Discussion
Amplicon sequencing as a powerful method of Wolbachia strain
determination
Strain determination is a key step in studying Wolbachiadistribution and host-shifting among a given host group that needs to be
performed using an efficient method. Given that infection with more than
one Wolbachia strain is common in various arthropod groups
(Werren et al. 1995; Perrot-Minnot et al. 1996; Hiroki et al. 2004;
Narita et al. 2007; Hou et al. 2020), strain determination methods
should be able to distinguish and identify strains in both singly and
multiply-infected samples. The traditional method of using Sanger
sequencing is not effective in dealing with co-infected arthropod
samples, and improvements such as using different primers and cloning
(Schuster 2008; Vo & Jedlicka 2014)) are costlier and more
labour-intensive, and also have limitations (Van Borm et al. 2003;
Schuler et al. 2011). High-throughput whole genome sequencing (WGS)
would seem to be the most accurate available methodology for strain
identification, but this approach has its own difficulties (Bleidorn &
Gerth 2018). First, given that Wolbachia is not culturable, it is
challenging to obtain genetic material enriched for Wolbachiarelative to host DNA, possibly resulting in low sequencing depth.
Second, even with high sequencing depth,
assembling Wolbachiagenomes can be difficult due to a high density of mobile elements (Wang
et al. 2019) and thus only draft genomes can be recovered. Finally, the
still relatively high costs of WGS make this approach less applicable in
large Wolbachia surveys. Due to these limitations, only 33Wolbachia annotated whole genomes have been publicly available on
GenBank so far (as of October 2021).
As suggested by Bleidorn and
Gerth (2018), instead of whole genomes, sequencing and assembling aWolbachia draft genome is sufficient for strain determination.
However, many more draft genomes should be publicly available first to
provide a reliable reference bank for strain determination. Although a
draft genome can indeed be mapped to the selective marker amplicons
(e.g. MLST), generating such data for large surveys is still time- and
cost-intensive. To overcome these technical obstacles, we suggest
Illumina multi-target amplicon sequencing as a middle-ground, efficient
and affordable method that can be applied to large surveys and is also
capable of dealing with multiple infections. In particular, the fiveWolbachia MLST genes along with wsp and 16S used in our
study appear to be well suited to distinguish between strains, as has
also been shown in a recent comparative study of available whole genomes
of Wolbachia (Wang et al. 2020).
Wolbachia diversity in scale
insects
This study revealed that a substantial portion of tested scale insects
are infected with more than one strain of Wolbachia (27% double
and 5% triple infected). We also found Wolbachia multiple
infections in associate species (including wasps and ants), indicating
co-infection might be a common phenomenon in most of these insect
groups. However, it is important
to caution that detecting a given Wolbachia strain in a given
host is not conclusive evidence of a stable infection, and laboratory
assays should be conducted to ascertain Wolbachia maternal
transmission and establishment within the host population (Chrostek et
al. 2017). Moreover, in the case of parasitoids and predators, a
detected strain may derive from their undigested prey rather than the
screened insect itself (Ross et al. 2020). Unfortunately, laboratory
rearing of collected samples is not feasible for large Wolbachiasurveys such as the current study. Therefore, any interpretation from
this type of data should be treated with caution.
Based on the MLST database (as of 31st August 2021), 24 strains with
complete MLST gene sequences had previously been reported from the
Australian fauna (https://pubmlst.org/organisms/wolbachia-spp).
Here, we report 63 new strains (belonging to 31 strain groups) for
Australia, including the first three Supergroup F strains in
Australasia. Apart from two strains (w Sph4.1 = ST 289, andw Cal = ST 357), none of the strains in the current study were
100% identical to any registered in the MLST database. As our sequenced
regions were slightly (~5%) smaller than the MLST
amplicons available on MLST online database, there is a possibility that
the two strains that were identical to the MLST profiles were different
in the remaining part of the gene fragments. We found w Sph1 to be
the most common and widely distributed strain group in Australia
(detected in seven scale insects, four wasps and one ant species). Based
on the phylogenetic tree of all reported strains in the MLST database
and the current study strains, there are six registered strains (STs)
within the w Sph1 strain group (Figure S2). These strains seem to
be globally distributed across various insect orders. For example, one
of the strains in this group, registered as ST=19, has been reported in
16 different host species belonging to four insect orders. This broad
host range may be an indicator of an extraordinary host-shifting ability
of w Sph1. Mostly based on the number of infected host species,
several Wolbachia strains have been reported with a similar
ability (e.g., HVR-2 in ants (Tolley et al. 2019), ST41 in Lepidoptera
(Ilinsky & Kosterin 2017), and w Hypera in weevils (Sanaei et al.
2019)). Among all the superspreaders, w Ri is one of the
best-studied Wolbachia strain groups that has rapidly (within
14,000 years) naturally infected five Drosophila species (Turelli
et al. 2018). w Ri can also be introduced to mosquitoes by
transinfection, corroborating this strain’s potential to infect new host
species (Fraser et al. 2017). Compared to w Ri, it seems thatw Sph1 has been reported in a higher number of host species that
are taxonomically more diversified (belonging to various insect orders).
