Factors shaping microbial communies associated with ecologically important bark beetles

Charles University, Faculty of Science Univerzita Karlova, Přírodovědecká fakulta Study programe: Botany Studijní program: Botanika Factors shaping microbial communies associated with ecologically important bark beetles Faktory ovlivňující složení mikrobiálních společenstev vázaných na ekologicky významné kůrovce Mgr. Karel Švec Doctoral Thesis / Disertační práce Supervisor / Školitel: doc. Mgr. Miroslav Kolařík, Ph.D. Praha 2025 2 Declaration Hereby, I declare that I have written the present Ph.D. thesis independently, using the listed references. The papers that form part of the thesis are co-authored, and author contributions are stated in the respective section. I have not used any part of this thesis to gain any other academic title. Prohlášení Prohlašuji, že jsem tuto disertační práci vypracovala nezávisle s použitím citované literatury. Publikace, které jsou součástí této práce mají spoluautory. Příspěvky jednotlivých autorů jsou uvedeny v příslušné sekci. Žádná část této práce nebyla použita pro získání jiného akademického titulu. In Prague / V Praze 01. 06. 2025 Mgr. Karel Švec „Die Natur ist ewig in neue und wunderbare Formen sich ergießend; wenn du ein o;enes Auge hast, wirst du täglich neue Wunder sehen. “ "Nature is ever pouring forth new and marvelous forms; she is an exhaustless source of wonders, and if you have an eye for it, you will see them every day." „Příroda je stále plná nekonečných zázraků; pokud máš oči, aby je viděly, a ducha, aby je pochopil. “ Johann Wolfgang von Goethe - Maxims and Reflections (posthumously published 1833) „Die Wissenschaft kann das letzte Rätsel der Natur nicht lösen. Und das liegt daran, dass wir selbst Teil der Natur sind und daher Teil des Geheimnisses, das wir zu lösen versuchen. “ "Science cannot solve the ultimate mystery of nature. And that is because, in the last analysis, we ourselves are part of nature and therefore part of the mystery that we are trying to solve." „Věda nemůže vyřešit ultimátní tajemství přírody. A to proto, že v poslední analýze jsme my sami součástí přírody, a proto i součástí tajemství, které se snažíme vyřešit. “ Max Planck - Where is Science Going? (1932) 3 Contents Declaration ............................................................................................................................. 2 Prohlášení ............................................................................................................................... 2 List of original publications: ..................................................................................................... 8 Publication 1 Proportions of taxa belonging to the gut core microbiome change throughout the life cycle and season of the bark beetle Ips typographus ................................ 8 Publication 2 New insight into the bark beetle Ips typographus bacteriome reveals unexplored diversity potentially beneficial to the host ........................................................... 8 Publication 3 Insight into the genomes of dominant yeast symbionts of European spruce bark beetle, Ips typographus ................................................................................................ 9 Manuscript 1 Metabolic synergy and complementarity in the Ips typographus holobiont .. 9 Manuscript 2 Organ specificity in taxonomically related ash bark beetles is accompanied by specific microbial communities ..................................................................................... 10 Poděkování ........................................................................................................................ 11 Acknowledgements ............................................................................................................... 11 List of abbreviations: ............................................................................................................. 12 Abstrakt ................................................................................................................................ 13 Klíčová slova: ..................................................................................................................... 13 Abstract ................................................................................................................................ 14 Keywords: .......................................................................................................................... 14 INTRODUCTION .................................................................................................................... 14 Symbiosis as a fundamental ecological and evolutionary process ....................................... 14 Using the term “Symbiosis” ............................................................................................ 16 Holobiont concept ......................................................................................................... 16 Symbiosis in insects .......................................................................................................... 17 Bark beetles as a model for studying insect–microbe symbioses ......................................... 18 Mutual benefits of bark beetle-microbe symbiosis ........................................................... 21 Intestinal microbiome of bark beetles ................................................................................. 22 Metabolic interactions in the bark beetle gut ....................................................................... 22 Research aims and objectives ............................................................................................ 23 Main results .......................................................................................................................... 25 4 Publication 1 – Proportions of taxa belonging to the gut core microbiome change throughout the lifecycle and season of the bark beetle I. typographus ................................................... 25 Diversity and Composition of the Gut Microbiome in I. typographus Across Life Stages and Seasons ......................................................................................................................... 25 Core Microbiome and Transmission Mechanisms ............................................................ 27 Our analysis identified a core microbiome of I. typographus that persisted throughout the beetle’s life cycle and across the seasons. The fungal core microbiome included Cyberlindnera sp., Kuraishia capsulata, K. molischiana, N. ambrosiae, O. neopini, O. ramenticola, Ophiostoma bicolor, Saccharomycopsis lassenensis, W. bisporus, and Yamadazyma scolyti, while the bacterial core included E. typographi, E. cedenensis, P. bohemica, Pseudoxanthomonas spadix, Rhizobium sp., Curtobacterium sp., Roseomonas sp., Serratia sp., Taibaiella sp., and unidentified species of the family Sphingobacteriaceae, Lachnospiraceae, and Enterobacteriaceae. ........................................................................ 27 ......................................................................................................................................... 27 Publication 2 – New insight into the bark beetle I. typographus bacteriome reveals unexplored diversity potentially beneficial to the host ........................................................................... 27 Bacteriome Diversity and Taxonomic Composition .......................................................... 27 Hydrolytic Enzyme Activity and Carbon Assimilation ........................................................ 27 Antifungal Potential of the Bacteriome ............................................................................ 28 Core Bacteriome and Key Microbial Species .................................................................... 28 Implications and Future Directions ................................................................................. 28 5 ......................................................................................................................................... 29 Publication 3 – Insight into the genomes of dominant yeast symbionts of European spruce bark beetle, Ips typographus .............................................................................................. 29 Genome Sequencing and Analysis of Yeasts in the I. typographus Gut Microbiome ........... 