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.