The toxins of vertically transmitted Spiroplasma

Abstract

Vertically transmitted (VT) microbial symbionts play a vital role in the evolution of their insect hosts. A longstanding question in symbiont research is what genes help promote long-term stability of vertically transmitted lifestyles. Symbiont success in insect hosts is due in part to expression of beneficial or manipulative phenotypes that favor symbiont persistence in host populations. In Spiroplasma, these phenotypes have been linked to toxin and virulence domains among a few related strains. However, these domains also appear frequently in phylogenetically distant Spiroplasma, and little is known about their distribution across the Spiroplasma genus. In this study, we present the complete genome sequence of the Spiroplasma symbiont of Drosophila atripex, a non-manipulating member of the Ixodetis clade of Spiroplasma, for which genomic data are still limited. We perform a genus-wide comparative analysis of toxin domains implicated in defensive and reproductive phenotypes. From 12 VT and 31 non-VT Spiroplasma genomes, ribosome-inactivating proteins (RIPs), OTU-like cysteine proteases (OTUs), ankyrins, and ETX/MTX2 domains show high propensity for VT Spiroplasma compared to non-VT Spiroplasma. Specifically, OTU and ankyrin domains can be found only in VT-Spiroplasma, and RIP domains are found in all VT Spiroplasma and three non-VT Spiroplasma. These domains are frequently associated with Spiroplasma plasmids, suggesting a possible mechanism for dispersal and maintenance among heritable strains. Searching insect genome assemblies available on public databases uncovered uncharacterized Spiroplasma genomes from which we identified several spaid-like genes encoding RIP, OTU, and ankyrin domains, suggesting functional interactions among those domain types. Our results suggest a conserved core of symbiont domains play an important role in the evolution and persistence of VT Spiroplasma in insects.

Full text

Frontiers in Microbiology 01 frontiersin.org
The toxins of vertically transmitted
Spiroplasma
LoganD.Moore * and MatthewJ.Ballinger
Department of Biological Sciences, Mississippi State University, Mississippi State, MS, United States
Vertically transmitted (VT) microbial symbionts play a vital role in the evolution
of their insect hosts. A longstanding question in symbiont research is what
genes help promote long-term stability of vertically transmitted lifestyles.
Symbiont success in insect hosts is due in part to expression of beneficial or
manipulative phenotypes that favor symbiont persistence in host populations. In
Spiroplasma, these phenotypes have been linked to toxin and virulence domains
among a few related strains. However, these domains also appear frequently in
phylogenetically distant Spiroplasma, and little is known about their distribution
across the Spiroplasma genus. In this study, wepresent the complete genome
sequence of the Spiroplasma symbiont of Drosophila atripex, a non-manipulating
member of the Ixodetis clade of Spiroplasma, for which genomic data are still
limited. We perform a genus-wide comparative analysis of toxin domains
implicated in defensive and reproductive phenotypes. From 12 VT and 31 non-VT
Spiroplasma genomes, ribosome-inactivating proteins (RIPs), OTU-like cysteine
proteases (OTUs), ankyrins, and ETX/MTX2 domains show high propensity for
VT Spiroplasma compared to non-VT Spiroplasma. Specifically, OTU and ankyrin
domains can befound only in VT-Spiroplasma, and RIP domains are found in all
VT Spiroplasma and three non-VT Spiroplasma. These domains are frequently
associated with Spiroplasma plasmids, suggesting a possible mechanism for
dispersal and maintenance among heritable strains. Searching insect genome
assemblies available on public databases uncovered uncharacterized Spiroplasma
genomes from which weidentified several spaid-like genes encoding RIP, OTU,
and ankyrin domains, suggesting functional interactions among those domain
types. Our results suggest a conserved core of symbiont domains play an
important role in the evolution and persistence of VT Spiroplasma in insects.
KEYWORDS
Spiroplasma, Ixodetis, ETX/MTX2, OTU-like cysteine protease, ribosome-inactivating
proteins, ankyrin, vertical transmission
Introduction
Gene duplications, losses, and horizontal transfers can facilitate dramatic shis in bacterial
lifestyle and capabilities (Romero and Palacios, 1997; Moran, 2002; Arnold etal., 2022). Gene
loss is a dominant feature of symbiont evolution due to the selective benets of removing
metabolically costly genes (McCutcheon and Moran, 2012; McCutcheon et al., 2019).
Alternatively, gene gain via horizontal transfer and duplication can lead to rapid adaptation
across symbiotic species. For example, several mutualistic soil bacteria with nitrogen-xing
capabilities have beneted from receiving “symbiont islands” and plasmids enriched with
nitrogen-xing genes (Sullivan and Ronson, 1998; Andrews etal., 2018). Vertically transmitted
symbionts are no exception to this phenomenon and can acquire novel phenotypes that benet
OPEN ACCESS
EDITED BY
Akiko Sugio,
Institut National de la Recherche Agronomique,
France
REVIEWED BY
Hiroshi Arai,
National Agriculture and Food Research
Organization (NARO), Japan
Emily Hornett,
University of Liverpool, UnitedKingdom
*CORRESPONDENCE
Logan D. Moore
ldm427@msstate.edu
RECEIVED 19 January 2023
ACCEPTED 19 April 2023
PUBLISHED 18 May 2023
CITATION
Moore LD and Ballinger MJ (2023) The toxins
of vertically transmitted Spiroplasma.
Front. Microbiol. 14:1148263.
doi: 10.3389/fmicb.2023.1148263
COPYRIGHT
© 2023 Moore and Ballinger. This is an open-
access article distributed under the terms of
the Creative Commons Attribution License
(CC BY). The use, distribution or reproduction
in other forums is permitted, provided the
original author(s) and the copyright owner(s)
are credited and that the original publication in
this journal is cited, in accordance with
accepted academic practice. No use,
distribution or reproduction is permitted which
does not comply with these terms.
