INTRODUCTION
Bark beetles (Curculionidae: Scolytinae) is an ubiquitous, taxonomically and ecologically highly diversified group of insects. They include more than 6,000 species that feed on various plant tissues (Kirkendall et al., 2015). Bark beetles are an integral part of forest ecosystems as they promote forest heterogeneity and resilience across multiple scales (Kulakowski et al., 2016). However, they cause increasingly severe outbreaks worldwide as a result of global climate change, insufficient forest management and introductions of new invasive species often linked to international trade (Hlasny et al., 2021). Thus, bark beetles are considered the most important threat to agricultural forests (Kirkendall & Faccoli, 2010; Hicke et al., 2013; 2016; Mezei et al., 2017).
The evolutionary success of bark beetles has been undoubtedly facilitated by a wide range of associations with microbial symbionts (Sun et al., 2013; Douglas, 2015; García-Fraile, 2018; Chakrabotry 2020; Dinkins-Bookwalter et al., 2015). Nature of the symbiosis is diverse, ranging from parasitism to mutualistic relationship (Six, 2012; Su et al. 2013) and microbiome fulfills multiple proposed functions. Firstly, these microorganisms enrich beetles’ diet with sterols, vitamins, essential amino acids, and nitrogenous compounds (Rivera et al., 2009; Hernandez-Garcia et al., 2018; Ibarra-Juarez et al., 2020; Fabryová et al., 2018; Veselská et al., 2018; García-Fraile, 2018; Bentz & Six 2006; Ayres, Wilkens et al., 2000). Secondly, they ease plant tissue colonization by detoxification of tree defense compounds (Hammerbacher et al., 2013; Giron et al., 2020), or by production of small molecules defending against pathogens, parasites, and predators (Conord, 2008; Berasategui et al., 2016; Giron et al., 2020). Thirdly, they ferment sugars in the tree phloem and convert them into beetle´s pheromones (Zhao et al., 2019). For that purpose, insects’ symbionts are a promising source of new bioactive compounds (VanMoll et al., 2021; Saati-Santamaría et al., 2018). Finally, some of the symbionts are phytopathogens that can necrotize healthy plant tissue, thereby not only increasing insects’ fitness but causing tree mortality (Li et al., 2022; Hofestetter, 2015).
Previous studies of the microbiome have studied a very different set of substrates, such as galleries or whole larvae or adults, or their mixture, not allowing to distinguish between ecto- and endosymbiotic microbial communities. When looking at the gallery system in more detail, it is clear that it is composed of a number of different niches. The life cycle of the beetle begins with the maternal beetles laying eggs and actively or passively introducing microorganisms into the system, which then grow into the surrounding plant tissues and are further consumed by larvae, which build their tunnels. Ectosymbiotic microorganisms proliferating around and inside galleries are the most conspicuous and studied, because they include tree-killing ophiostomatoid fungi. However, bark beetles, like other herbivores, also have endosymbiotic communities in their guts, which are then in direct interactions (e.g. detoxification, nutrient supply, protection, pheromone production) with ingested plant matter (Douglas 2015; Engel & Moran 2013; Giron et al. 2017). Several studies, mostly on Dendroctonus beetles and bacteria (e.g. Hou et al. 2022), have investigated the structure and composition of bark beetle gut microbial communities. More work is needed to understand how specific or labile gut communities are, and how their structure and composition relates to functions required by the beetle host (reviewed in Six et al. 2013; Engel, P. & Moran 2013).
European bark beetle, Ips typographus is currently the most serious pest of spruce forests (Biedermann et al., 2019). Its distribution follows in its entirety the area of its host treePicea abies which has a continuous range in Scandinavia, north-eastern Europe and western Russia, and central Europe.Ips typographus has one to three generations annually depending on temperature. Predictions suggest that the number of complete generations will increase as a result of global climate change (Jakoby, et al. 2019; Biedermann, et al. 2019). Despite the economic significance of I. typographus and the known importance of microorganisms on bark beetles´ ecology, we have only limited knowledge of the composition, ecological functions and seasonality of the microorganismal community associated with I. typographu s. Current knowledge is based almost exclusively on cultivation approaches, often focused on ophiostomatoid fungi only. Thus, baseline data on the total spectrum of associated microorganisms is missing. Ips typographus transmits microorganisms via gut and body surface, as it lacks any specialized phoretic structure (Bentz, et al. 2019). Ophiostomatoid fungi such asGrosmannia penicillata , Ophiostoma bicolor andEndoconidiophora polonica , which participate in the detoxification of spruce defense compounds (Hammerbacher et al., 2013; Zhao et al., 2019), are reported as dominant and stable associates. They are followed by a variety of other filamentous fungi and yeasts such asOgataea , Pichia , Candida , Kuraishia andCryptococcus (see Linnakoski et al., 2012 for review). Bacteriome has so far been studied sporadically and only few taxa such asSerratia liquefaciens (Muratoglu et al., 2009), Erwinia typographi (Skrodenytė-Arbačiauskienė et al., 2012),Staphylococcus , and Pseudomonas (Berasategui et al., 2016, Peral-Aranega et al., 2020, Saati-Santamaria et al., 2021) were reported. So far, only two studies based on fungal and bacterial DNA metabarcode sequencing respectively have been published (Chakraborty et al., 2020a, 2020b). However, these studies were focused only on large scale comparison of gut-associated microbiome among several bark beetle species, including I. typographus . Thus, our study is the first which brings detailed analyses of I. typographus microbiome throughout its life cycle and seasons. It is surprising that for such a fundamental forest pest, we do not have a clear idea of the complete structure of microbial communities (both intestinal and ectosymbiotic) and their changes during the life cycle and seasons (i.e. in different generations during the year). Due to sampling design biases, there is also a lack of information about which organisms the bark beetle vectors and which it acquires from the environment, via environmental filtering.
Here we report the first rigorous description of the core microbiome of I. typographus throughout its life cycle and two generations using a combination of cultivation technique, DNA and RNA metabarcode sequencing and transmission electron microscopy. For that purpose, we collected parental adults (beetle-finding galleries), larvae, pupae and young adults (teneral adults) from the two generations of the same year (spring and summer generation). Precise taxonomic identification allowed us to distinguish taxa mostly at the species level, which is essential for the interpretation of the ecology and biology of the particular symbiont. According to the current state of art, there are multiple definitions of core microbiome (Berg et al., 2020; Risely, 2020). In the present study, dominant species from the DNA and RNA metabarcode sequencing were assigned to belong among the core microbiota. We found that even though the taxonomic composition of core microbiota is rather similar, the proportions of the dominant taxa varies throughout the life cycle and seasons. Fungal microbiome is dominated by yeasts (mainlyWickerhamomyces bisporus , Nakazawaea ambrosiae andKuraishia molischiana ). Bacterial microbiome is dominated by orders Enterobacteriales, mainly by Erwinia typographi , Pseudomonadales and Pseudoxanthomonadales.