Transmission electron microscopy (TEM)
In some insect specific structures for vertical transport of endosymbionts were formed during evolution (e.g. Xue et al. 2014, Matsuura et al. 2018). It is not known whether Ips typographusgut microbiome is transmitted vertically or is acquired horizontally from the environment. We used TEM for direct observation of Ips typographus gut in purpose to examine possible formation of biofilm on intestinal wall. We examined all three parts of the beetle’s gut (foregut, midgut, and hindgut); however, we did not find any sign of polymicrobial biofilm formation on the beetle’s tissues. We found bacterial and fungal cells as a part of chyme together with clean epithelia and microvilli without any signs of formed residual biofilms (Fig. 5).
DISCUSSION
Ips typographus is a serious spruce pest that largely impacts the European landscape. Albeit known importance of microorganisms on bark beetle fitness, sparse information is available about the microorganismal community associated with I. typographus (Chakraborty et al. 2020a, 2020b). Our study is the first that describes the fungal and bacterial intestinal microbiome of the spruce bark beetle I. typographus throughout its whole life cycle in two subsequent generations. Combination of DNA metabarcode analyses with cultivation and subsequent molecular identification of pure cultures enables us to identify the dominant fungal and bacterial taxa mostly up to the species level. As the DNA metabarcode analysis is biased by capturing total persisting environmental DNA, even DNA from already dead cells eaten by insects (Gifford at al.,2014), their functional dominance was further confirmed by RNA metaborcode analysis. The intestinal microbiome was also observed directly in the gut by Transmission Electron Microscopy (TEM) to examine potential formation of biofilm structure.
In some herbivorous insects as well as bark beetles, gut communities are fairly specific and highly resistant to perturbation (e.g.Dendroctonus ponderosae, Adams et al. 2013). In other cases, the bacterial community appears to be more dynamic, mostly food derived and differing between host populations (D. valens , Adams et al. 2010). It is known that host plants fundamentally influence gut microbiome of insect herbivores (Jones et al. 2019, Šigut et al. 2022) and intestinal microorganisms could be acquired horizontally from the environment (Kikuchi et al. 2007). On the other hand, specific internal structures like bacteriocytes, mycetocytes (Douglas 1998), or fat body cells (Xue, Zhou et al. 2014) allowing vertical transmission have also been described in insects.I. typographus belongs among bark beetles that lack any specific external morphological structures for transmission of associated microorganisms and visualization of its gut epithelium is scarce (Takov et al., 2012). Thus, it is not clear to which extent are microorganisms transferred vertically via gut and body surface or recruited de novo in a new host.
Overall microbial intestinal α-diversity was low, which is typical for bark beetle associated communities (e.g. Briones-Roblero et., 2017; Barcoto et al., 2020), with a few dominant species. At the same time, the intestinal microbiome of I. typographus represents a subset of species endophytically residing in uninfested spruce phloem. Lower microbial diversity in gut compared to body surface of bark beetleDendroctonus valens (Lou et al., 2014) also indicates that only a subset of species from the environment enters into the insect gut. In addition, we did not observe any specific structure or biofilm formation in the beetle gut by TEM analysis. This suggests non-specific distribution of microorganisms in gut lumens and their attachment to the digested plant residuals. We thus propose that the intestinal microbiome of I. typographus is mostly recruited from the plant tissue and formed by environmental filtering which selects for species that can cope with the stressful environment of insect gut (Appel & Maines 1995; Douglas, 2015; Engel &Moran 2013). Future feeding experiments may answer this hypothesis. Only exceptions for above mentioned observations were mutualistic filamentous fungi Ophiostoma bicolor andEndoconidiophora polonica(Jankowiak and Hilszczanski 2005; Linnakoski et al. 2016; Repe et al. 2013). These fungi were found in very low numbers in fresh uncolonized spruce phloem; however, their proportions increase throughout life cycle and finally they dominate in phloem adjected to bark beetles’ galleries at the end of I. typographus development. These fungi create sticky conidia as an adaptation for transmission on beetles’ body surface (Harrington, 2005) and our data support vertical transmission of these fungi into a new host.