29 Carbohydrate-Active Enzymes (CAZymes) and Plant Cell Wall Degradation ...................... 29 Detoxification Mechanisms in Yeasts .............................................................................. 29 Extracellular Enzyme Activities and Functional Implications ............................................ 30 Symbiotic Roles in Nutrient Synthesis and Detoxification ................................................. 30 ......................................................................................................................................... 31 Manuscript 1 – Metabolic synergy and complementarity in the Ips typographus holobiont .... 31 Metatranscriptome Assembly and Taxonomic Composition ............................................. 31 Functional Diversity and Holobiont Complementarity ...................................................... 31 Despite the dominance of the beetle’s transcriptome in the dataset, bacteria and fungi contribute equally to the overall functional diversity within the holobiont. The PERMANOVA analysis revealed significant di;erences in life stages (p = 0.004), though the variability within groups was greater than between life stages, especially between larvae and adult beetles. This indicates that the microbial community plays a more pivotal role than life-stage-specific variations in shaping the functional outputs of the holobiont. .............................................. 31 Microbial Contributions to Degradation of Plant and Fungal Cell Wall Polymers ................ 31 Nitrogen Utilization within the Holobiont ......................................................................... 32 6 Nitrogen acquisition is critical for the beetle, given the nitrogen-limited environment in Picea abies phloem. Our results show that bacterial symbionts facilitate nitrogen acquisition, primarily through nitrate reduction to ammonia. The beetle’s microbiota also contributes to the conversion of uric acid into usable nitrogen sources, such as ammonia. Bacterial taxa like Delftia, Erwinia, and Cupriavidus are key players in this process. Fungi seem to rely on the ammonia produced by bacteria, scavenging it rather than contributing directly to ammonia conversion. This nitrogen cycle illustrates the essential role of microbial collaboration in overcoming the beetle’s nutritional constraints. .............................................................. 32 Cross-Kingdom Cooperation in Amino Acid Metabolism .................................................. 32 Our analysis further revealed the intricate cooperation between beetle, bacteria, and fungi in amino acid metabolism. Essential amino acids such as histidine and lysine are synthesized by bacteria, with fungi playing a supporting role. However, the beetle largely depends on its symbiotic microbes for the synthesis of amino acids such as branched-chain amino acids (valine, leucine, isoleucine) and phenylalanine. This highlights the interdependence of the beetle and its microbial partners in synthesizing and interconverting amino acids. Notably, bacterial taxa such as Erwinia, Delftia, and Phyllobacterium, as well as fungal taxa like Sordariomycetes (including Ophiostoma), are key contributors to these pathways. .............. 32 Vitamin Co-Metabolism in the Holobiont ......................................................................... 32 Conclusion .................................................................................................................... 33 ......................................................................................................................................... 33 Manuscript 2 - Organ specificity in taxonomically related ash bark beetles is accompanied by specific microbial communities ......................................................................................... 33 Diversity and Taxonomic Composition ............................................................................. 33 Distinct fungal communities were linked to the beetle species: ........................................ 34 Community Structure and Di;erentiation ........................................................................ 34 Physiological Adaptations of Symbiotic Fungi .................................................................. 34 Conclusion .................................................................................................................... 35 General discussion ................................................................................................................ 35 The specific requirements of life under the bark and the choice of a suitable model species ...................................................................................................................................... 35 Observed microbial diversity ........................................................................................... 36 Identifying the core microbiome of I. typographus ............................................................ 37 Acquisition and Localization of Gut Symbionts ................................................................ 38 Metabolic complementarity within the I. typographus holobiont ....................................... 39 Gut micobiome benefits – direct study approaches ......................................................... 40 7 Mycobiome benefits – Genomic study of I. typographus major fungal symbionts ............... 43 Ecological filtering and symbiont divergence in 2 non-mycangial Ash bark beetles ............... 45 Simplified Conclusions .......................................................................................................... 46 Funding ................................................................................................................................. 47 References ............................................................................................................................ 47 8 List of original publications: This thesis is based on the following five publications: Publication 1 Proportions of taxa belonging to the gut core microbiome change throughout the life cycle and season of the bark beetle Ips typographus Tereza Veselská & Karel Švec*, Martin Kostovčík, Ezequiel Peral-Aranega, Paula Garcia-Fraile, Barbora Křížková, Václav Havlíček, Zaki Saati-Santamaría, Miroslav Kolařík (2023). FEMS Microbiology Ecology 99 (8), fiad072 doi: 10.1093/femsec/fiad072 *Tereza Veselská &Karel Švec – shared first author position Authors contribution: Tereza Veselská (Conceptualization, Formal analysis, Investigation, Methodology, Visualization, Writing – original draft), Karel Švec (Conceptualization, Formal analysis, Investigation, Methodology, Visualization, Writing – original draft), Martin Kostovčík (Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Writing – original draft), Ezequiel Peral-Aranega (Formal analysis, Investigation), Paula Garcia-Fraile (Funding acquisition, Writing – review & editing), Barbora Křížková (Investigation), Václav Havlíček (Investigation), Zaki Saati-Santamaría (Formal analysis, Investigation), and Miroslav Kolařík (Conceptualization, Funding acquisition, Resources, Supervision, Validation, Writing – review & editing). Publication 2 New insight into the bark beetle Ips typographus bacteriome reveals unexplored diversity potentially beneficial to the host Ezequiel Peral-Aranega, Zaki Saati-Santamaría, Miguel Ayuso-Calles, Martin Kostovčík, Tereza Veselská, Karel Švec, Raúl Rivas, Miroslav Kolařík, Paula García-Fraile. (2023). Environmental Microbiome doi: 10.1186/s40793-023-00510-z Authors contribution: Ezequiel Peral-Aranega (Investigation, Methodology, Software, Visualization, Writing – review & editing), Zaki Saati-Santamaría (Formal analysis, 9 Investigation, Software, Writing – review & editing), Miguel Ayuso-Calles (Investigation), Martin Kostovčík (Investigation), Tereza Veselská (Investigation, Resources), Karel Švec (Investigation, Resources), Raúl Rivas (Resources, Supervision, Validation), Miroslav Kolařík (Conceptualization, Investigation, Project administration, Resources, Supervision, Validation), Paula García-Fraile (Conceptualization, Data curation, Formal analysis, Funding acquisition, Methodology, Resources, Supervision, Validation, Visualization, Writing – original draft preparation). Publication 3 Insight into the genomes of dominant yeast symbionts of European spruce bark beetle, Ips typographus Tian Cheng, Tereza Veselská, Barbora