239. Maeda, H. and N. Dudareva (2012). "The Shikimate Pathway and Aromatic Amino Acid Biosynthesis in Plants." Annual Review of Plant Biology, Vol 63 63: 73-105. Marmulla, R. and J. Harder (2014). "Microbial monoterpene transformations—a review." Frontiers in Microbiology 5: 346. Morales-Jiménez, J., A. Vera-Ponce De León, A. García-Domínguez, E. Martínez-Romero, G. Zúñiga and C. Hernández-Rodríguez (2013). "Nitrogen- Fixing and Uricolytic Bacteria Associated with the Gut of Dendroctonus rhizophagus and Dendroctonus valens ( Curculionidae: Scolytinae)." Microbial Ecology 66(1): 200-210. Morales-Jimenez, J., G. Zuniga, H. C. Ramirez-Saad and C. Hernandez-Rodriguez (2012). "Gut- Associated Bacteria Throughout the Life Cycle of the Bark Beetle Dendroctonus rhizophagus Thomas and Bright (Curculionidae: Scolytinae) and Their Cellulolytic Activities." Microbial Ecology 64(1): 268-278. Morris, C. E., D. C. Sands, B. A. Vinatzer, C. Glaux, C. Guilbaud, A. Buffiere, S. Yan, H. Dominguez and B. M. Thompson (2008). "The life history of the plant pathogen Pseudomonas syringae is linked to the water cycle." ISME J 2(3): 321-334. Mustaparta, H. (1975). "Behavioral responses of pine weevil Hylobius abietis L. (Col. Curculionidae) to odors activatin different groups of receptor cells " Journal of Comparative Physiology 102(1): 57-63. Månsson, P. E. and F. Schlyter (2004). "Hylobius pine weevils adult host selection and antifeedants: feeding behaviour on host and non-host woody scandinavian plants." Agricultural and Forest Entomology 6(2): 165-171. Nilsson, P. and N. Cory (2015). Forest statistics 2015. S. Swedish University of Agricultural Sciences. Umeå, SLU.

39 Nordenhem, H. and G. Nordlander (1994). "Olfactory oriented migration through soil by root living Hylobius abietis (L) larvae (Col. Curculionidae)." Journal of Applied Entomology- Zeitschrift Fur Angewandte Entomologie 117(5): 457-462. Nordlander, G. (1990). "Limonene inhibits attraction to alpha-pinene in the pine weevils Hylobius abietis and H. pinastri " Journal of Chemical Ecology 16(4): 1307-1320. Nordlander, G. (1991). "Host finding in the pine weevil Hylobius abietis - Effects of conifer volatiles and added limonen " Entomologia Experimentalis Et Applicata 59(3): 229-237. Nordlander, G., H. Nordenhem and H. Bylund (1997). "Oviposition patterns of the pine weevil Hylobius abietis." Entomologia Experimentalis Et Applicata 85(1): 1-9. Nyasembe, V. O., D. P. Tchouassi, C. M. Mbogo, C. L. Sole, C. Pirk and B. Torto (2015). "Linalool oxide: generalist plant based lure for mosquito disease vectors." Parasites & Vectors 8. Paine, T. D., K. F. Raffa and T. C. Harrington (1997). "Interactions among scolytid bark beetles, their associated fungi, and live host conifers." Annual Review of Entomology 42: 179-206. Petersson, M. and G. Örlander (2003). "Effectiveness of combinations of shelterwood, scarification, and feeding barriers to reduce pine weevil damage." Canadian Journal of Forest Research-Revue Canadienne De Recherche Forestiere 33(1): 64-73. Pichersky, E., J. P. Noel and N. Dudareva (2006). "Biosynthesis of plant volatiles: Nature's diversity and ingenuity." Science 311(5762): 808-811. Raffa, K. F. (2014). "Terpenes Tell Different Tales at Different Scales: Glimpses into the Chemical Ecology of Conifer - Bark Beetle - Microbial Interactions." Journal of Chemical Ecology 40(1): 1-20. Ralph, S. G., H. Yueh, M. Friedmann, D. Aeschliman, J. A. Zeznik, C. C. Nelson, Y. S. N. Butterfield, R. Kirkpatrick, J. Liu, S. J. M. Jones, M. A. Marra, C. J. Douglas, K. Ritland and J. Bohlmann (2006). "Conifer defence against insects: microarray gene expression profiling of Sitka spruce (Picea sitchensis) induced by mechanical wounding or feeding by spruce budworms (Choristoneura occidentalis) or white pine weevils (Pissodes strobi) reveals large-scale changes of the host transcriptome." Plant Cell and Environment 29(8): 1545-1570. Rieske, L. K. (2000). "Pine weevil (Coleoptera : Curculionidae) population monitoring in Christmas trees using volatile host compounds." Journal of Entomological Science 35(2): 167-175. Risticevic, S., J. R. Deell and J. Pawliszyn (2012). "Solid phase microextraction coupled with comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry for high-resolution metabolite profiling in apples: Implementation of structured separations for optimization of sample preparation procedure in complex samples." Journal of Chromatography A 1251: 208-218. Rizzi, A., E. Crotti, L. Borruso, C. Jucker, D. Lupi, M. Colombo and D. Daffonchio (2013). "Characterization of the bacterial community associated with larvae and adults of Anoplophora chinensis collected in Italy by culture and culture-independent methods." BioMed research international 2013: 420287-420287. Ruiu, L. (2015). "Insect Pathogenic Bacteria in Integrated Pest Management." Insects 6(2): 352- 367.

