Introduction
Wolbachia is one of the
best-known groups of heritable endosymbionts, widely distributed in
arthropods and some nematodes (Hertig 1936; Sironi et al. 1995; Werren
1997). These bacteria form one of the most abundant and diverse groups
of symbionts on earth: an estimated 40-60% of arthropod species are
infected with Wolbachia strains (Zug & Hammerstein 2012; Weinert
et al. 2015). Their ability to induce reproduction manipulations
(Rousset et al. 1992; Werren et al. 2008) and their application in
vector born disease control (Kambris et al. 2009; Hoffmann et al. 2015;
Ross et al. 2019) are key aspects of Wolbachia that have been
studied extensively in the last two decades.
Similar to many other symbionts,
the current distribution of Wolbachia results from three major
processes: co-diversification with the host clade, shifting between host
species, and symbiont loss (Thompson 1987; Charleston & Perkins 2006).
Although co-speciation is common among Wolbachia strains
belonging to supergroups C and D in their nematode hosts (Bandi et al.
1998; Fenn & Blaxter 2004) and certain strains of supergroup F
infecting bed bugs (Balvín et al. 2018), many studies have failed to
find evidence of codiversification between Wolbachia strains of
supergroups A/B and arthropods (e.g., in fig wasps (Shoemaker et al.
2002), ants (Frost et al. 2010), butterflies (Ahmed et al. 2016), bees
(Gerth et al. 2013), and collembolans (Ma et al. 2017)). In the absence
of co-diversification, host-shifting is the alternative hypothesis to
explain the current distribution of Wolbachia (reviewed in
Sanaei et al. (2021a)).Wolbachia shift hosts when a given strain infects a novel
arthropod species, mostly through horizontal transfer (Boyle et al.
1993; Heath et al. 1999) and possibly occasionally through hybridisation
(Turelli et al. 2018; Cooper et al. 2019). The possibility of host-shift
events in Wolbachia has been confirmed through numerous
transinfection studies when a strain is artificially introduced to an
uninfected species (reviewed in Hughes & Rasgon, 2014), and the
existence of “super spreader strains” that infect host species which
are phylogenetically distantly related (e.g., ST41 strain type in
Lepidoptera (Ilinsky & Kosterin 2017)). Physical transfer ofWolbachia from donor to recipient species is the first step of
host-shifting, achieved via various ”routes of transfer” and usually
facilitated by a biological vector or a suitable environmental medium
(Vavre et al. 2003; Riegler et al. 2004). Routes of transfer reported so
far include prey-predator interactions (Le Clec’h et al. 2013),
host-parasite interactions (Cook & Butcher 1999; Vavre et al. 1999;
Ahmed et al. 2015) and sharing a common food resource (Li et al. 2017).
Host phylogeny and ecological connectivity are thought to be the two
main factors determining Wolbachia host shifting. As
phylogenetically closely related species are similar in many respects,
including their intercellular environment and immunology (Perlman &
Jaenike 2003), it is expected
that a given symbiont will shift more easily between them than between
distantly related species (Charleston & Robertson 2002). This
assumption, referred to as the “phylogenetic distance effect” (PDE)
(Longdon et al. 2011; Engelstädter & Fortuna 2019), may partly explain
host shifts of Wolbachia across closely related species. In spite
of limited case studies which indicated the presence of PDE in part of
the host phylogeny (e.g., in fig wasps (Shoemaker et al. 2002), fungus
growing ants (Frost et al. 2010), bees (Gerth et al. 2013) and
Collembolans (Ma et al. 2017)), the influence of PDE on Wolbachiahost shifting is not clear. Overlapping geographic distributions of host
species is another possible explanatory factor. Sharing a common habitat
and consequently potential ecological interactions may lead to several
direct and indirect physical contacts between a given donor and
recipient host and, therefore, also increase the probability ofWolbachia host shifting. Indeed, several case studies documented
host-shift events between host species that share the same habitat,
e.g., in a rice field community (Kittayapong et al. 2003) and a mushroom
habitat (Stahlhut et al. 2010)).
Here, we use scale insects as a model system to gain a better
understanding of Wolbachia host shifting. With more than 8200
described species and 24 families, the superfamily of scale insects
(Coccoidea) is globally distributed (Gullan & Cook 2007; García Morales
et al. 2016). Like many other members of the suborder Sternorrhyncha,
such as aphids, whiteflies and psyllids, scale insects exclusively feed
on plants and some are considered serious agricultural pests (Kondo et
al. 2008). Scale insects have been recorded in ecological associations
with a range of arthropods species. In particular, many are usually
observed in close interactions with ants through trophallaxis (where the
honeydew produced by the scale insects is consumed by ants) (Hölldobler
et al. 1990; Buckley & Gullan 1991; Gullan et al. 1993). Despite
several similarities with other hemipterans, Sanaei et al. (2021b) found
that most species are predicted to have low to intermediateWolbachia prevalence, in contrast to a u-shaped distribution
(most species have a very low or very high prevalence) predicted for
other groups (Hilgenboecker et al. 2008). Also, a positive correlation
between Wolbachia infection in scale insects and their associate
ants indirectly points to a plausible route of transfer (Sanaei et al.
2021b). These preliminary results provided a broad view of theWolbachia infection dynamic in scale insects and thus inspired us
to investigate Wolbachia strain diversity and consequently
host-shifting in scale insects.
Studying of Wolbachia host-shifting requires using and developing
new methodologies. To overcome technical problems associated with Sanger
sequencing (see Discussion), we adopted Illumina multi-target amplicon
sequencing techniques to determine the Wolbachia strains in scale
insects and their associate species. Using this effective methodology,
we revealed the strain diversity and composition (including both single
and multiple infections) in scale insects. Using phylogenetic trees of
both scale insects and their Wolbachia strains, and the
geographic distribution range of each scale insect species, we assessed
which factors (phylogeny or geography) best explain host-shifting
events. Finally, by determining Wolbachia strains in individual
scale insects and directly associated individuals of other species, we
identified plausible routes of horizontal transfer.