The cultivation technic captured only around 11% of fungal and 9% of bacterial species identified by DNA metabarcoding as many species are hard or still impossible to cultivate (Six, 2003; Lou e al., 2014; Hiergeist et al., 2015; Wang et al., 2020). However, these species belong to the dominantly present species in DNA metabarcode analysis. We were able to identify functionally active microorganisms from RNA metabarcode analysis up to genera level in Bacteria and mostly to family level in Fungi. Functional analysis revealed similar dominant taxa; however, the proportions of some rare bacterial and fungal classes in DNA metabarcode analysis were increased, see below.
Fungal microbiome was dominated by yeasts (Ascomycota: Saccharomycetales), which indicates their important function in I. typographus gut. The most dominant yeasts are Wickerhamomyces bisporus, Nakazawaea ambrosiae, Kuraishia molischiana, Ogataea ramenticola, Cyberlindnera, Yamadazyma scolyti and Meyerozyma guilliermondii. Meyerozyma guilliermondii was captured by us only from cultivation and it was also recorded in the microbiome of ambrosia beetle Platypus koryoensis (Yun et al. 2015), where also its enzymatic capability of plant tissue degradation was detected.Wickerhamomyces bisporus was previously found in association withI. typographus (Giordano et al. 2012) and is also the dominant yeast species found on phoretic mites of the same beetle species (Linnakoski et al., 2021). Species of Wickerhamomyces have been also reported from galleries and guts of wood-boring insects (Hui et al. 2013; Ninomiya et al. 2013), indicating their common association with beetles. The importance of yeasts in bark beetles’ ecosystem was proposed earlier based on the cultivation technic (Beck, 1922; Siemaszko, 1929; Shifrine & Phaff, 1956) and also emphasized in recent molecular studies (Chakraborty et al. 2020, Ibarra-Juarez et al., 2020). Some gut yeasts can convert host tree defensive chemicals to beetle pheromones; however, the insect is not dependent upon them for this function (Hunt and Borden 1990). Yeasts provide a variety of benefits in several insect systems (Ganter, 2006; Rohlfs & Kurschner, 2010). Nevertheless, the roles yeasts play in bark beetle systems remain unclear (Six, 2013). The second most abundant order was Sordariomycetes, which was mainly represented by symbiotic Ophiostoma bicolor andEndoconodiophora polonica(Jankowiak & Hilszczanski 2005, Linnakoski et al., 2016; Repe et al., 2013). Their abundance in the gut microbiome gradually increases throughout I. typographus life cycle. This may be due to the fact that their frequency also increases over time in infested phloem, which in turn affects the composition of the intestinal biota. Another filamentous fungi, Morchella importuna (Pezizomycetes), was also significantly more abundant at the end of the beetle development. The significance of Pezizomycetes was further highlighted in RNA metabarcode analysis in which this class took 2.9 % of total fungal reads. That may also point to succession in beetle’s galleries which are at first dominated by yeasts which are only after some time accompanied by filamentous fungi.