Scheepmaker, J. W. A. and T. M. Butt (2010). "Natural and released inoculum levels of entomopathogenic fungal biocontrol agents in soil in relation to risk assessment and in accordance with EU regulations." Biocontrol Science and Technology 20(5): 503-552. Schlyter, F. (2007). Semiochemicals in the life of bark feeding weevils. Bark and wood boring insects in living trees in Europe, a synthesis. F. Lieutier, K. R. Day, A. Battisti et al. The Netherlands, Springer: 351-364. Schlyter, F., G. Birgersson, J. A. Byers, J. Lofqvist and G. Bergstrom (1987). "Field response of spruce bark beetle,Ips typographus, to aggregation pheromone candidates." J Chem Ecol 13(4): 701-716. Schlyter, F., O. Smitt, K. Sjodin, H. E. Hogberg and J. Lofqvist (2004). "Carvone and less volatile analogues as repellent and deterrent antifeedants against the pine weevil, Hylobius abietis." Journal of Applied Entomology 128(9-10): 610-619. Schulz, S. and J. S. Dickschat (2007). "Bacterial volatiles: the smell of small organisms." Natural Product Reports 24(4): 814-842. Schwab, W., R. Davidovich‐Rikanati and E. Lewinsohn (2008). "Biosynthesis of plant‐derived flavor compounds." The Plant Journal 54(4): 712-732. Solbreck, C. (1980). "Dispersal distances of migrating pine weevils, Hylobius abietis, Coleoptera: Curculionidae " Entomologia Experimentalis et Applicata 28(2): 123-131. Sreekumar, R., Z. Al-Attabi, H. C. Deeth and M. S. Turner (2009). "Volatile sulfur compounds produced by probiotic bacteria in the presence of cysteine or methionine." Letters in Applied Microbiology 48(6): 777-782. Staffan Lindgren, B. and D. R. Miller (2002). "Effect of Verbenone on Five Species of Bark Beetles (Coleoptera: Scolytidae) in Lodgepole Pine Forests." Environmental Entomology 31(5): 759-765. Stead, D. E. and e. al. (2003). Pseudomonas syringae and related pathongens. Dordrecht/Boston/London, Kluwer academic publishers. Sunnerheim, K., A. Nordqvist, G. Nordlander, A. K. Borg-Karlson, C. R. Unelius, B. Bohman, H. Nordenhem, C. Hellqvist and A. Karlen (2007). "Quantitative structure-activity relationships of pine weevil antifeedants, a Multivariate approach." Journal of Agricultural and Food Chemistry 55(23): 9365-9372. Tilles, D. A., G. Nordlander, H. Nordenhem, H. H. Eidmann, A. B. Wassgren and G. Bergstrom (1986). "Increased release of host volatiles from feeding scars - A major cause of field aggregation in the pine weevil Hylobius abietis (Coleoptera, Curculionidae) " Environmental Entomology 15(5): 1050-1054. Tilles, D. A., K. Sjodin, G. Nordlander and H. H. Eidmann (1986). "Synergism between ethanol and conifer host volatiles as attractants for the pine weevil, Hylobius abietis (L) (Coleoptera, curculionidae)." Journal of Economic Entomology 79(4): 970-973. Turgeon, J. J., A. Roques and P. Degroot (1994). "Insect fauna of coniferous seed cones - Diversity, host-plant interactions, and management " Annual Review of Entomology 39: 179-212. Unelius, C. R., G. Nordlander, H. Nordenhem, C. Hellqvist, S. Legrand and A. K. Borg-Karlson (2006). "Structure-activity relationships of benzoic acid derivatives as antifeedants for the pine weevil, Hylobius abietis." Journal of Chemical Ecology 32(10): 2191-2203.