TYPE Original Research
PUBLISHED 18 May 2023
DOI 10.3389/fmicb.2023.1148263

Moore and Ballinger 10.3389/fmicb.2023.1148263
Frontiers in Microbiology 02 frontiersin.org
a heritable lifestyle via the transfer of symbiosis-supporting genes
(Ballinger et al., 2019; Manzano-Marin et al., 2020; Wat er wort h
etal., 2020).
Vertically transmitted microbial symbionts can strongly inuence
the life history traits of their insect hosts to maintain stable vertical
transmission (VT) (Moran etal., 2008). For example, the facultative
endosymbiont Wolbachia can defend against viral infections in their
Drosophila hosts (Teixeira et al., 2008) but can also be penetrant
reproductive parasites of Drosophila, using manipulative phenotypes
like male-killing and cytoplasmic incompatibility to favor their
transmission to subsequent generations. Heritable symbionts are
ubiquitous among insects (Moran etal., 2008), and yet, a central
question remains for many heritable microbes—what gene eectors
promote vertical transmission and how do they arise?
Spiroplasma is a genus of helical, cell wall-less bacteria estimated
to infect up to 7% of terrestrial arthropod species (Duron etal., 2008).
e genus is phylogenetically structured into three large clades—the
Apis clade, the Citri-Chrysopicola-Mirum clade, and the Ixodetis
clade (Gasparich et al., 2004). Despite their rich diversity, all
Spiroplasma are non-free-living bacteria, infecting the gut or
hemolymph of invertebrates and/or the phloem of plants. At ecological
timescales, Spiroplasma infections are transmitted horizontally by
ingestion or maternally via the cytoplasm of mature oocytes (Herren
et al., 2013). e Apis clade contains several insect pathogens,
including Spiroplasma taiwanense and Spiroplasma culicicola of
mosquitoes and Spiroplasma apis of European honey-bees (Mouches
etal., 1982; Humphery-Smith etal., 1991; Bolaños etal., 2015). No VT
Spiroplasma have been described in the Apis clade, while both VT and
non-VT Spiroplasma have been reported in the Citri-Chrysopicola-
Mirum clade (hereaer referred to as the Citri clade) and the Ixodetis
clade (Jiggins etal., 2000; Haselkorn, 2010; Ballinger etal., 2018;
Binetruy etal., 2019; Vera-Ponce León etal., 2021).
Heritable Spiroplasma have been described across insect orders,
and are well-studied in vinegar ies, butteries, beetles, aphids and
parasitoid wasps. Some members of the Citri clade defend Drosophila
hosts against parasitic nematodes and parasitoid wasps (Xie etal.,
2010; Jaenike etal., 2010bcc Haselkorn and Jaenike, 2015). Others in
this clade are both male-killers and defensive symbionts of Drosophila
(Montenegro etal., 2005; Xie etal., 2014). Reproductive manipulation
is also common among Ixodetis clade Spiroplasma. Ixodetis
Spiroplasma of the buttery Danaus chrysippus (sChrys) and the
ladybird beetle Anisosticta novemdecimpunctata are eective VT male-
killers (Jiggins etal., 2000; Tinsley and Majerus, 2006) and the recently
discovered Spiroplasma symbiont that causes cytoplasmic
incompatibility in the parasitoid wasp Lariophagus distinguendus
(sDis) is also an Ixodetis clade member (Pollmann etal., 2022).
Cytoplasmic incompatibility is a form of reproductive manipulation
whereby toxic or modied sperm of symbiont-infected fathers kills the
ospring of uninfected mothers, thus driving symbiont spread
through the population. As defensive symbionts, Ixodetis Spiroplasma
have been shown to confer resistance to fungal infections in their
aphid hosts (Łukasik etal., 2013). Indeed, VT strains are present in
divergent Spiroplasma clades, yet little is known about the genetic
mechanisms supporting their host-associated phenotypes beyond
those of the Poulsonii group in the Citri clade. Within the Poulsonii
group, symbiont toxins have been implicated in both defensive and
reproductive phenotypes (Hamilton et al., 2016; Ballinger and
Perlman, 2017; Harumoto and Lemaitre, 2018).
Toxic peptides are known eectors of defensive and reproductive
phenotypes across symbiotic bacterial taxa (Gillespie etal., 2018;
Oliver and Perlman, 2020; Massey and Newton, 2022). Hamiltonella
defensa utilizes phage-encoded toxins to defend aphid hosts against
parasitoid wasps (Degnan and Moran, 2008). Wolbachia symbionts
employ a toxin-antitoxin or host modication system to inict
cytoplasmic incompatibility in their hosts (Beckmann etal., 2017;
LePage etal., 2017; Horard etal., 2022; Kaur etal., 2022). Within the
Citri-based Poulsonii group, including Spiroplasma strains infecting
Drosophila hydei, Drosophila neotestacea, and Drosophila melanogaster
(sHyd, sNeo, and MSRO respectively), defensive strains utilize
ribosome-inactivating protein toxins (RIPs) to contribute to defense
against parasitoid wasps and parasitic nematodes (Hamilton etal.,
2016; Ballinger and Perlman, 2017). RIPs cause irreversible damage to
a key enzymatic residue of ribosomes, resulting in cell death (Stirpe,
2004). RIP gene families are remarkably diverse in Citri clade
Spiroplasma, ostensibly due to extensive horizontal transmission,
duplication, loss, and recombination (Ballinger etal., 2019; Gerth
etal., 2021). Spiroplasma poulsonii strain MSRO is a reproductive
manipulator that encodes a multidomain toxin called Spaid that
causes male-killing in Drosophila (Harumoto and Lemaitre, 2018).