Although the w Ri group has an extensive genomic diversity
(Ishmael et al. 2009; Turelli et al. 2018), low variation has been
observed within its MLST profiles
(https://pubmlst.org/organisms/wolbachia-spp). Four strains have
been reported in the w Ri group and only one strain (ST=17) has
been reported in more than one species of Drosophila (based on
MLST website as of 31st August 2021). Therefore, w Sph1 might have
a higher diversity than w Ri and may therefore have a potential to
be artificially introduced to other insects for human applications
(e.g., controlling vector born disease). However, transinfection studies
are necessary to ascertain the host-shifting ability of w Sph1 in
laboratory conditions.
Phylogenetic distance effect can explain
host-shifting
As is typical of Wolbachia infection in an arthropod family,
non-independence between the scale insect and Wolbachiaphylogenetic trees were not statistically supported, i.e. no signal of
congruence was detected between them. Instead, the current distribution
of Wolbachia in scale insects was most likely shaped by
host-shifting. Among many potential factors determining host shifts, it
seems that host phylogeny and geographic distributions are two major
players (Sanaei et al. 2021a). Combining data from 25 transinfection
studies, Russell et al. (2009) showed that there is a positive
correlation between host phylogenetic relatedness and success of theWolbachia transinfection. In addition, by focusing only on a part
of the host phylogenetic tree, several studies uncovered a pattern of
host-shifting among closely related species (Haine et al. 2005; Guz et
al. 2012; Turelli et al. 2018). On the other hand, the observation of
identical Wolbachia strains in species that live in the same area
points to a role of geography in host-shifting (Kittayapong et al. 2003;
Stahlhut et al. 2010; Morrow et al. 2014; Gupta et al. 2021). The
relative contributions of the host phylogenetic and geographic distance
effect on Wolbachia host shifts are poorly understood. Here, we
tried to evaluate these two factors in Wolbachia host shifting by
using a powerful statistical method. The results of our GAMM indicate
that host shifts in scale insects can be mainly explained by the
phylogenetic distance effect (host shifting is more feasible between
closely related species compared to distantly related) (Figure 3). This
result is in line with numerous examples of finding the sameWolbachia strain group in congeneric species (e.g.,wHypera1 in the genus Hypera (Coleoptera) (Sanaei et al.
2019), w Lev in the genus Lutzomyia (Diptera) (Vivero et
al. 2017), ST19 in the genus Bicyclus (Lepidoptera) (Duplouy &
Brattström 2018)).
Horizontal
transfer of parasites/symbionts among closely related species can
generate a phylogenetic signal similar to host-parasite co-speciation
(De Vienne et al. 2007). However, there is indirect evidence advocatingWolbachia sharing patterns in scale insects that can be explained
best by recent host-shifting. In contrast to horizontal transmission
which occurs rapidly, co-speciation happens in an evolutionary timeframe
which allows Wolbachia genes to be mutated. By investigatingWolbachia infection in Nasonia species complex, it is
estimated that Wolbachia MLST genes mutation rate is one third of
their host nuclear genes (from nine single copy nuclear regions)
(Raychoudhury et al. 2009). Although this ratio can be slightly
different among various host species and Wolbachia strains (see
also Conner et al. (2017)), it can be adopted as a tool to distinguish
co-diversifications from recent host shifting. Given that the lowest
pairwise distance between host species nuclear genes that we have in our
dataset is 2%, in the case of Wolbachia co-speciation, at least
17 bp differences (out of 2608 bp) should be observed between two
closely related strains. We infer host-shift events based on sharing
either identical strains or identical strain groups (which includes
strains with up to only 5bp differences across all Wolbachiaamplicons) (File S1). In addition,
in
73% of determined host-shift
events in scale insects, shared Wolbachia strains have identical
wsp which is less conserved compared to the MLST genes.
Interestingly, the significant
impact of host phylogenetic distance on Wolbachia strain group
sharing was not mirrored by statistically significant tests for host andWolbachia phylogenetic tree non-independence (Parafit and Paco),
as might have been expected under host shifting with a PDE. We speculate
that these tests for tree independence are not sufficiently powerful to
detect minor departures from tree independence as caused by host
shifting under a PDE. It would be useful to verify this using computer
simulations of host shifting with PDE (e.g., de Vienne et al. (2007);
Engelstädter & Fortuna (2019)).
Hybridisation
between closely related species may lead to introgression ofWolbachia into a new species by vertical transmission. This has
been demonstrated in the Nasonia species complex (Raychoudhury et
al. 2009) and some species of Drosophila (Turelli et al. 2018;
Cooper et al. 2019).
Introgression can be considered a
special case of host shifting (referred to as ‘hybridisation-mediated
host shifts’ by Sanaei et al.,
(2021a), but also has
similarities to cospeciation. Since this type of host shifting can only
occur between closely related species, it is expected to produce a PDE
signal. We believe, however, that
hybridization is unlikely to explain the observed patterns of strain
group sharing in our dataset. There are only two con-generic species in
our dataset that share the same Wolbachia strain
(Cystococcus pomiformis /C. echiniformis andEriococcus sp1 /E. sp2 ), and hybridisation between species
from different genera seems unlikely. Given that Wolbachia and
mitochondria are co-transmitted, introgression of Wolbachia would
lead to mtDNA hitchhiking and hence be expected to leave a signature of
similar mtDNA sequences (Jiggins 2003; Cooper et al. 2019; Miyata et al.
2020). By contrast, none of our
congeneric species have very similar COI sequences. (The difference
between C. pomiformis and C. echiniformis is 6.5%, and the
difference between E. sp1 and E. sp2 is 3%).