Bacterial class Gammaproteobacteria was found to dominate the bacterial microbiome of fungus growing insects (Barcoto et al., 2020). This predominance was also described in bark beetles (Hernandez-Garcia et al., 2018; Chakraborty et al., 2020) and confirmed in the present study. However, our functional analysis highlighted the importance of other bacterial classes: Betaproteobacteria (22% of reads), Actinomycetia (8% of reads) and Alphaproteobacteria (7% of reads). Interestingly, some of the most active microbes (higher abundance in metatranscriptomic samples) are not among the most abundant taxa identified in the metabarcoding analyses. For instance, Klebsiella ,Enterobacter , and Phyllobacterium , have been found as the three most active genera in I . typographus. It has been suggested that Klebsiella spp. may fix nitrogen within other bark beetle species and insects (Yaman et al., 2010). Enterobacterspecies have been isolated from other bark beetles, such as the great spruce bark beetle (Dendroctonus micans ) (Morales-Jiménez et al., 2012). Finally, as far as we know, there is not any report that suggests the presence nor any function of Phyllobacterium spp. within bark beetles; however, it is a common plant endophytic genus, which harbors strains with the ability to promote the growth of spruce trees (Anand et al., 2006). The activity of these microbes should be further investigated to understand their roles within the I. typographusholobiont. The microbiome was dominated by order Enterobacteriales, especially by species Erwinia typographi, which is common in intestinal communities as it is able to grow in almost anaerobic conditions in the gut and is resistant against high concentrations of monoterpene myrcene (Skrodenytė-Arbačiauskienė et al., 2012). Other abundant taxa were Pseudomonas bohemica (Pseudomonadales) and Pseudoxanthomonas (Pseudoxanthomonadales). These genera were previously isolated from I. typographus and seem to be also common associates of other bark beetles (Briones-Roblero et., 2017; García-Fraile, 2018; Hernandez-Garcia et al., 2018; Saati-Santamaría et al., 2018; Barcoto et al., 2020; Chakraborty et al., 2020; Peral-Aranega et al., 2020; Saati-Santamaría et al., 2021). Micrococcus luteusisolated by us from I. typographus gut, was previously isolated from oral secretion of bark beetle Dendroctonus rufipenis where it helps with inhibition of growth of antagonistic filamentous fungi (Cardoza et al., 2006). Overall, inferred functions of these bacteria are degradation of lignocellulose, detoxification of plant secondary metabolites, protection against fungal pathogens and metabolism of diverse nutrients (García-Fraile et al., 2018; Ibarra-Juarez et al., 2020; Barcoto et al., 2020).
We found significant change in microbial communities throughout the life cycle of I. typographus . Some fungal species are more abundant in certain developmental stages, e.g. Kuraishia molischiana andNakazawaea ambrosiae in larval and pupal stages orSaccharomycopsis lassenensis in parental adult stage. Developmental stages also differ in the number of fungal ASVs, concretely we observed a drop in fungal ASVs number in the larval and pupal life stages compared to parental and teneral stages. This phenomenon was also observed by Lou et al. (2014) in yeast communities of bark beetle Dendroctonus valens and by González-Serrano et al. (2020) in bacterial communities of moth Brithys crini . The first drop in fungal diversity in larval stage may be caused by loss of species transmitted in guts of parental adults from the previous hosts and the second drop in pupal stage may be linked with metamorphosis and construction of a new intestinal system (González-Serrano et al., 2020).
Season has a statistically significant effect on microbial communities. Communities differ in proportion of dominantly associated microbes rather than in change in species composition. Similarly to Jankowiak and Hilszczanski (2005), Louca et al. (2016), and Bang-Andreasen et al. (2020), we suppose that environmental conditions shape the composition and structure of microbiomes. Filamentous fungi associated withIps typographus have similar capability in detoxification of plant secondary metabolites and thus have interchangeable functions (Zhao et al., 2019) and similar situation can take place in the yeast and bacterial community, when individual species have interchangeable functions and their proportion are driven by environmental conditions.
To conclude, this study is the first to show the composition of the core gut microbiome of spruce pest I. typographus based on cultivation and DNA/RNA metabarcode sequencing. We found it is changing throughout the life cycle of the beetle with the drop in fungal diversity in larval and pupal stages. Saccharomycetous yeasts and bacteria from the family Enterobacteriaceae dominate the intestinal microbiome. We also detected changes in proportions of the dominant taxa in respect to season. We propose that these species have interchangeable functions in bark beetles’ habitat and their proportions are driven by environmental conditions. Future studies may focus on functional analyses of the core microbiome of I. typographus to address our hypothesis.