Spaid consists of three integral domains including ankyrin repeat
domains, an OTU-like cysteine protease domain (OTU) with
predicted function in deubiquitination, and a C-terminal
transmembrane domain. OTUs and ankyrin domains are also
associated with type V cifB-like proteins of CI-inducing Wolbachia,
suggesting they can beadapted for dierent reproductive phenotypes
in diverse systems (Martinez etal., 2021). While toxins have been
identied to play an essential role in maintaining symbiosis in these
heritable Citri clade Spiroplasma, far less is known about eector genes
supporting the diverse Ixodetis Spiroplasma phenotypes. However,
recent advances in genomic representation among these strains
(Yeoman etal., 2019; Martin etal., 2020; Vera-Ponce León etal., 2021;
Pollmann etal., 2022) facilitates an in-depth comparative analysis of
toxin gene content in this enigmatic clade.
In this study, wereport the genome sequence of a Drosophila-
associated Ixodetis clade member, the Spiroplasma symbiont of
Drosophila atripex (sAtri). is strain does not induce male-killing or
cytoplasmic incompatibility in Drosophila (Haselkorn etal., 2009) but
does provide protection against parasitoid wasps in the family
Figitidae (T. Chris Amuwa, unpublished results). Weidentify a diverse
set of toxin and virulence domains in the genome, including the same
domains contributing to defense and reproductive manipulation in
the Citri clade. Weconducted a genus-wide survey of 43 Spiroplasma
genomes across all three clades to characterize the distribution of
toxin domains between heritable and non-heritable Spiroplasma. Our
results uncover a striking association of RIPs, OTUs, ankyrin, and
ETX/MTX2 domains with heritable strains, suggesting that these
domains play an important role in promoting VT symbiosis
across Spiroplasma.
Results
Genome of sAtri
Using a hybrid long and short read sequencing strategy,
weproduced a closed genome assembly of 1.27 Mb for sAtri. e

Moore and Ballinger 10.3389/fmicb.2023.1148263
Frontiers in Microbiology 03 frontiersin.org
genome is covered to 29X depth by short reads
(Supplementary Figure S1). Phylogenomic analysis nests sAtri with
strong support among other Ixodetis clade endosymbiont members
that infect a wide range of insect hosts (Figure 1). Genome
completeness based on single copy orthologs was estimated by
BUSCO at 98% (Table 1). Missing orthologs from the genome
completeness analysis include the genes rplL and rsmL. Weidentied
rplL in the sAtri genome with a manual BLAST search but were unable
to identify rsmL. While rsmL (rRNA ribose-2’-O-methyltransferase)
has a conserved role in bacterial translation, it is absent from all
currently available Ixodetis clade genomes (Pollmann etal., 2022). e
hybrid genome assembly includes eight putative plasmids, which were
assigned as such based on gene content, coverage depth, and
circularity (Supplementary Tables S1, S2). Repeat content is 10.73%,
but only nine transposable elements could beannotated. Of the 1,313
putative protein coding genes, 639 could beassigned a functional
annotation by prokka. sAtri encodes complete ATP synthase and
glycolysis pathways, as well as transport components for glucose,
fructose, GlcNAc, glycerol, and possibly mannose. Summaries of
functional predictions and carbon metabolism pathway components
are shown in Supplementary Figure S2A and Supplementary Table S3.
Amino acid and lipid biosynthesis pathways are incomplete or absent,
as reported previously for hemolymph-dwelling Spiroplasma species,
in both the Citri and Ixodetis clades (Paredes etal., 2015; Yeoman
etal., 2019; Vera-Ponce León etal., 2021). In addition to the PTS genes
for carbon import (Supplementary Figure S2B), ABC membrane
proteins for transport of phosphate (ptsABCS), nucleosides (bmpA,
nupABC), and micronutrients (ecfA1, ecfA2) could beidentied. A
comparative summary of membrane transport, biosynthesis, and
metabolism genes in sAtri and two other Ixodetis clade genomes is
present in Supplementary Table S3. sAtri also encodes a Type II-C
CRISPR/Cas9 system which has not been previously reported in a
heritable Spiroplasma. No matches between sAtri spacers and known
phages or phage regions were identied. However, several of the
spacers in sAtri are exact matches to sequences present on sAtri
plasmids, suggesting a possible interaction with CRISPR/Cas9 (e.g.,
plasmid copy number suppression). sAtri spacers also match to
non-phage genomic sequences of other Ixodetis Spiroplasma, though
it is unclear if those sequences are associated with plasmids.
