Introduction
It is now widely recognized that animals live in a microbial world, and
that many aspects of animal biology, ecology and evolution are a product
of their symbioses with microorganisms (McFall-Ngai et al., 2013). In
invertebrates, these symbioses may be particularly intimate, and involve
transmission of the microbe from parent to offspring (Hurst, 2017). The
alignment of host reproduction with symbiont transmission produces a
correlation between the fitness interests of the parties, reflected in
symbionts evolving to play a number of physiological roles within the
host, from defence (Jaenike, Unckless, Cockburn, Boelio, & Perlman,
2010; Łukasik, Guo, van Asch, Ferrari, & Godfray, 2013; Teixeira,
Ferreira, & Ashburner, 2008) through to core anabolic and digestive
functions (Douglas, 2009; Rio, Attardo, & Weiss, 2016). However, the
maternal inheritance of these microbes has led to the retention of
parasitic phenotypes associated with distortion of reproduction, with
symbiont phenotypes including biases towards daughter production and
cytoplasmic incompatibility (Hurst & Frost, 2015). These diverse
individual impacts alter the ecology and evolution of the host, in terms
of diet, dynamics of interaction with natural enemies, sexual selection
and speciation.
Heritable symbioses have evolved on multiple occasions amongst microbial
taxa. In some cases, the microbial lineage is limited to a single clade
of related animal hosts, such as Buchnera in aphids
(Munson, Baumann,
& Kinsey, 1991). In other cases, particular heritable microbes are
found across a wide range of arthropod species. Wolbachiarepresents the most common associate, considered to infect nearly half
of all species (Zug & Hammerstein, 2012), and this commonness is a
function in part of the ability of Wolbachia to transfer to a
broad range of new host species and spread within them (host shift
events) (Siozios, Gerth, Griffin, & Hurst, 2018; Turelli et al., 2018).
Aside Wolbachia , other microbes are found commonly as heritable
symbionts of arthropod hosts (Duron et al., 2008). Cardinium andRickettsia , for instance, have been estimated at being present in
13-55% and 20-42% of species respectively
(Weinert,
Araujo-Jnr, Ahmed, & Welch, 2015).
In this paper, we address the diversity and commonness of symbioses
between Rickettsia and arthropods. The Rickettsia have
increasingly been recognized as a genus of bacteria with diverse
interactions with arthropods
(Perlman, Hunter,
& Zchori-Fein, 2006; Weinert, Werren, Aebi, Stone, & Jiggins, 2009).
First discovered as the agents underlying several diseases of humans
vectored by haematophagous arthropods (da Rocha-Lima, 1916; Ricketts,
1909), our understanding of the group changed in the 1990s with the
recognition that Rickettsia were commonly arthropod symbionts
(Chen, Campbell,
& Purcell, 1996; Fukatsu & Shimada, 1999; Werren et al., 1994).Rickettsia were recognized first as male-killing reproductive
parasites (Hurst,
Walker, & Majerus, 1996; Werren et al., 1994) and then later as
beneficial partners
(Hendry, Hunter,
& Baltrus, 2014; Himler et al., 2011; Łukasik et al., 2013).
Following this extension of our understanding ofRickettsia -arthropod interactions, a new clade ofRickettsia was discovered from work in Torix leeches
(Kikuchi &
Fukatsu, 2005; Kikuchi, Sameshima, Kitade, Kojima, & Fukatsu, 2002).
This clade was sister to all other Rickettsia genera, with no
evidence to date of any strain having a vertebrate pathogen phase. The
host range for Torix Rickettsia is broader than that for other
members of the genus, going beyond arthropods to include leech and
amoeba hosts
(Dyková,
Veverková, Fiala, Macháčková, & Pecková, 2003; Galindo et al., 2019;
Kikuchi & Fukatsu, 2005). Targeted PCR based screening have revealed
Torix group Rickettsia as particularly common in three groups
with aquatic association: Culicoides biting midges, deronectid
beetles and odonates
(Küchler, Kehl and
Dettner, 2009; Pilgrim et al., 2017; Thongprem et al.2020). However, some previous hypothesis-free PCR screens that aimed to
detect Rickettsia in arthropods have likely missed these
symbioses, due to divergence of the marker sequence and mismatch with
the primers
(Weinert, 2015).
During our previous work on Torix Rickettsia in biting midges
(Pilgrim et al., 2017), we became aware of the presence ofRickettsia CoxA sequences deposited in Genbank that derived from
studies where the intended target of amplification/sequencing was
cytochrome c oxidase I (COI ), the mitochondrial equivalent
of CoxA . These deposits derived from studies using mtDNA for
phylogeographic inference (Lagrue, Joannes, Poulin, & Blasco-Costa,
2016), or in barcoding based species identification approaches
(Ceccarelli, Haddad, & Ramírez, 2016; Řezáč, Gasparo, Král, &
Heneberg, 2014). Non-target amplification of Rickettsia COI using
mitochondrial COI barcoding primers has been reported in spiders
(Ceccarelli et al., 2016; Řezáč et al., 2014) and freshwater amphipods
(Lagrue et al., 2016; Park & Poulin, 2020). Furthermore, we have noted
two cases in our lab where amplicons obtained for mtDNA barcoding of an
insect have, on sequence analysis, revealed Rickettsia COIamplification (Belli group Rickettsia from Collembola, and Torix
group Rickettsia from Cimex lectularius bedbugs). Previous
work had established barcoding approaches may amplify COI fromWolbachia symbionts (Smith et al., 2012), and the data above
indicate that non-target Rickettsia COI may be likewise amplified
during this PCR amplification for mitochondrialCOI .
In this paper, we use three approaches to reveal the diversity and
commonness of Torix Rickettsia in arthropods. First, we probed
the contaminant bin of the Barcode of Life Data System (BOLD) forRickettsia sequences and used the template from these projects to
define the diversity of Rickettsia observed using a multilocus
approach. Second, we screened DNA templates from multiple individuals
from 169 arthropod species for Rickettsia presence using PCR
assays that function more broadly than previously utilized in screens.
Within this, we included a wider range of aquatic taxa, to investigate
if the previous work highlighting particular aquatic taxa as hotspots
for Rickettsia symbiosis (water beetles, biting midges,
damselflies) reflects a wider higher incidence in species from this
habitat. Finally, we used bioinformatic approaches to examine the
Sequence Read Archive (SRA) depositions for one individual from 1,342
arthropod species for the presence of Rickettsia and used this as
a means of estimating the relative balance of Torix group to otherRickettsia within symbioses.