Discussion
Symbiotic interactions between hosts and microbes are important drivers
of host phenotype, with symbionts both contributing to, and degrading,
host performance. Heritable microbes are particularly important
contributors to arthropod biology, with marked attention focused onWolbachia , the most common associate (Hilgenboecker, Hammerstein,
Schlattmann, Telschow, & Werren, 2008; Zug & Hammerstein, 2012).
Members of the Rickettsiales, like Wolbachia , share an
evolutionary history with mitochondria (Ferla, Thrash, Giovannoni, &
Patrick, 2013; Wang & Wu, 2015), such that a previous screen of BOLD
submissions of mtDNA submissions observed Wolbachia as the main
bacterial contaminant associated with DNA barcoding
(Smith et al.,
2012). However, our BOLD screen found that Rickettsia were more
likely to be amplified than Wolbachia (0.41% vs 0.17% of
deposits). Furthermore, Torix group Rickettsia were
overrepresented in barcode misamplifications (95%) when compared to
other groups within the genus. A comparison of the most commonly used
barcoding primers to Wolbachia and Rickettsia genomes
suggest homology of the forward primer 3’ end was likely responsible for
this bias towards Torix Rickettsia amplification. To gain a
clearer understanding of the relative balance of Torix group to otherRickettsia within symbioses and habitats, a targeted screen and
bioinformatic approach was also undertaken. Through these three screens,
a broad range of host diversity associated with Torix Rickettsiawas uncovered.
As the in silico and empirical evidence suggests Rickettsia
COI amplification is not uncommon
(Řezáč et al.,
2014; Ceccarelli et al., 2016; Park & Poulin, 2020), why has this
phenomenon not been described more widely before? The conduction of a
previous large-scale non-target COI study using BOLD submissions
(Smith et al.,
2012), revealed only Wolbachia hits. This screen involved
comparison to a Wolbachia -specific reference library and was thus
likely to miss Rickettsia . Additionally, there has been a lack of
Torix Rickettsia COI homologues to compare barcodes to until
recently, where a multilocus identification system, including COIwas devised
(Pilgrim et al.,
2017). Indeed, out of the contaminant dataset received in this study,
some of the Rickettsia contaminants were tentatively described by
BOLD as Wolbachia due to the previous absence of publicly
available Rickettsia COI to compare.
Although Rickettsia will only interfere with barcoding in a
minority of cases (~0.4%), it is likely that alternate
screening primers for some studies will need to be considered. In a
demonstration of how unintended Rickettsia amplifications can
affect phylogeographic studies relying on DNA barcoding, aRickettsia COI was conflated with the mtDNA COI of a
species of freshwater amphipod, Paracalliope fluvitalis(Lagrue et al.,
2016). Subsequently, supposed unique mtDNA haplotypes were allocated to
a particular collection site, whereas this merely demonstrated the
presence of Torix Rickettsia in host individuals in this lake.
Contrastingly, non-target Rickettsia amplification can also allow
for the elucidation of a novel host range of the symbiont
(Ceccarelli et
al., 2016; Park & Poulin, 2020; Řezáč et al., 2014) and this has been
exemplified with our probing of BOLD.
Previously, several host orders have been associated with TorixRickettsia , including Araneae, Coleoptera, Diptera, Hemiptera and
Odonata (Goodacre et al., 2006; Küchler, Kehl and Dettner, 2009;
Machtelinckx et al., 2012; Martin et al., 2013; Thongprem et al. 2020).
However, newly uncovered host orders from this study include Gastropoda
(snails), Trichoptera (caddis flies) and Trombidiformes (mites) (Table
2). Caution needs to be taken when interpreting what these newly found
associations mean, as mere presence of Rickettsia DNA does not
definitively indicate an endosymbiotic association. Indeed, parasitism
or ingestion of symbiont-infected biota can also result in PCR detection
(Le Clec’h et al., 2013; Plantard et al., 2012; Ramage et al., 2017).
Additionally, by calculating barcode success rate at an order level,
Hemiptera were deemed to fail barcoding (either lack of amplification
and/or quality sequence) more commonly than Diptera despite having a
similar estimated Rickettsia prevalence (Table S8). As an
increased barcoding failure rate is correlated with non-targetCOI amplification, it is probable there is a higher overall
proportion of Torix Rickettsia -associated Diptera than Hemiptera
in BOLD and likely in
nature.
Model-based estimation techniques suggest Rickettsia are present
in between 20-42% of arthropod species
(Weinert et al.,
2015). However, targeted screens often underestimate the incidence ofRickettsia hosts due to various methodological biases including
small within-species sample sizes (missing low-prevalence infections)
and the use of non-conserved primers
(Weinert, 2015).
Importantly, the inclusion and exclusion of specific ecological niches
can also lead to a skewed view of Rickettsia symbioses. A
previous review of Rickettsia bacterial and host diversity by
Weinert et al.
(2009) suggested a
possible (true) bias towards aquatic taxa in the Torix group. In
accordance with this, our targeted screen demonstrated TorixRickettsia infections were more prevalent in aquatic arthropod
species compared to terrestrial. However, our observed
over-representation of Torix group Rickettsia (14/16 strains)
contrasts with Weinert’s findings which show a predominance of Belli
infections and is likely due to the latter study’s absence of screened
aquatic taxa. Furthermore, through the additional use of a
bioinformatics approach, our SRA search appears to confirm that Belli
and Torix are two of the most common Rickettsia groups among
arthropods. Overall, these multiple screening methods suggest TorixRickettsia are more widespread than previously thought and their
biological significance underestimated.