sAtri encodes a diverse set of toxins and
virulence genes
e sAtri genome assembly reveals similarities to and departures
from other Spiroplasma with regard to toxin and virulence gene
content. ree RIP domain-containing proteins are present, two of
which encode a signal peptide for secretion. RIP toxins contribute
to defense against natural enemies in some Citri clade members
(Hamilton etal., 2016; Ballinger and Perlman, 2017). e sAtri RIPs
are phylogenetically distant from one another (Figure2) and from
other non-Ixodetis Spiroplasma RIPs with less than 30% similarity
at the amino acid level. We also identied RIPs in the genome
assemblies of the Spiroplasma symbiont of Danaus chrysippus
(sChrys) and the Spiroplasma symbiont of Cephus cinctus (sCinc),
and retrieved the RIPs reported in the Spiroplasma symbiont of
Lariophagus distinguendus (sDis) and the Spiroplasma symbiont of
Dactylopius coccus (sCoccus). Examining the phylogenetic
relationships of Ixodetis clade RIPs reveals them to bepolyphyletic,
grouping separately with distinct clades of Citri RIPs. is is
consistent with ancient ancestry or horizontal transfer within the
genus (Ballinger etal., 2019). sAtri encodes two ETX/MTX2 toxins,
both of which possess signal peptides for secretion. ETX/MTX2
toxins are β pore-forming toxins that inict cytocidal activity against
their target cells and are noted for their insecticidal activity (Palma
etal., 2014; Moar etal., 2017). Like sAtri RIPs, these toxins are
diverse, grouping separately with other Ixodetis clade-encoded ETX/
MTX2 toxins (Supplementary Figure S3). ETX/MTX2 toxins are
numerous and widespread in Spiroplasma but their potential role in
host-symbiont relationships is unknown. sAtri encodes 11 total
genes containing ankyrin domains (Supplementary Figure S4) and
a separate gene with an OTU-like cysteine protease. While not
inherently toxic, ankyrin and OTU domains are associated with
genes involved in host manipulation, and work in conjunction to
cause male-killing by MSRO and possibly cytoplasmic
incompatibility by Wolbachia (Pan etal., 2008; Nguyen etal., 2014;
Harumoto and Lemaitre, 2018; Martinez etal., 2021). e OTU of
sAtri is highly conserved among other heritable Ixodetis members
(Supplementary Figure S5) and does not share an open reading
frame with ankyrin domains. Despite the presence of these domains,
no sex-ratio distortion has been observed in previous studies
(Haselkorn et al., 2009) or in our lab stock (T. Chris Amuwa,
unpublished results).
sAtri also encodes virulence genes that are scarcely found or not
yet described in the Spiroplasma genus. ese include an adenylate
cyclase domain annotated by HMMER as anthrax toxin LF subunit.
is domain is also found on the edema factor of Bacillus anthracis
anthrax toxin. e edema factor is itself an adenylate cyclase toxin that
causes large increases in the ubiquitous signaling molecule cAMP,
resulting in severe disruptions to cellular processes (Tang and
Guo, 2009).
sAtri encodes two metalloproteases of the M60 family which are
implicated in virulence and infection outcomes in some bacteria and
viruses (Nakjang et al., 2012; Belousov et al., 2018). M60-like
0.002
Spiroplasma of Lariophagus distinguendus
Spiroplasma of Dactylopius coccus
Spiroplasma of Cephus cinctus
Spiroplasma of Drosophila atripex
Spiroplasma of Danaus chrysippus
Spiroplasma of Dermanyssus gallinae
1
0.958
0.865
1
MK
CI
D
FIGURE1
Phylogeny of heritable Ixodetis Spiroplasma. Midpoint-rooted
phylogeny of 67 genes in ten syntenous blocks (72 kb) from available
heritable Ixodetis genomes shows relationship of sAtri to its closest
neighbors. Five gene loci from the Ixodetis Spiroplasma of
Dermanyssus gallinae were used in alignment to provide additional
context for phylogeny grouping. Known phenotypes are indicated
with abbreviations: CI = cytoplasmic incompatibility and MK = male
killing. Node labels indicate ML-like FastTree support values.

Moore and Ballinger 10.3389/fmicb.2023.1148263
Frontiers in Microbiology 04 frontiersin.org
peptidases, also commonly referred to as enhancins, are well studied
in, but not restricted to, insect pathogenic dsDNA viruses in the
family Baculoviridae and function by binding to and degrading
mucin layers that protect epithelial tissues (Ishimwe etal., 2015).
A conserved core of toxin domains in
heritable Spiroplasma
e discovery that the Drosophila symbiont sAtri harbors
multiple functional domains previously reported in distantly related
heritable Citri clade symbionts motivated a more detailed analysis of
domain distributions across the genus Spiroplasma. Wefocused the
scope of this analysis on RIP, OTU and ankyrin domains due to their
role in defensive and reproductive manipulation phenotypes. Wealso
analyzed ETX/MTX2 domains across Spiroplasma which are found
widespread across this genus with little understanding of their role.
VT-capable strains were determined based on a number of qualifying
characteristics including evidence of transovarial transmission, PCR
detection in hemolymph, ovaries and/or eggs, and presence of
reproductive manipulation phenotypes (Table2). VT Spiroplasma
strains investigated in this study and their associated hosts, taxonomy
and phenotypes are listed in Table3. OTU and ankyrin domains can
befound only in VT Spiroplasma across the Citri and Ixodetis clade
(Figure3). RIP domains are found in all VT Spiroplasma and in three
non-VT Spiroplasma. ETX/MTX2 domains are more widely
TABLE1 Genome assembly statistics of Spiroplasma.
Genome Size (bp) Contigs %GC N50 CDS tRNAs
Spiroplasma of Drosophila atripex 1,271,056 1 23.7 Closed 1,506 27
Spiroplasma of Lariophagus distinguendus†† 1,163,832 198 24.3 14,219 1,175 27
Spiroplasma of Cephus cinctus 713,566 145 24.9 5,160 754 23
Spiroplasma of Danaus chrysippus†1,745,430 12 23.7 215,399 1,782 27
Spiroplasma of Dactylopius coccus (DCF) 1,195,508 286 23.7 6,014 1,253 27
Spiroplasma poulsonii strain MSRO†,‡ 1,883,005 1 26.4 Closed 2,217 31
†Male-killing strain.
††CI-inducing strain.
‡Citri clade.
