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.