Křížková, Karel Švec, Václav Havlíček, Marc Stadler, Miroslav Kolařík. (2023). Froners in Microbiology…. doi: 10.3389/fmicb.2023.1108975 Authors contribution: Tian Cheng (Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Visualization, Writing – original draft), Tereza Veselská (Conceptualization, Formal analysis, Investigation, Writing – review & editing), Barbora Křížková (Investigation), Karel Švec (Investigation, Resources, Writing – review & editing), Václav Havlíček (Investigation, Resources), Marc Stadler (Funding acquisition, Supervision), Miroslav Kolařík (Funding acquisition, Supervision, Validation). Manuscript 1 Metabolic synergy and complementarity in the Ips typographus holobiont Zaki Saati Santamaria & Karel Švec & Martin Kostovčík, Tereza Veselská, Miroslav Kolařík Currently under review in ISME journal – 6/25 Preprint available on Research square; doi: 10.21203/rs.3.rs-5950784/v1 Authors contribution: Zaki Saati Santamaria (Conceptualization, Formal analysis, Investigation, Methodology, Software, Visualization, Writing – original draft), Karel Švec (Conceptualization, Formal analysis, Investigation, Methodology, Resources, Software, Visualization, Writing – review & editing), Martin Kostovčík (Investigation, Methodology, Software), Tereza Veselská (Investigation, Methodology, Writing – review & editing), Miroslav Kolařík (Conceptualization, Funding acquisition, Supervision, Validation, Writing – review & editing). 16 Mutualism might be obligatory for the host, the symbiont, both, or none. The extensively examined instances revolve typically around extremely specialized partnerships, wherein both entities can exist solely in each other's presence (Moran, 2006). Di;erences in the tightness of coexistence between the host and symbiont lead to di;erences in genome evolution of the symbiont as well as of the host (Moran, 2006). In the evolutionary process, some of these tightly associated organisms experienced the loss of genes responsible for encoding enzymes essential in the biosynthesis of numerous vital organic compounds(e.g. riboflavin pathway in Aphids (Monnin et al., 2020)). Consequently, animals dependent on symbionts face limitations in their capacity to produce various compounds necessary for cellular function and exhibit restrictions in utilizing diverse energy sources (Moran, 2006). Using the term “Symbiosis” In this dissertation thesis and our attached publications, the term “symbiosis” is used primarily to refer to mutualistic interactions between bark beetles (Curculionidae: Scolytinae) and their microbial partners (bacteria and fungi). Although in ecological literature, the concept of symbiosis broadly encompasses all types of close interspecific interactions, including parasitism, commensalism, and predation (Boucher, 2016; Douglas, 2021; Thorpe, 1953), in this work it is applied specifically to interactions that provide functional benefits to both partners, i.e., mutualisms. This focus reflects the common usage in insect–microbe research, where the term “symbiont” typically refers to microbial partners involved in nutrient provision, detoxification, or defense (Biedermann & Vega, 2020; Moran, 2006), all of which contribute to the ecological success of the host. Holobiont concept In recent years, the concept of the “holobiont” has become an important ecological and evolutionary framework for understanding the relationships between multicellular organisms and their associated microbiota (Gilbert et al., 2012; Mindell, 1992). In this dissertation, the bark beetle holobiont is understood as the integrated unit formed by the insect host together with its symbiotic microorganisms, including bacteria, yeasts, and filamentous fungi (Six, 2013). This concept emphasizes the close metabolic and ecological interdependence between the beetle and its microbial partners, which enables the colonization of nutritionally challenging habitats (Moran, 2007), and the exploitation of otherwise inaccessible resources (Klepzig & Six, 2004). The holobiont perspective also highlights the role of microbes in modulation of the host physiology, including detoxification, immune interactions, and ecological adaptation (Biedermann & Vega, 2020; Moran, 2007). In non-mycangial bark beetles, the holobiont is shaped 17 by a combination of vertical as well as horizontal transmission routes, which influences the stability and flexibility of these associations across environmental gradients and life stages (Biedermann & Vega, 2020; Moran, 2007). Symbiosis in insects Microbial symbiosis is widespread among insects and plays a fundamental role in insect behavior and life strategies (Biedermann & Vega, 2020; Hosokawa & Fukatsu, 2020; Lu et al., 2016; Vidal et al., 2021). Insects are often strict dietary specialists, shaped by a long-term coevolution with microbes, which has driven the development of specialized phoretic strategies and organs, including the formation of mycangia (specialized organs adapted for transport of symbiotic fungi) (Calevro et al., 2023). Obligate symbiosis between insects and microbes is an attractive and wellstudied topic, e.g., in Atta leaf-cutter ants and termites (Schultz & Brady, 2008). Ambrosia beetles are representatives of obligate symbiosis within bark beetles, where mycangia play a crucial role (Skelton et al., 2019). This group of bark beetles is not studied in our work and will therefore be mentioned here only marginally, despite being considered an example of one of the most successful symbiotic relationships among bark beetles and their fungal symbionts (Skelton et al., 2019). The less stringent type of association is facultative symbiosis, which is not strictly required for host survival, and its typical attributes are conditional beneficence, horizontal transmission, and dynamic interactions compared to obligate symbiosis (Klepzig & Six, 2004; Moran, 2006; Six & Klepzig, 2021). feature Obligatory Symbionts Non - Obligatory Symbionts Dependency a host cannot survive without Host survives Transmission strictly vertical Vertical + horizontal Genome reduced Less reduced 18 It has been reported that nutritional benefits to hosts are commonly promoted by long-term coevolution and genome stasis of microbial symbionts (Moran, 2006). This phenomenon has been described in the aphid–Buchnera system, where, over evolutionary time, bacteria have undergone significant genome reduction, stabilizing in a state that specifically supports the nutritional needs of the host ((Mondal et al., 2023; Moran, 2006, 2007; Moran & Telang, 1998; Moran & Yun, 2015; Ummah et al., 2020)) see table 1. Tab 1. Feature comparison based on: (Baumann, 2005; Chen et al., 2017; Ferrari & Vavre, 2011; Mondal et al., 2023; Moran, 2006; Moran & Telang, 1998; Moran & Yun, 2015) The mechanism by which microbial symbionts are carried is a key feature important for understanding their mode of action. Various food specialists, such as ambrosia beetles, have developed (thanks to long coevolution – Mayers 2020) specific organs called “mycangia” for the sophisticated retention and phoresy of their vital microbial symbionts (Skelton et al., 2019). These specific organs are also found in some aggressive bark beetles (Six et al., 2011; Six & Wingfield, 2011). Other bark beetles, including I. typographus or Hylesinus species, studied in the following publications, rely on less specific means for symbiont transmission. These non-mycangial bark beetles are commonly associated with Ophiostomatoid fungi or fungi from the genus Geosmithia (B. J. Bentz et al., 2019; Biedermann et al., 2019a; B. R. Jankowiak, 2005). These ectosymbionts are passively carried on the cuticle surface (in tiny pits or hairs). Adaptation of filamentous fungi for this trait involves the massive production of sticky and highly adhesive conidia and ascospores or yeast growth within a viscous extracellular matrix (e.g. Saccharomycetales). These adaptations help them adhere to the beetles’ cuticle. This significantly enhances the probability of fungi being transferred by the bark beetle to the new habitat (B. J. Bentz et al., 2019; Biedermann et al., 2019a; Hammerbacher et al., 2013; Six & Wingfield, 2011). Alternatively, symbiotic microbes can be carried in the gut lumen. These gut symbionts are typically represented by bacteria and yeast (Saccharomycetales)(Baños-Quintana et al., 2024). Bark beetles as a model for studying insect–microbe symbioses Bark beetles represent a highly diverse and ecologically significant group of