0.4
sNeo (Drosophila neotestacea) RIP1 WP_127093322
sNigra (Megaselia nigra) RIP5b
sNigra (Megaselia nigra) RIP6 WP_126820955
Spiroplasma eriocheiris RIP2 WP_047791253
Escherichia coli Shiga toxin subunit A WP_062860140
sScab (Myrmica scabrinodis) RIP1 SAMN08120314; Contig 25; 10916 12094
Spiroplasma sabaudiense RIP1 WP_025251437
sDis (Lariophagus distinguendus) RIP1 WP_252319696
sNeo (Drosophila neotestacea) RIP2 ASM46792
sVan (Myrmica vandeli) RIP1 SAMN0812031; Contig 24351; 424-977
sNeo (Drosophila neotestacea) RIP3 ASM46791
sNigra (Megaselia nigra) RIP7 WP_126821022
sChrys (Danaus chrysippus) RIP1 WP_174479925
sMoj (Drosophila mojavensis) RIP1 MBH8624287
sHyd (Drosophila hydei) RIP1 WP_198049692
MSRO (Drosophila melanogaster) RIP1 WP_040093770
sRubra (Myrmica rubra) RIP1 LJ130441.1, LJ130442.1
sHyd (Drosophila hydei) RIP2 MBH8623170
sNigra (Megaselia nigra) RIP3 WP_126821462
Spiroplasma mirum RIP1 WP_025317327
sNigra (Megaselia nigra) RIP5a
Spiroplasma eriochieris RIP1 WP_079450805
sNigra (Megaselia nigra) RIP2 WP_126821476
sCinc (Cephus cinctus) RIP1 TLF25546
sNeo (Drosophila neotestacea) RIP4 WP_158676203
sAtri (Drosophila atripex) RIP3
sNigra (Megaselia nigra) RIP4 WP_126821293
sAtri (Drosophila atripex) RIP1
sRiversi (Nebria riversi) RIP1 WP_215826391
sCoccus (Dactylopius coccus) RIP1 MBP1525224
Spiroplasma sabaudiense RIP2 WP_025251436
sAtri (Drosophila atripex) RIP2
sSue (Jemadia suekentonmiller) RIP1 DWDU01007497; 330-5375
Spiroplasma sabaudiense RIP3 WP_025250933
MSRO (Drosophila melanogaster) RIP2 WP_040093936
MSRO (Drosophila melanogaster) RIP4 WP_040092751
sChrys (Danaus chrysippus) RIP2 WP_174479900
MSRO (Drosophila melanogaster) RIP5
sChrys (Danaus chrysippus) RIP3 WP_174481319
MSRO (Drosophila melanogaster) RIP3
sRiversi (Nebria riversi) RIP2 WP_215825920
sNigra (Megaselia nigra) RIP1 WP_126821049
0.76
0.78
0.67
0.76
1
0.73
0.84
0.78
0.84
0.95
0.82
0.93
0.77
0.85
1
0.95
0.9
0.75
0.75
0.78
0.87
0.9
1
0.91
0.66
1
0.86
WP_126821432
MK D
MKD
MK
MK
D
D
D
MK
D
D
CI
D
P
P
P
MK D
D
D
D
FIGURE2
Ixodetis Spiroplasma encode diverse RIPs. FastTree phylogeny built from a MAFFT alignment of Spiroplasma RIP domain amino acid sequences and
rooted to Escherichia coli shiga toxin subunit A. Ixodetis clade Spiroplasma possess multiple RIP-domain bearing proteins with a distribution explained
by gains, losses, duplications, and horizontal transfers. Red shading indicates Apis clade, orange shading indicates Citri clade and yellow shading
indicates Ixodetis clade. Abbreviations indicate known phenotypes of the strain from which each domain was extracted: (P: pathogenic, MK: male-
killing, CI: cytoplasmic incompatibility-inducing, D: defensive). sAtri RIP domains are highlighted in bold. Support values greater than or equal to 0.60
are shown.

Moore and Ballinger 10.3389/fmicb.2023.1148263
Frontiers in Microbiology 05 frontiersin.org
distributed among members of the Citri and Ixodetis clades,
including most VT Spiroplasma and a few non-VT Spiroplasma. All
four domains are almost entirely absent from the Apis clade except
for three RIPs in Spiroplasma sabaudiense and a single ETX/MTX2
copy in Spiroplasma culicicola. Interestingly, no VT strains have been
described in the Apis clade (Bolaños etal., 2015). Weused BayesTraits
soware to identify correlations between distributions of toxin
domains and heritability under two dierent models—one that
assumes independent evolution and one that assumes dependent
evolution. We nd that all four domains are signicantly better
represented under a dependent model of evolution compared to an
independent model (Table4). For comparison, weused the same
approach to determine if a correlation existed between heritability
and other putative virulent Spiroplasma genes including glpO
(glycerol 3 phosphate oxidase), chiA (chitinase), and spi (spiralin)
(Alexeev etal., 2012; Chang etal., 2014; Duret etal., 2014). Wend
little to no support that the distribution of these domains across
Spiroplasma are associated with heritability (Table 4;
Supplementary Figure S6).
VT-specific domains vary in copy number
and origin
While RIP, OTU, ankyrin, and ETX/MTX2 domains are
distributed across diverse heritable Spiroplasma, the number of genes
possessing these domains can vary drastically, even between closely
related strains (Figure4). For example, the number of genes possessing
RIP domains varies both within and between clades, likely due to gene
duplications and horizontal gene transfers. e number of genes
possessing ankyrin domains are consistent within clade but show
extreme copy number variation between clades characterized by
ankyrin enrichment in Ixodetis Spiroplasma. Conversely, the number
of OTU and ETX/MTX2 copies is relatively consistent within and
between clades. All four of these domains can befound on plasmids
(Harumoto and Lemaitre, 2018; Ballinger etal., 2019). In some cases,
a given strain’s entire domain repertoire can only befound on plasmids
such as ankyrins and OTUs in MSRO, and ETX/MTX2in Spiroplasma
citri. In contrast, only one domain could be found within an
endogenous phage region (ETX/MTX2 of MSRO), suggesting
TABLE2 Evidence of vertical transmission.
