Introduction
An increasing number of studies demonstrate ubiquity and high diversity
of insect-associated microbiomes (Douglas, 2015; Engel, & Moran, 2013).
These microbial communities, composed of various pathogens, commensals
and random contaminants, can serve as natural sources of beneficial
symbiotic bacteria. In some insects they give rise to highly
specialized, maternally-transmitted mutualists called primary symbionts
(P-symbionts), which contribute to the host’s metabolism (Douglas,
1989). However, depending on richness and dynamics, the microbiomes
usually contain several symbiotic bacteria in various evolutionary
stages. In their typical form, P-symbionts are readily recognized by
several features (since they are indispensable mutualists): they are
universally present in all individuals, as a rule inhabiting specialized
host’s organs called bacteriomes (Baumann, 2005), and their genomes are
significantly reduced with a strong AT bias (Moran, 1996). One specific
feature of P-symbionts is their co-phylogeny with the host (Chen, Li, &
Aksoy, 1999; Clark, Moran, Baumann, & Wernegreen, 2000; Sauer,
Stackebrandt, Gadau, Holldobler, & Gross, 2000). For example, two of
the most studied P-symbionts, Buchnera in aphids andWigglesworthia in tsetse flies, were acquired at the beginning of
their hosts’ diversification and strictly mirror their entire phylogeny
(Chen et al., 1999, Clark et al., 2000). Other P-symbionts are
restricted to some of the host’s lineages, indicating that they are
either recently acquired symbionts or remnants of an ancient symbiont
lost in some of the host lineages (Bennett & Moran, 2013). In some
insects, several different P-symbionts may coexist and/or can be
accompanied by various secondary symbionts (S-symbionts). The latter are
less modified, retain more free-living-like characteristics, and some
are supposed to be the intermediate stages of evolution towards obligate
symbionts. Wigglesworthia represents a typical example of this as
it is often accompanied by the S-symbionts Sodalis glossinidiusand Wolbachia (Aksoy, 2000). The complexity of symbiotic
associations is obviously due to an ongoing process of symbiont
acquisition/loss/replacement, which is well known from several
bacteria-insect models and has a well-developed theoretical background
(Bennett & Moran, 2015). The theoretical work postulates that after a
certain amount of coevolutionary time, the symbiotic bacterium becomes
too degenerated and functionally inadequate, and it has to be replaced
(or accompanied) by another symbiont. While it would be interesting to
see how the microbiome diversity and dynamics relate to the complexity
of symbiosis in different insect groups, there is very little
information available today. The majority of studies on insects and
their P- and S- symbionts relies on metagenomic information and
phylogenetic reconstructions, likely missing a substantial part of
microbiome diversity. The introduction of amplicon approaches recently
demonstrated that this method can significantly improve our insight into
microbiome composition, even in extensively studied model systems
(Doudoumis et al., 2017; Gauthier, Outreman, Mieuzet, & Simon, 2015;
Manzano-Marin, Szabo, Simon, Horn, & Latorre, 2017; Meseguer et al.,
2017).
Amongst hematophagous (blood-feeding) insects which live exclusively on
vertebrate blood, sucking lice of the order Anoplura, with more than 500
spp. (Light, Smith, Allen, Durden, & Reed, 2010), are the most ancient
and diversified group. Accordingly, they possess a high diversity of
symbiotic bacteria (Allen, Burleigh, Light, & Reed, 2016; Boyd, Allen,
de Crécy-Lagard, & Reed, 2014; Boyd et al., 2016; Fukatsu et al., 2009;
Hypsa & Krizek, 2007). Depending on interpretation, the 16S rRNA
gene-based phylogenies for the available taxa suggest 5-6 independent
symbiotic lineages. However, none of them is a universal louse symbiont
distributed across the whole order (e.g. like Buchnera in
aphids). The distribution of louse symbionts suggests a relatively
recent origin of each lineage and hence a high rate of
acquisition/loss/replacement processes. Moreover, compared to the
extensively screened phytophagous groups, only a small fraction of
sucking lice diversity has been investigated. The actual number of
symbiotic lineages is therefore likely to be much higher. Of the
currently known lineages, genomic data are only available for four;
three of them showing clear signatures of P-symbionts: Riesiaspp., Puchtella pedicinophila , and Legionellapolyplacis (Table 1). Correspondingly, each of these lineages has
been found in two to four related host species as a result of
co-phylogenetic processes. The fourth lineage, the Sodalis -like
symbiont from Proechinophthirus fluctus , possesses a
significantly larger genome exceeding 2 Mbp, and GC content 50%, which
the authors interpret as possible evidence of recent replacement of a
more ancient and now extinct endosymbiont (Boyd et al., 2016). The
diversity and distribution of the known symbionts in sucking lice thus
indicate that this insect group has been undergoing particularly dynamic
acquisition, loss, and replacement of symbionts. In this study, we
analyze the background of these processes by combining genomic and
amplicon approaches across several populations of the louse generaPolyplax and Hoplopleura . We reveal a new symbiotic
lineage related to the genera Neisseria and Snodgrassella(the latter being a symbiont of bees). We show that these bacteria
established their symbiotic relationships independently with the two
louse genera, and we prove their intracellular localization in host’s
bacteriocytes. Based on the phylogeny-dependent diversity of the
microbiome profiles, we suggest rapid microbiome changes at the host
population level, possibly underlying the dynamic processes of symbiont
acquisition, loss, and replacement in these blood sucking insects.