insects, with approximately 6000 described species worldwide (Kirkendall et al., 2015; Wood & Bright, 1992). Their life strategies range from primary bark beetles, capable of killing healthy trees, to secondary Functional Role core metabolism (e.g. nutrition) Defense, stress tolerance, communication 19 species colonizing weakened or dead trees, and saprophytic species that exploit decaying plant tissue (Kirkendall et al., 2015; Paine et al., 1997). Bark beetles play a crucial role in shaping of forest ecosystems, serving as major disturbance agents and contributing to forest dynamics, nutrient cycling, and biodiversity (Seidl et al., 2017; Six, 2020). Bark beetles themselves are an important agent of disturbance of forest ecosystems, and their massive outbreaks often occur in forest ecosystems weakened by unfavorable weather conditions, such as ongoing drought or, conversely, storms accompanied by winds, or (in the case of production forests) by inappropriate management (B. Bentz et al., 2009; Davis et al., 2020; Hlásny et al., 2019, 2021a; Kulakowski, 2016; Lundquist, 2019; Rodman et al., 2021). This makes the topic of bark beetle outbreaks an intensely studied and important issue from a human socioeconomic perspective. This situation is compounded by global climate change, which, in conjunction with inappropriate management, creates a positive feedback loop that worsens the situation (Baños-Quintana et al., 2024; Biedermann et al., 2019a; Hlásny et al., 2019; Jaime et al., 2023) (see Fig. 1.), not only in Europe but also in North America (B. Bentz et al., 2009; Fettig et al., 2022; Six, 2020) and Asia(Karpov et al., 2024) . Bark beetles are recognized as key drivers of ecosystem structure and function in temperate and boreal forests. They contribute to forest heterogeneity and biodiversity by causing tree diebacks, which create opportunities for the growth of understory plants (Kulakowski, 2016) and consequently promote the presence of pollinators (Davis et al., 2020). On the other hand, they can cause extensive tree mortality during the mentioned outbreaks, which impacts carbon storage, nutrient cycling, and water resource provisioning. The fundamental impact of bark beetles on ecosystems has been described as a “knock-on e;ect,” referring to their ability to trigger chain reactions within the forest systems (Biedermann et al., 2019a; Jaime et al., 2023; Six & Wingfield, 2011). Some authors point to the increased risk associated with environmental change and bark beetle outbreaks, especially in silvicultural monocultural forests (see Fig. 1). The risk of destructive outbreaks can be supported by common inadequate forest management, as well as the introduction of new, potentially invasive species through international trade (B. J. Bentz et al., 2010; Biedermann et al., 2019b; Hlásny et al., 2021a; Mezei et al., 2017). 20 Fig. 1. (Biedermann et al., 2019a) Precise “map” of the complex relationships and dynamics between the bark beetles, host (tree host) and their associated microbes, predators, parasites and the environment itself. Original description in Biedermann 2019: “(I) Major climatic variables aAected by climate change at a macroand regional scale. (II) The most important variables relating to the properties of individual host trees and trees at a landscape scale. (III) Main population phases (non-outbreak, build-up, outbreak, collapse) of an eruptive insect species. (IV) Major biotic variables associated with an eruptive insect species, plus intraspecific eAects (phenotype, genotype, and intraspecific competition). Arrows are exemplary of the Ips typographus system. An arrow from one of the boxes in group I, II, or IV to one of the boxes in group III would indicate a direct eAect on the population phases 21 of the beetle. Arrows connecting multiple boxes and eventually pointing to one of the four population phases would indicate an indirect eAect. The grey arrows represent hypotheses that have yet to be tested and thus mirror gaps in our knowledge of the I. typographus. The absence of an arrow between boxes implies that there is probably no eAect of one variable on another in the I. typographus”. Mutual benefits of bark beetle-microbe symbiosis One of the most fascinating aspects of bark beetle biology is their cooperation with various microbial partners, including filamentous fungi, yeasts, and bacteria (Hofstetter et al., 2015; Hosokawa & Fukatsu, 2020; Mondal et al., 2023; Moran, 2006; Six & Wingfield, 2011). These symbionts perform various functions, such as facilitating nutrient acquisition, degrading host plant defences, providing detoxification capabilities, and mediating aggregation and host recognition (Boone et al., 2013; Hulcr & Stelinski, 2017; Shamjana et al., 2024). Fungal associates, particularly species of Ophiostoma, Ceratocystiopsis, Grosmannia, or Geosmithia, play an essential role in the nutritional ecology of bark beetles and contribute to the success of tree colonization (Kirisits, 2004; Kolařík & Hulcr, 2023; Paine et al., 1997; Zhao et al., 2019). The transmission of symbionts in bark beetles is highly variable. It includes both specialized structures, such as mycangia, which ensure vertical transmission of fungal partners, and horizontal acquisition of microbes from galleries or the surrounding environment (Hulcr & Stelinski, 2017; Klepzig & Six, 2004). Interestingly, some bark beetles without mycangia still maintain consistent associations with yeasts or fungi, highlighting the flexibility and evolutionary importance of these partnerships (Lehenberger et al., 2021). Bark beetles have therefore emerged as an important model system for studying insect–microbe symbioses, providing insights into the ecological, functional, and evolutionary dynamics of complex multipartite interactions. Their ecological relevance, wide range of symbiotic strategies, and increasing availability of genomic resources make them an exceptional system for nderstanding the role of microbes in insect adaptation and diversification. In this dissertation, three bark beetle species (I. typographus, H. crenatus and H. fraxini) were selected to study various aspects of symbiosis between ecologically important European bark beetles and their microbial symbionts. I. typographus was chosen as a model species, as its genome (Powell et al., 2021) and transcriptomes (Andersson et al., 2013; Ramakrishnan et al., 2022) are already available. Its associated microbial communities have been extensively studied through traditional cultivation-based methods over several decades (Fabryová et al., 2018; García-Fraile, 2018; R. Jankowiak, 2005; R. Jankowiak & Hilszczański, 2005; Linnakoski 22 et al., 2012). Moreover, its impact on the environment, economics and human populations is immense (Biedermann et al., 2019b). In contrast, both Hylesinus crenatus and H. fraxini, although belonging to the same genus, were selected to represent contrasting symbiotic strategies. Hylesinus crenatus is known to be associated with ophiostomatoid fungi and attacks ash (Fraxinus sp.) tree trunks, whereas H. fraxini is strictly associated with the Geosmithia genus and colonizes smaller ash branches (Strzałka et al., 2021). This combination of species enables a comparative analysis across di;erent symbiotic associations, host tree species, and ecological strategies, providing a robust framework for investigating the ecological and evolutionary roles of microbial symbionts in bark beetles (Kirschner, 2001). Intestinal microbiome of bark beetles The intestinal microbiome of bark beetles represents a diverse and dynamic community of microorganisms, including bacteria, yeasts, and filamentous fungi. Bacterial genera frequently documented in the guts of bark beetles include Enterobacter and Rahnella(Morales-Jiménez et al., 2012a), Pseudomonas (Adams et al., 2013a; Fabryová et al., 2018), Serratia and Stenotrophomonas (Fabryová et al., 2018; Saati-Santamaría et al., 2021. The yeast microbiome is often represented by species of Candida, Cyberlindnera, Ogataea and Wickerhamomyces (Boone et al., 2013). These communities of gut-microbiome are shaped by both environmental acquisition and vertical transmission (Bracewell & Six, 2015; Six, 2013; Six & Biedermann, 2023; Zhou et al., 2016), with specific taxa consistently reported across di;erent bark beetle species and life stages (Adams et al., 2013a). The composition of the gut microbiome is influenced by host tree species (Morales-Jiménez et al., 2012a), developmental stage (Boone et al., 2013), and ecological conditions (Zhou et al., 2016), reflecting the complex interactions between beetles, their diet, and their microbial partners. Metabolic interactions in the bark beetle gut Microbial symbionts in the gut of bark beetles perform a variety of metabolic functions that support host survival in nutritionally challenging environments. These include the degradation of recalcitrant plant polymers such as cellulose, hemicellulose, and lignin (Adams et al., 2013a), as well as the hydrolysis of chitin derived from fungal and insect material (Boone et al., 2013). Symbionts contribute to nitrogen cycling through processes such as nitrogen fixation and ammonium assimilation (Morales-Jiménez et al., 2012a), and they play roles in the recycling of nitrogenous waste products such as uric acid (Zhou et al., 2016), thereby providing essential amino acids to the host (Morales-Jiménez et al., 2012a). Additionally, microbial communities 23 in the beetle gut are involved in the biosynthesis of B vitamins, sterols, and fatty acids (Boone et al., 2013), which are crucial for development and reproduction. Some bacteria and yeasts also participate in the detoxification of plant secondary metabolites, including terpenoids and phenolic compounds (Adams et al., 2013a), reducing the chemical defenses of host trees and facilitating beetle colonization (Zhou et al., 2016). Importantly, recent research has shown that gut bacteria regulate glucose transport in bark beetle larvae by inducing gut hypoxia and secreting riboflavin, which activates host hypoxia-inducible factors to upregulate glucose transporters, thereby promoting nutrient absorption and larval development (Liu et al. 2024). Research aims and objectives The main objective of this dissertation is to increase our current understanding of the functioning of mutualistic relationships between bark beetles and their associated microorganisms, integrating ecological, genomic, and functional perspectives. This work represents an almost decade-long e;ort to elucidate the dynamics, evolution, and complexity of this symbiotic multitrophic system, approached primarily from the standpoint of environmental ecology rather than pest management. Specifically, three bark beetle species were selected to explore various aspects of symbiosis between ecologically important European bark beetles and their microbial symbionts. I. typographus was chosen as a “well-known” model species due to the availability of genomic and transcriptomic resources and because its associated microbial communities have been extensively studied using cultivation-based approaches. In contrast, H. crenatus and H. fraxini were selected to represent two closely related species with contrasting symbiotic strategies, thereby broadening our understanding of symbiosis in less-explored non-model systems. While initial research has highlighted the importance of microbial symbionts in bark beetles, key aspects such as defining the core microbiome and understanding its succession across the life cycle have remained largely unaddressed, leaving several critical knowledge gaps. A general understanding is lacks explanation of how the multitrophic environment of the bark beetle gut functions, what specific roles bacteria and fungi play, and how microbial community composition changes across the host life cycle and influences gut-associated processes. More broadly, there is limited knowledge of the evolutionary adaptations that enable microbial symbionts to function as mutualists in the bark beetle gut environment. These gaps are particularly relevant in the case of I. typographus, where dominant yeast and bacterial symbionts have been described, but their functional and ecological roles remain poorly understood. In H. crenatus and H. fraxini, the ecological drivers shaping microbial communities are largely unknown, and it remains to be 24 verified what specific features drive the associations of bark beetles either with genus Geosmithia or ophiostomatoid fungi (an ecological, paraphyletic group of several ascomycetous genera from orders Ophiostomatales, Microascales, Hypocreales and Saccharomycetales). This dissertation resolves the addressed knowledge gaps by combining “traditional” cultivation-dependent and cultivation-independent (DNA/RNA metabarcoding) approaches, genomic and functional analyses, combined with physiological experiments and various types of microscopy. The main aims of this dissertation are to: • Describe the composition of microbiomes in 3 important bark beetle species. – Define the core gut microbiome of I. typographus and describe its seasonal and ontogenetic dynamics. – Characterize the microbial communities associated with ash bark beetles, H. fraxini and H. crenatus. • Evaluate the metabolic activities and functional ecological roles of dominant fungal and bacterial symbionts in I. typographus gut microbiome. • Present a taxonomic microbial analysis of I. typographus across di;erent life stages using culture-dependent and -independent techniques. • Study potential ecological roles of the I. typographus microbiome, identifying key taxa involved in nutrient acquisition and pathogen defense. • Sequence and analyze genomes of major symbiotic yeasts associated with I. typographus. • Investigate environmental factors driving microbiome specialization within Hylesinus bark beetle species. • Evaluate trophic interactions within the gut ecosystems of bark beetles. • Determine the metabolic roles of bacteria and fungi in the gut of I. typographus and related species. 25 Main results This dissertation brings together 3 publications in IF journals and 2 submitted manuscripts. All of them focus on microbial communities (fungi and bacteria) associated with their hosts - bark beetles a;ecting European forest ecosystems. Publications 1-3 and Manuscript 1 study the European spruce bark beetle I. typographus, the succession process of its gut microbiome and selected features of dominant microbes present in the mentioned system. Manuscript 2 focuses on the question of preference of dominant fungal symbiont (Geosmithia associated type versus Ophiostomatoid fungi associated type) in 2 closely related species of single genus (Hylesinus fraxini versus H. crenatus), evaluating the composition of their associated communities and physiological features of symbiotic fungi (Ophisotoma and Geosmithia species) to explain this rigid association scheme of “symbiont preference”. In the following paragraphs, I will present and comment on selected results from each article. Publication 1 – Proportions of taxa belonging to the gut core microbiome change throughout the lifecycle and season of the bark beetle I. typographus Diversity and Composition of the Gut Microbiome in I. typographus Across Life Stages and Seasons We used molecular methods, including eDNA and eRNA metabarcoding as well as traditional cultivation techniques, to examine the active gut microbiomes of I. typographus. This dual approach allowed us to capture a comprehensive view of the microbial community across the beetle's entire life cycle, from parental adults to larvae, pupae, and teneral adults. We also 32 and fungal cell walls. Our data indicate that while bacterial gut symbionts play a dominant role in polymer degradation, the entire holobiont participates in completing these complex metabolic processes. For instance, chitin degradation is facilitated by both the beetle and its microbial partners, with similar contributions from fungi and bacteria. Bacteria initiate Xylan and pectin degradation, but beetle and fungi further support these processes. Lignin degradation is primarily bacterial, with eukaryotes utilizing by-products. Notably, the bacterial taxa Erwinia, Delftia, and Phyllobacterium drive most of these catabolic activities. On the other hand, Ascomycota, particularly Ogataea yeast, contribute to glucan degradation, while the beetle helps to facilitate cellulose breakdown. Nitrogen Utilization within the Holobiont Nitrogen acquisition is critical for the beetle, given the nitrogen-limited environment in Picea abies phloem. Our results show that bacterial symbionts facilitate nitrogen acquisition, primarily through nitrate reduction to ammonia. The beetle’s microbiota also contributes to the conversion of uric acid into usable nitrogen sources, such as ammonia. Bacterial taxa like Delftia, Erwinia, and Cupriavidus are key players in this process. Fungi seem to rely on the ammonia produced by