Strain (host) Evidence of vertical transmission References
sAtri (Drosophila atripex) Transgenerational transmission
PCR detection in hemolymph
Genome sequenced from host ovaries
T. Chris Amuwa (unpublished results) and this study
MSRO (Drosophila melanogaster) Transgenerational transmission
Detection in hemolymph via transinfection
experiments
FISH visualization in eggs
FISH visualization of ovaries
Reproductive manipulation
Pool etal. (2006) and Harumoto and Lemaitre (2018)
Herren et al. (2013)
sNeo (Drosophila neotestacea) Transgenerational transmission
Detection in hemolymph via transinfection
experiments
Jaenike etal. (2010a) and Haselkorn and Jaenike (2015)
sHyd (Drosophila hydei) Transgenerational transmission
Detection in hemolymph via transinfection
PCR detection in eggs (unpublished)
Mateos etal. (2006) and Xie etal. (2010)
sMoj (Drosophila mojavensis) Transgenerational transmission Haselkorn etal. (2013)
sVan (Myrmica vandeli) PCR detection in hemolymph
High infection frequency (92.3%)
Host specicity in sympatry
Ballinger etal. (2018)
sScab (Myrmica scabrinodis) PCR detection in hemolymph
High infection frequency (91.9–97.6%)
Host specicity in sympatry
Ballinger etal. (2018)
sNigra (Megaselia nigra) Transgenerational transmission (unpublished)
PCR detection in hemolymph (unpublished)
High infection frequency (73.3–80.2%)
Ballinger etal. (2019)
sRiversi (Nebria riversi) PCR detection in egg, larval and adult stages Weng etal. (2021)
sChrys (Danaus chrysippus) Transgenerational transmission
Reproductive manipulation
Martin etal. (2020)
sCoccus (Dactylopius coccus) PCR detection in ovaries Vera-Ponce León etal. (2021)
sDis (Lariophagus distinguendus) Transgenerational transmission
FISH visualization in ovaries
Reproductive manipulation
Pollmann etal. (2022)

Moore and Ballinger 10.3389/fmicb.2023.1148263
Frontiers in Microbiology 06 frontiersin.org
Spiroplasma phages do not explain the distribution of RIP, OTU,
ankyrin, or ETX/MTX2 domains across the genus.
VT-specific domains source diverse
recombinant genes
Across Citri and Ixodetis-clade Spiroplasma, weobserve multiple
structural variants of RIPs, OTUs, ankyrins, and ETX/MTXs within
the same open reading frame (Figure5). In a particularly interesting
case, weuncovered two Spaid-like toxins from the genome of the
Spiroplasma endosymbiont of Drosophila hydei (strain sHyd1, NCBI
BioProject PRJNA274591) and a Spiroplasma genome extracted from
whole-genome sequence data of the South American buttery Jemadia
suekentonmiller (sSue). ese open reading frames include ankyrin
domains, an OTU domain, and the Spaid C-terminal transmembrane
domain. Toward the N-terminus, these proteins are also equipped
with two RIP domains which together possess all the conserved active
site residues of a single RIP domain. ese Spaid-like toxins are
present across several Spiroplasma genomes extracted from hesperid
buttery assemblies (Supplementary Figure S7).
Spiroplasma with VT-associated domains
are widespread across Hesperiidae
butterflies and other insects
We further investigated the presence of Spiroplasma in insect
genome assemblies using MSRO Spaid, and FtsZ and rpoB proteins
from Apis, Citri, and Ixodetis clade taxa as queries against whole
genome shotgun (WGS) sequence databases on NCBI and identied
Citri clade Spiroplasma genomic sequences in several South
American members of the Hesperiidae buttery family. A few of
these Spiroplasma infections are also reported in an unpublished
preprint (Medina et al., 2020; Manzano-Marin etal., 2020). ese
Hesperiidae-infecting Spiroplasma strains group closely to the
heritable Spiroplasma strains infecting Drosophila mojavensis and
Megaselia nigra (sMoj and sNigra, respectively) (Figure 6).
Metagenomic binning of Spiroplasma contigs reveals this novel group
of Spiroplasma is also equipped with RIPs, OTUs, ankyrins, and ETX/
MTX2 domains. Phylogenetic analysis of Ixodetis clade Spiroplasma
extracted from WGS sequences reveals an association with diverse
insects including two ants, a buttery, a springtail and a drosophilid
(Figure6). e presence of RIPs, OTUs, ankyrins, and ETX/MTX2
domains is variable among these novel Ixodetis strains (Figure6). For
example, the Spiroplasma genome extracted from a Monomorium
pharaonic ant assembly (sPharaoh) is estimated to be98% complete
(Supplementary Table S4) and only encodes a single ETX/MTX2
domain. Alternatively, Ixodetis clade Spiroplasma genomes extracted
from the assemblies of the buttery Colias croceus (sCroceus) and the
drosophilid Zaprionus kolodkinae (sKolod) both encode RIPs, OTUs,
ankyrins, and ETX/MTX2 domains.