41 Wada-Katsumata, A., L. Zurek, G. Nalyanya, W. L. Roelofs, A. Zhang and C. Schal (2015). "Gut bacteria mediate aggregation in the German cockroach." Proceedings of the National Academy of Sciences of the United States of America 112(51): 15678-15683. van der Hoeven, R., G. Betrabet and S. Forst (2008). "Characterization of the gut bacterial community in Manduca sexta and effect of antibiotics on bacterial diversity and nematode reproduction." Fems Microbiology Letters 286(2): 249-256. Warner, R. E. (1966). "A Review of the Hylobius of North America, with a New Species Injurious to Slash Pine (Coleoptera: Curculionidae)." The Coleopterists Bulletin 20(3): 65-81. Vas, G. and K. Vekey (2004). "Solid-phase microextraction: a powerful sample preparation tool prior to mass spectrometric analysis." J Mass Spectrom 39(3): 233-254. Vasudevan, S. and S. V. Bhat (2011). "Biotransformation of isoeugenol catalyzed by growing cells of Pseudomonas putida." Biocatalysis and Biotransformation 29(4): 147-150. Wermelinger, B. (2004). "Ecology and management of the spruce bark beetle Ips typographus - a review of recent research." Forest Ecology and Management 202(1-3): 67-82. Xu, L., Q. Lou, C. Cheng, M. Lu and J. Sun (2015). "Gut-Associated Bacteria of Dendroctonus valens and their Involvement in Verbenone Production." Microbial Ecology 70(4): 1012- 1023. Yamada, Y., T. Kuzuyama, M. Komatsu, K. Shin-ya, S. Omura, D. E. Cane and H. Ikeda (2015). "Terpene synthases are widely distributed in bacteria." Proceedings of the National Academy of Sciences of the United States of America 112(3): 857-862. Zhang, Z. Y. and J. Pawliszyn (1993). "Headspace solid-phase microextraction." Analytical Chemistry 65(14): 1843-1852. Zhao, T. (2011). Conifer Chemical Defense: Regulation of Bark Beetle Colonization Doctoral, Royal Institute of Technology. Zhao, T., K. Axelsson, P. Krokene and A.-K. Borg-Karlson (2015). "Fungal Symbionts of the Spruce Bark Beetle Synthesize the Beetle Aggregation Pheromone 2-Methyl-3-buten-2- ol." Journal of Chemical Ecology 41(9): 848-852. Örlander, G., G. Nordlander, K. Wallertz and H. Nordenhem (2000). "Feeding in the crowns of Scots pine trees by the pine weevil Hylobius abietis." Scandinavian Journal of Forest Research 15(2): 194-201.

Author’s contributions

The following is a description of my contribution to Publications I to III and Appendix A and B, as requested by KTH.

Paper I: I performed most of the chemical analyses and wrote major parts of the manuscript.

Paper II: I contributed in the planning, organized the pine weevil collection and dissected the major part of the pine weevils.

Paper III: I planned most of the experiment, performed all of the chemical sampling, all the chemical analyses, and wrote a major part of the manuscript.

Appendix A: I performed parts of the chemical sampling and analyses.

Appendix B: I performed most of the analyses of the chemical data and wrote the major part of the manuscript.