bacteria, scavenging it rather than contributing directly to ammonia conversion. This nitrogen cycle illustrates the essential role of microbial collaboration in overcoming the beetle’s nutritional constraints. Cross-Kingdom Cooperation in Amino Acid Metabolism Our analysis further revealed the intricate cooperation between beetle, bacteria, and fungi in amino acid metabolism. Essential amino acids such as histidine and lysine are synthesized by bacteria, with fungi playing a supporting role. However, the beetle largely depends on its symbiotic microbes for the synthesis of amino acids such as branched-chain amino acids (valine, leucine, isoleucine) and phenylalanine. This highlights the interdependence of the beetle and its microbial partners in synthesizing and interconverting amino acids. Notably, bacterial taxa such as Erwinia, Delftia, and Phyllobacterium, as well as fungal taxa like Sordariomycetes (including Ophiostoma), are key contributors to these pathways. Vitamin Co-Metabolism in the Holobiont Our data also shed light on the complex division of labor in vitamin co-metabolism within the holobiont. Microbial symbionts, particularly bacteria and fungi, contribute significantly to the biosynthesis of essential vitamins such as folate (B9), biotin (B7), and vitamins B3, B5, B2, B1, and B6. For example, folate biosynthesis is primarily driven by bacteria, with fungi and beetle 33 scavenging and utilizing the by-products. Similarly, biotin synthesis is largely bacterial, with beetle and fungi recycling biotin from biocytin. We suggest that particular bacteria play an important role in the metabolism of several vitamins, with bacteria and fungi providing essential precursors and modifications. This metabolic network ensures that the beetle’s nutritional needs are met, further emphasizing the cooperative nature of the beetle holobiont. Conclusion The metatranscriptomic data reveals a highly integrated and cooperative system within the bark beetle holobiont, where bacteria, fungi, and the beetle itself complement each other’s metabolic functions. The beetle relies on its microbial symbionts for essential processes such as degradation of complex polymers, nitrogen utilization, amino acid biosynthesis, and vitamin metabolism. The findings underscore the importance of cross-kingdom cooperation in sustaining the nutritional and metabolic needs of the holobiont, o;ering a deeper understanding of the functional roles of its microbial partners. The interplay between the beetle and its microbiota is a key factor in its survival and adaptation in its ecological niche. Manuscript 2 - Organ specificity in taxonomically related ash bark beetles is accompanied by specific microbial communities Diversity and Taxonomic Composition We investigated the total microbial communities associated with two closely related Ash bark beetle species, H. fraxini (infesting thin branches) and H. crenatus (infesting tree trunks), using a combination of NGS metabarcoding (markers: 16S, ITS, TEF1α) and cultivation approaches. Across all datasets, we obtained over 3 million sequencing reads and characterized 134 fungal species from cultivation. Rarefaction analyses indicated near-complete sampling, particularly for beetle-associated samples, though intact phloem samples were slightly under-sampled. Bacterial communities were dominated by Proteobacteria, Bacteroidota, and Actinobacteria, with beetle samples harboring mainly Enterobacterales and intact phloem showing greater 34 taxonomic diversity. Fungal communities were strongly dominated by Ascomycota across beetles and phloem, with some Basidiomycota more prevalent in H. fraxini samples. Distinct fungal communities were linked to the beetle species: H. crenatus primarily associated with Saccharomycetales and Ophiostomatales (e.g., Ophiostoma hylesinum and Ogataea spp.). H. fraxini exhibited a broader fungal community, including Geosmithia spp., Candida spp., and other diverse fungi, with almost no Ophiostomatales detected. Cultivation data confirmed metabarcoding results but also revealed a significant fraction of undescribed fungal taxa (particularly among yeasts). Bacterial taxa such as Erwinia spp. and Sphingobacteriaceae were characteristic for beetle tissue samples, while Hymenobacter and Sphingomonas dominated control samples (phloem). Community Structure and Di;erentiation Beta-diversity analyses (Robust Aitchison PCA made in DEICODE toolbox) clearly separated microbial communities of H. crenatus and H. fraxini based on fungi and bacteria, with dominant fungal species such as O. hylesinum and Geosmithia flava significantly influencing di;erentiation. Larval and both tunneling and overwintering adult stages showed consistent symbiont profiles, but larvae displayed lower fungal biodiversity than adults. Physiological Adaptations of Symbiotic Fungi We examined physiological traits of key fungi under varying abiotic conditions. pH tolerance: O. hylesinum preferred more acidic conditions (optimum pH 4), whereas Geosmithia species tolerated wider pH ranges, favoring slightly neutral conditions. Osmotolerance: Geosmithia species showed higher salt tolerance compared to Ophiostomatales. Temperature: No significant di;erence between groups; most fungi grew optimally at 25–30°C. Oxygen levels: All tested species preferred low-oxygen (microaerophilic) conditions. Tolerance to plant secondary metabolites: Most fungi grew well with condensed tannins and flavonoids, but tannic acid inhibited many strains except G. flava. 35 Conclusion Despite living on the same host tree, H. fraxini and H. crenatus maintain distinct microbial communities. Our experimental results suggest that the ecological di;erentiation of their symbionts may be linked to microhabitat-specific abiotic factors, particularly pH and water availability, rather than solely to beetle-mediated selection. Our findings highlight the complex interplay of host organ specificity, environment, and microbial physiology in shaping beetleassociated microbiomes. General discussion The specific requirements of life under the bark and the choice of a suitable model species When bark beetles enter the environment beneath tree bark, they encounter a challenging habitat characterized by hard-to-degrade polysaccharides, toxic plant secondary metabolites, insu;icient amount of essential nutrients required for their development and often an oxygenpoor atmosphere combined with rapidly fluctuating temperatures and water activity levels (Boone et al., 2013; Gandhi & Hofstetter, 2021; García-Fraile, 2018; Hagen, 2002; Linnakoski et al., 2017; Morales-Jiménez et al., 2012b). Additionally, they face competition from numerous microorganisms that also rely on the limited available nutrients. To successfully colonize host trees, bark beetles form symbiotic relationships with diverse microbial communities (Biedermann & Vega, 2020). The European spruce bark beetle, I. typographus, is a major pest of spruce trees in the Palearctic region. This aggressive bark beetle causes the notorious outbreaks in silvicultures throughout Europe, causing enormous economic losses (Hlásny et al., 2021a, 2021b) . Furthermore, it can be assumed that climate change will further intensify the impact of bark beetles on forest ecosystems (Biedermann et al., 2019a; García-Fraile, 2018). As a result of its socio-economic impact on human communities, it has a significant e;ect on the environment, thus subsequent attention of the scientific community; its genome is already available (Powell et al., 2021) and transcriptome as well (Ramakrishnan et al., 2022). Although previous research has explored the microbial communities associated with this beetle species (Chakraborty, Ashraf, et al., 2020; Chakraborty, Modlinger, et al., 2020a; Fabryová et al., 2018; Fang et al., 2020; Moussa et al., 2024), our study is the first comprehensive description of both bacterial and fungal gut microbiota across the beetle’s entire developmental cycle and spanning two successive generations. 