Discussion
In this study, wereport a conserved core of toxin and virulence
domains associated with heritable Spiroplasma. As one of the most
successful and diverse arthropod-associated heritable symbionts on
Earth (Duron etal., 2008), the mechanisms that support Spiroplasma
persistence across ecological timescales are of great interest and yet
largely unknown. Previous work has demonstrated that Spiroplasma
exploit conserved yolk protein import machinery to invade maturing
oocytes, providing a possible explanation for their eective vertical
transmission in diverse arthropods (Herren et al. 2013). But how
Spiroplasma navigates numerous other obstacles required for growth
and persistence in host hemolymph throughout host development is
not well understood. While toxins are oen viewed through the lens
of pathogenesis, they can also mediate and manipulate host processes
to maintain symbiont persistence. For instance, RIPs and an
OTU-ankyrin-bearing Spaid toxin promote Spiroplasma persistence
by supporting defensive and reproductive phenotypes, respectively
(Hamilton etal., 2016; Ballinger and Perlman, 2017; Harumoto and
Lemaitre, 2018). Our assembly of the Ixodetis Spiroplasma genome
from Drosophila atripex and accompanying comparative analyses
reveal that the presence of these domains is highly conserved across
TABLE3 Vertically transmitted strains of Spiroplasma in study.
Host Spiroplasma strain Strain taxonomy Phenotype
Drosophila melanogaster MSRO Citri-Poulsonii Male-killing; defensive
Drosophila neotestacea sNeo Citri-Poulsonii Defensive
Drosophila hydei sHyd Citri-Poulsonii Defensive
Myrmica vandeli sVan Citri –
Myrmica scabrinodis sScab Citri –
Drosophila mojavensis s Moj Citri –
Megaselia nigra sNigra Citri –
Nebria riversi sRiversi Unplaced –
Dactylopius coccus sCoccus Ixodetis –
Danaus chrysippus sChrys Ixodetis Male-killing
Lariophagus distinguendus sDis Ixodetis Cytoplasmic incompatibility
Drosophila atripex sAt ri Ixodetis Defensive
Dash (−) indicates a phenotype has not yet been published.

Moore and Ballinger 10.3389/fmicb.2023.1148263
Frontiers in Microbiology 07 frontiersin.org
diverse heritable Spiroplasma. RIPs, OTUs and ankyrin domains are
specic to VT Spiroplasma, and are rare (RIPs) or entirely absent
(OTUs and ankyrins) in non-VT Spiroplasma genomes. ese toxin
and virulence domains may play a role in facilitating and maintaining
a vertically transmissible lifestyle across heritable Spiroplasma as has
been demonstrated for a small number of Citri clade members.
Finally, ETX/MTX2 toxins are more widespread in non-VT Citri and
Ixodetis Spiroplasma compared to RIPs, OTUs, and ankyrins but are
OTUAnkRIP
0.06
Spiroplasma chrysopicola
Spiroplasma platyhelix
Spiroplasma alleghenense
sScab (Myrmica scabrinodis)
sCinc (Cephus cinctus)
Spiroplasma corruscae
Spiroplasma cantharicola
sCoccus (Dactylopius coccus)
Spiroplasma litorale
sAtri (Drosophila atripex)
Spiroplasma diminutum
sHyd (Drosphila hydei)
sSep (Trachymyrmex septentrionalis)
Spiroplasma tabanidicola
sRiversi (Nebria riversi)
Spiroplasma culicicola
sVan (Myrmica vandeli)
Spiroplasma sp. NBRC
sMoj (Drosophila mojavensis)
sNigra (Megaselia nigra)
Spiroplasma eriocheiris
Spiroplasma melliferum
sNeo (Drosophila neotestacea)
Spiroplasma sp. BIUS-1
Spiroplasma sabaudiense
Spiroplasma syrphidicola
Spiroplasma apis
Spiroplasma mirum
Spiroplasma clarkii
sDis (Lariophagus distinguendus)
Spiroplasma sp. ChiS
Spiroplasma citri
Spiroplasma floricola
Spiroplasma chinense
Spiroplasma gladiatoris
Spiroplasma helicoides
MSRO (Drosophila melanogaster)
Spiroplasma taiwanense
Spiroplasma turonicum
Spiroplasma monobiae
Mycoplasma mycoides
Spiroplasma phoeniceum
sChrys (Danaus chrysippus)
Spiroplasma kunkellii
1
APIS CITRI IXODETIS
CI
D
MK
MK
D
D
P
P
P
P
P
P
P
P
P
ETX/MTX2
D
FIGURE3
Phylogenetic distribution of select toxins and virulence domains in the genus Spiroplasma. FastTree phylogeny built from MAFFT nucleotide alignments
of concatenated Spiroplasma ftsZ, rpoB and gyrB and rooted to M. mycoides. RIP, OTU, ankyrin, and ETX/MTX domains are preferentially distributed
among VT Spiroplasma. Small black dots next to nodes indicate FastTree support values greater than 75%. Large colored circles to the right of branch
labels indicate at least one domain copy is present in the genome. Black dash indicates no domain copies present in the genome. Red text is used for
VT Spiroplasma and black text is used for non-VT Spiroplasma. Abbreviations indicate known phenotypes of the strain from which each domain was
extracted: (P: pathogenic, MK: male-killing, CI: cytoplasmic incompatibility-inducing, D: defensive).
TABLE4 Model comparisons for describing toxin and virulence domains across heritable Spiroplasma.
Domain Dependent model
likelihood score
Independent model
likelihood score
Chi score df value of p*
RIP −29.855283 −40.389039 21.067512 4 0.000307
OTU −26.546300 −37.764235 22.43587 4 0.000164
ankyrin −26.036137 −35.577156 19.082038 4 0.000757
ETX/MTX2 −33.930099 −44.915624 21.97105 4 0.000203
glpO −44.902777 −46.963108 4.056606 4 0.398347
chiA −37.674589 −40.678499 6.00782 4 0.198552
spiralin −32.183710 −37.138576 9.91 4 0.041971
*Value of p threshold has been set at 0.001.

Moore and Ballinger 10.3389/fmicb.2023.1148263
Frontiers in Microbiology 08 frontiersin.org
still highly correlated with a VT lifestyle based on BayesTraits analysis.