Previous studies have used either one or two markers to identify the
relatedness of strains found in distinct hosts. In this study, we use
the multilocus approach developed in Pilgrim et al. (2017) to understand
the affiliation of Torix Rickettsia from diverse invertebrate
hosts. Our analysis of Torix strains indicates that closely related
strains are found in distantly related taxa. Closely relatedRickettsia are also found in hosts from different niches and
habitats – for instance, the Rickettsia strains found in
terrestrial blood feeders do not lie in a single clade, but rather are
allied to strains found in non-blood feeding host species. Likewise,
strains in phloem feeding insects are diverse rather than commonly
shared.
The distribution of Torix Rickettsia across a broad host range
suggests host shifts are occurring between distantly related taxa. It is
notable that parasitoid wasps are commonly infected withRickettsia and have been associated with enabling symbiont host
shifts (Ahmed et al., 2015; Vavre, Fleury, Lepetit, Fouillet, &
Bouletreau, 1999). Aside from endoparasitoids, it is also possible that
plant-feeding can allow for endosymbiont horizontal transmission
(Caspi-Fluger et al., 2012; Gonella et al., 2015; S.-J. Li et al.,
2017). For example, Rickettsia horizontal transmission has been
demonstrated in Bemisia whiteflies infected by phloem-feeding
(Caspi-Fluger et al., 2012; Y.-H. Li et al., 2017). Finally,
ectoparasites like the Torix-infected water mites of the
Calyptostomatidae family, could also play a role in establishing novelRickettsia -host associations, as feeding by mites has been
observed to lead to host shifts for other endosymbiont taxa (Jaenike,
Polak, Fiskin, Helou, & Minhas, 2007). Indeed, if multiple horizontal
transmission paths do exist, this could account for the diverse plethora
of infected taxa, as well as arthropods identified in this study which
harbour more than one strain of symbiont (Morrow, Frommer, Shearman, &
Riegler, 2014; Vavre et al., 1999).
The finding that Torix Rickettsia are associated with a broad
range of invertebrates leads to an obvious question: what is the impact
and importance of these symbiotic associations? Previous work has
established Torix Rickettsia represent heritable symbionts and it
is likely that this is true generally. There have, however, been few
studies on their impact on the host. In the earliest studies
(Kikuchi &
Fukatsu, 2005; Kikuchi et al., 2002), Torix spp. leeches infected
with Rickettsia were observed to be substantially larger than
their uninfected counterparts. Since then, the only observation of note,
pertaining to the Torix group, is the reduced ballooning (dispersal)
behaviour observed in infected Erigone atra money spiders
(Goodacre et al.,
2009). Overall, the incongruencies in host and Torix Rickettsiaphylogenies (suggesting a lack of co-speciation and obligate mutualism),
along with the lack of observed sex bias in carrying the symbiont,
indicate facultative benefits are the most likely symbiotic relationship
(Jaenike, 2012;
Weinert, 2015). However, Rickettsia induction of thelytokous
parthenogenesis should not be discounted in Torix infected parasitoid
wasps identified in this study
(Giorgini,
Bernardo, Monti, Nappo, & Gebiola, 2010; Hagimori, Abe, Date, & Miura,
2006). To add to the challenge of understanding Torix Rickettsiasymbioses, the challenges of laboratory rearing of many TorixRickettsia hosts has led to difficulties in identifying model
systems to work with. However, the large expansion of our Torix group
host knowledge can now allow for a focus on cultivatable hosts (e.g
phloem-feeding bugs).
A particularly important group for study are haematophagous host
species. Our discovery of Rickettsia -associated tabanid and
simulid flies, alongside Anopheles plumbeus mosquitoes, add to
existing blood-feeders previously identified as Torix group hosts which
include sand flies (Kaili Li et al., 2016; Reeves, Kato, & Gilchriest,
2008), fleas (Song et al., 2018), ticks (Floris et al., 2008) bed bugs
(Potts, Molina, Sheele, & Pietri, 2020) and biting midges (Pilgrim et
al., 2017). Some Rickettsia strains are known to be transmitted
to vertebrates via haematophagy
(Parola, Paddock,
& Raoult, 2005). However, there is no evidence to date for vertebrate
pathogenic potential for the Torix group. Despite this, TorixRickettsia could still play a significant role in the ecology of
vectors of disease. A key avenue of research is whether these
endosymbionts alter vectorial capacity, as found for other associations
(Bourtzis et al.,
2014; Hoffmann, Ross, & Rašić, 2015). In contrast to the widely
reported virus blocking phenotype observed in Wolbachia -infected
vectors (Moreira
et al., 2009; van den Hurk et al., 2012; Walker et al., 2011),Rickettsia have been associated with a virus potentiating effect
in Bemisia white flies vectoring Tomato yellow leaf curl virus
(Kliot, Cilia,
Czosnek, & Ghanim, 2014). Additionally, we uncovered aRickettsia -infected psyllid (Cacopsylla melanoneura ) which
is a vector of Phytoplasma mali (apple proliferation)
(Tedeschi,
Visentin, Alam, & Bosco, 2003). Thus, the question of TorixRickettsia vector-competence effects is clearly of widespread
relevance and deserves further attention.
To conclude, we have shown that large-scale DNA barcoding initiatives of
arthropods can include non-target amplification of TorixRickettsia . By examining these non-target sequences, alongside a
targeted screen and SRA search, we have uncovered numerous previously
undetected host associations. Our findings lay bare multiple new avenues
of inquiry for Torix Rickettsia symbioses.