36 In contrast to Ips, Hylesinus represents a chronically understudied genus of bark beetles from hardwoods. Regardless of its unexplored nature, H. crenatus and H. faxini represent an ideal species pair for answering the fundamental ecological questions related to the microhabitat adaptations through a specific symbiotic microbiota (Strzałka et al., 2021). Observed microbial diversity Our results revealed low microbial α-diversity within the intestinal community of bark beetles, a pattern commonly observed in bark beetle-associated microbiota (Barcoto et al., 2020; Briones-Roblero et al., 2017). In agreement with findings reported by (Chakraborty, Modlinger, et al., 2020a, 2020b) and (Baños-Quintana et al., 2024; Fang et al., 2020), our data confirmed the dominance of Gammaproteobacteria-Enterobacterales (namely E. typographi, P. bohemica, and P. typographi) within the bacterial microbiome of I. typographus. Although the previous studies (Chakraborty, Ashraf, et al., 2020; Chakraborty, Modlinger, et al., 2020a performed in Rouchovany, Czechia) and (Fang et al., 2020 performed in Jingouling Forest Farm, China) used di;erent region of the rRNA region for metabarcoding, our studies sampled in Křivoklátsko Protected Landscape Area, Czech Republic, targeted the V5–V6 region. This methodological di;erence may introduce biases in taxonomic resolution and community composition (Claesson et al., 2010). Interestingly, a potentially novel genus within the order Enterobacterales was consistently detected across all developmental stages in our dataset and was also reported in (Fang et al., 2020). It should be noted that (Fang et al., 2020) did not include teneral adults in their analysis and separated adult males and females. Despite these di;erences, they similarly observed a high abundance of Erwinia during the larval stage, which declined during pupation, whereas Pseudoxanthomonas showed the opposite trend. In contrast,(Chakraborty, Ashraf, et al., 2020) reported Rahnella and Raoultella as dominant taxa, neither of which were detected in our amplicon data, nor did they report the potentially undescribed Enterobacterales genus found in our study. However, genera such as Pseudomonas, Acinetobacter, and Streptococcus were consistently detected across all studies (Chakraborty, Ashraf, et al., 2020; Fang et al., 2020). Based on recently published works (Baños-Quintana et al., 2024; Chakraborty et al., 2023; Khara et al., 2024; Moussa et al., 2024) all following the publication of our studies, we can conclude that a similar bacterial community was found to be associated with I. typographus on a very large geographic area, which may indicate the stability of this symbiosis. The fungal community was primarily composed of yeasts belonging to the class Saccharomycetes , predominantly Genera (Kuraishia, Nakazawaea, Wickerhamomyces, Ogataea) and Cryptococcus from class Tremellomycetes, followed by filamentous fungi from 37 class Sordariomycetes, particularly species known as symbiotic partners of I. typographus, such as Ophiostoma bicolor and Endoconidiophora polonica (R. Jankowiak, 2005; Linnakoski et al., 2012; Repe et al., 2013a). While DNA-based metabarcoding and cultivation experiments primarily identified members of the class Gammaproteobacteria, our RNA-based metabarcoding approach additionally emphasized the significant presence and activity of Betaproteobacteria, Actinomycetia, and Alphaproteobacteria. Specifically, the genera Klebsiella, Enterobacter, and Phyllobacterium emerged as the most metabolically active taxa according to RNA metabarcoding results. Although fungal community composition appeared consistent between DNA and RNA metabarcoding analyses, significant discrepancies were observed within the bacterial microbiome. These di;erences may reflect the well-documented concept that species abundance does not necessarily correlate with functional activity or dominance (Risely, 2020). Enterobacter and Erwinia strains have previously been isolated from other bark beetle species, including the important species of the Dendroctonus valens (Morales-Jiménez et al., 2009). Similarly, family Enterobacteriaceae is reported to be a major bacterium in the Dendroctonus rhizophagus gut (Morales-Jiménez et al., 2012b). To the best of our knowledge, the presence or potential functional role of Phyllobacterium spp. has not yet been documented in bark beetles. However, this genus is frequently identified as an endophyte in plants, with certain strains capable of promoting the growth of Picea sp. (Anand et al., 2007). Future research should therefore explore the metabolic activities and ecological roles of these microbes within the I. typographus holobiont. Identifying the core microbiome of I. typographus Although the composition of the intestinal microbiome undergoes substantial changes during the lifecycle and across generations of I. typographus, we identified a stable core community consistently present in the beetle gut. This core microbiome includes yeast species such as Cyberlindnera sp., K. molischiana, K. capsulata, N. ambrosiae, W. bisporus, O. ramenticola, Ogataea neopini, S. lassenensis, and Y. scolyti, as well as the filamentous fungus O. bicolor. Among bacteria, we detected the consistent presence of E. typographi, E. cedenensis, Pseudomonas bohemica, Pseudoxanthomonas spadix, and strains from genera Curtobacterium, Roseomonas, Serratia, Taibaiella, and other unidentified species belonging to the families Sphingobacteriaceae, Lachnospiraceae, and Enterobacteriaceae. Most of these core microbial taxa have already been reported as associates of various bark beetle species, suggesting their 38 widespread occurrence in bark beetle habitats (Baños-Quintana et al., 2024; Barcoto et al., 2020; Briones-Roblero et al., 2017; Chakraborty, Ashraf, et al., 2020; Chakraborty et al., 2023; Chakraborty, Modlinger, et al., 2020a; García-Fraile, 2018; Giordano et al., 2013; HernándezGarcía et al., 2018; Linnakoski et al., 2021; Lou et al., 2014; Moussa et al., 2024; Saati-Santamaría et al., 2018, 2021). These results suggest that a stable core of gut microbiome (bacteria and fungi) persists throughout I. typographus development, providing essential metabolic support regardless of environmental fluctuation. In some plant-feeding insects, including bark beetles, the gut microbiome is very specific and resistant to changes (Adams et al., 2013b). In others, the microbiome changes more frequently, mostly based on their diet, and can di;er between insect populations(Adams et al., 2010; Jones et al., 2019; Šigut et al., 2022). This variability suggests that insects pick up their gut microbes directly from the environment (Kikuchi et al., 2007). We propose that the core gut microbiome of I. typographus originates from microbes naturally living inside healthy spruce phloem - the beetle's main food source. Our analysis of α-diversity (Shannon index) and cultured bacterial communities in (Peral-Aranega et al., 2023) showed that the composition of the I. typographus bacteriome changes across its life stages. Bacterial diversity was highest in larvae, decreased in pupae, remained low in teneral adults, and increased again in mature adults. This pattern was not always observed in our other study (Veselská et al., 2023) or (Fang et al., 2020), but it is similar to what has been reported in Dendroctonus rhizophagus, although teneral adults were not included in that study (MoralesJiménez et al., 2012a). We hypothesized that these changes are likely caused by metamorphosis, which alters the gut environment and a;ects which bacteria can survive. However, some bacterial taxa were present in all stages, suggesting they may play important roles throughout development. Most of the bacteria found in I. typographus are likely acquired from the phloem; however, it is also possible that some are passed from adults to their o;spring, as observed in other insect species (Baños-Quintana et al., 2024; Bright & Bulgheresi, 2010). Acquisition and Localization of Gut Symbionts At the same time, our TEM analysis did not reveal any specialized microbial structures or biofilm formation in the gut of I. typographus, which may indicate that microorganisms are likely transiently passing through the digestive tract along with plant material rather than being permanently retained. Therefore, we suggest that the gut microbiome of I. typographus is predominantly acquired from ingested plant tissues, with environmental filtering favoring microbial taxa adapted to survive under the harsh gut conditions (Appel & Maines, 1995; Douglas, 39 2014; Engel & Moran, 2013). Further feeding experiments would be valuable to test this hypothesis. An exception to this general pattern was observed for the mutualistic filamentous fungi O. bicolor and Endoconidiophora polonica (B. R. Jankowiak, 2005; R. Jankowiak et al., 2009; Linnakoski et al., 2016; Repe et al., 2013b), whose proportions in the gut microbiome increased progressively throughout the beetle’s development. This trend suggests microbial succession analogous to that documented in other insects, e.g. galleries of the ambrosia beetle Xyleborus aAinis (Ibarra-Juarez et al., 2020), where initial colonizers such as bacteria and yeasts are gradually replaced or supplemented by filamentous fungi. Both O. bicolor and E. polonica are actively transported by bark beetles into new host trees (Biedermann et al., 2019c), aligning with our observations of their relatively low abundance in intact spruce phloem compared to their increased presence in phloem adjacent to bark beetle galleries by the end of the beetle’s development. Metabolic complementarity within the I. typographus holobiont Our previous studies have shown that the gut-associated bacterial and fungal symbionts of I. typographus possess genomic potential for nutrient provisioning, detoxification, and complex carbohydrate degradation(Cheng et al., 2023; Fabryová et al., 2018; Veselská et al., 2023). (Fabryová et al., 2018) is not part of this dissertation, but it is also a publication performed by our broader team. However, the level to which these microbes interact functionally as a cooperative system remained unresolved. In this dissertation, we build on these foundational insights and present the first metatranscriptomic evidence for metabolic complementarity among the members of the I. typographus holobiont (Saati-Santamaría et al.2025, unpubl. -here referred to as “Manuscript 1”). These analyses revealed a pronounced division of labor among bacterial and fungal taxa: bacterial symbionts such as Erwinia, Delftia, and Phyllobacterium were transcriptionally active in pathways involved in nitrogen cycling - specifically nitrate/nitrite reduction and uric acid catabolism, while fungal taxa, including Ogataea and Kuraishia, contributed to urea and ammonia assimilation (Saati-Santamaría et al.2025, unpubl.-here referred to as “Manuscript 1”).). This functional pattern reflects a cross-kingdom interdependence, where metabolic products from one group serve as substrates for another. While such microbial cooperation was previously hypothesized in gallery biofilms (Barcoto et al., 2020; Ibarra-Juarez et al., 2020), our study provides the first functional evidence for this integration at the gut level. Moreover, our metatranscriptomic results confirm and extend genome-based predictions from our genomic study of yeast symbionts (Cheng et al., 2023), which demonstrated the presence 40 of biosynthetic pathways for vitamin B6 and essential amino acids, but according to the transcriptome, they rely on intermediates from bacteria. Perhaps this is because they “save” and take what is in excess from the environment. We also observed that bacterial taxa were transcriptionally active in biosynthetic pathways for thiamine (B1), riboflavin (B2), biotin (B7), and pyridoxal derivatives. At the same time, yeasts appeared to complement or scavenge these intermediates (Saati-Santamaría et al., unpubl.). These observations are further supported by enzymatic assays from our culture-based study (Peral-Aranega et al., 2023), which demonstrated that strains of Pseudomonas and Bacillus exhibited strong hydrolytic and antifungal activity against bacterial isolates across various stages of beetle development. The yeast strains examined according to sequenced genomes should be capable of breaking down pectin, but our in vitro tests did not confirm this ability, or only inconclusively. Filamentous fungal symbionts are known to break down cellulose, which has been confirmed by our transcriptomic data, unlike pectin, which in our system is probably degraded only mostly by bacteria. Among other things, this shows that the presence of a pathway in the fungal genome may not always manifest itself under in vivo conditions, as observed, for example, in the work of (Kjærbølling et al., 2020). Our transcriptomic findings also align with previous work highlighting the dynamic nature of the I. typographus gut microbiome (Veselská et al., 2023), where temporal shifts in taxonomic composition were observed but masked potential functional redundancy among community members. The current metatranscriptomic dataset resolves this ambiguity by showing that taxa di;er in abundance may still contribute consistently to critical functions. Taken together, these results demonstrate that the I. typographus holobiont functions not merely as a host-associated microbial assemblage, but as an ecologically and metabolically integrated consortium. This organization likely confers adaptive advantages in the nutrient-limited and chemically defended phloem environment (Zhao et al., 2019). It represents a model of microbial synergy with relevance beyond bark beetle systems. Gut micobiome benefits – direct study approaches While metatranscriptomic results revealed functionally active metabolic interactions, we further validated the ecological potential of bacterial symbionts through in vitro functional assays. Only a limited number of studies have investigated the composition and ecological significance of the bacterial community associated with the bark beetle I. typographus. In our studies, we examine the diversity of the beetle’s bacteriome across di;erent developmental stages by integrating data from both cultured isolates and metabarcoding analyses. Furthermore, we performed in vitro experiments to assess the metabolic capabilities of the isolated strains 41 and to explore their potential contributions to the beetle's ecological interactions, dynamics and functional redundancy in the gut microbiome across the life cycle of I. typographus (Cheng et al., 2023). We observed considerable shifts in the overall microbial composition throughout the developmental stages of I. typographus. Some fungi showed increased abundance at specific life stages; for example, yeasts K. molischiana and N. ambrosiae dominated during larval and pupal stages, while S. lassenensis was more abundant in parental adults. Additionally, spring adult beetles exhibited a high proportion of unique ASVs. Since I. typographus adults may overwinter beneath the bark or in forest litter, this could temporarily enrich their gut microbiome with microbial species originating from environments beyond spruce phloem. However, these externally acquired microorganisms seem to diminish or disappear during subsequent developmental phases. We also recorded a reduction in the number of fungal ASVs in larvae and pupae compared to parental adults and teneral stages. A similar decline has previously been noted by (Lou et al., 2014) in yeasts associated with the bark beetle Dendroctonus valens, and by (González-Serrano et al., 2020) in bacteria from the moth Brithys crini. The initial fungal diversity decrease at the larval stage may reflect the loss of parental adult-transmitted microbes originating from previous hosts, whereas the second reduction during the pupal stage might be linked to physiological changes accompanying metamorphosis and reformation of the gut (González-Serrano et al., 2020). Furthermore, seasonality had a significant influence on the microbial community structure. Seasonal variations primarily a;ected the relative proportions of dominant microbial taxa rather than their presence or absence. The filamentous fungi associated with I. typographus likely share similar functional roles, such as detoxifying plantderived secondary metabolites, making them functionally interchangeable (Zhao et al., 2019). Consequently, in agreement with (Bang-Andreasen et al., 2019; R. Jankowiak, 2005; Louca et al., 2017) we propose that I. typographus associates with functionally similar microbial taxa, whose community composition and structure are primarily shaped by environmental conditions (Banos, 2024; Chakraborty, 2024). We also investigated the potential ecological functions of the isolated bacterial strains concerning two key aspects of I. typographus biology: digestion of complex carbon sources and protection against fungal entomopathogens, both traditionally assumed to be benefits mediated by bacteria (Peral-Aranega et al., 2023). Several bacterial isolates demonstrated the ability to hydrolyze CMC, xylan, pectin, and starch, all of which are components of the inner bark (PeralAranega et al., 2020, 2023, Saati-Santamaría et al.2025, unpubl. -here referred as “Manuscript 1”). Notably, Bacillus tequilensis and P. typographi strains isolated from larvae and adults 48 Andersson, M. 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