As previously stated, the role of ETX/MTX2 toxins in host-
Spiroplasma interactions is unknown but their propensity for
VT-Spiroplasma may suggest a role in maintaining a VT lifestyle
similar to RIPs, OTUs and ankyrin domains (i.e., defense or
host manipulation).
We nd that similar toxin evolutionary processes previously
reported in Poulsonii clade strains (Ballinger etal., 2019; Gerth
etal., 2021) are mirrored in toxin domain-containing proteins of
the Ixodetis clade, suggesting conservation of dominant toxin
evolution mechanisms across heritable strains throughout the
genus. Citri clade RIPs experience duplications, losses, and domain
sAtri (Drosphila atripex)
Spiroplasma melliferum
sScab (Myrmica scabrinodis)
Spiroplasma sp. ChiS
sNeo (Drosophila neotestacea)
Spiroplasma eriocheiris
Spiroplasma culicicola
sDis (Lariophagus distinguendus)
Spiroplasma sp. NBRC
Mycoplasma mycoides
sCoccus (Dactylopius coccus)
sChrys (Danaus chrysippus)
MSRO (Drosophila melanogaster)
sMoj (Drosophila mojavensis)
Spiroplasma sabaudiense
sCinc (Cephus cinctus)
Spiroplasma mirum
Spiroplasma citri
sRiversi (Nebria riversi)
sVan (Myrmica vandeli)
sNigra (Megaselia nigra)
sHyd (Drosophila hydei)
624 8
062 4 8 10 12
0
RIP copy # Ankyrin copy #
2 4
0
OTU copy #
24
0
ETX/MTX2 copy #
0.05
FIGURE4
Number of toxin and virulence domains varies across Spiroplasma genus. FastTree phylogeny built from MAFFT nucleotide alignment of concatenated
Spiroplasma ftsZ, rpoB, and gyrB sequences and rooted to M. mycoides. All Spiroplasma present in the phylogeny encode at least one domain copy of
RIP, OTU, ankyrin, or ETX/MTX2. Bar graphs show the number of domain copies present in each genome for each domain type. Double-helix circles
indicate at least one of the domains are found on a plasmid. Phage illustrations indicate domains are found within a phage region of the genome. Red
shading represents Apis clade, orange shading represents Citri clade and yellow shading represents Ixodetis clade. Red color text is used for VT
Spiroplasma and black color text is used for non-VT Spiroplasma. Small black dots indicate FastTree support values above 0.75.
sRiversi (Nebria riversi)
sSue (Jemadia suekentonmiller)
sChrys (Danaus chrysippus)
ETX/MTX2
RIP
OTU
MSRO (Drosophila melanogaster) *Spaid toxin*
sNigra (Megaselia nigra)
PQQ
DnaJ
sAtri (Drosophila atripex)
Ank (PF13637)
Ank (PF12796)
Ank (PF00023)
200 aa
sHyd (Drosophila hydei)
Signal Peptide
Hydrophobic region
-JXYY01000041; 4552 - 9630
-WP_105629072
-DWDU01007497; 333 - 5285
-WP_215825920
-WP_126821430
-WP_174481319
Ank (PF13606)
-WP_241460302
sHyd (Drosophila hydei)
(Harumoto and Lemaitre, 2018)
(Gerth et al., 2021)
FIGURE5
Dynamic variants of toxin and virulence genes across Spiroplasma. Scaled representation showing diverse array of Spiroplasma toxin-bearing proteins
identified in this study. Orange shading indicates Citri clade and yellow shading indicates Ixodetis clade. A bracket is used for sHyd and sSue to
highlight that the two separated RIP domains make up a whole RIP domain. Spaid toxin responsible for male-killing by MSRO is shown above.

Moore and Ballinger 10.3389/fmicb.2023.1148263
Frontiers in Microbiology 09 frontiersin.org
swapping events that have accompanied an explosion in RIP
diversity. Similarly, Ixodetis RIPs are polyphyletic and RIP
representation diers between strains. For example, a RIP from the
Ixodetis endosymbiont of the buttery Danaus chrysippus groups
closely with the enigmatic double RIP domain-possessing proteins
recently uncovered from sHyd1 and sSue (Figure5; Gerth etal.,
2021). Ankyrin domain-containing proteins are especially
numerous in members of the Ixodetis clade, ranging from six to
fourteen copies across members of this clade. Believed to originate
in eukaryotes, ankyrin domains are more commonly found in
heritable microbes than other bacterial taxa (Jernigan and
Bordenstein, 2014), and may facilitate a variety of microbe-host
interactions. is makes the expansion of ankyrin domains in
Ixodetis particularly interesting, especially given the broad range of
insect hosts they infect (Figure1).
Dynamic evolution is also demonstrated in the varied number
of genes that possess these domains (i.e., RIP, OTU, ankyrin, and
ETX/MTX2)—even among closely related strains—indicating that
duplications, gains, and losses are common generators of their
diversity in Spiroplasma. RIP, OTU, ankyrin, and ETX/MTX2
domains are also present on plasmids, possibly contributing
functional roles that favor their dispersal and persistence through
the genus. Plasmids oen confer adaptive phenotypes to their
bacterial hosts to help drive their own spread among bacterial
populations (Dimitriu etal., 2021). Interestingly, no RIP, OTU, or
ankyrin domains are present within the diverse plasmids of non-VT
Citri clade Spiroplasma. For example, some Spiroplasma citri strains
can have upwards of nine unique plasmids (Rattner etal., 2021).
Given the promiscuity of plasmids across Spiroplasma¸ the lack of
RIP, OTU, and ankyrin domains on plasmids in non-heritable
strains suggests that they may have little adaptive function in
that lifestyle.
Not only do RIPs, OTUs, and ankyrin domains exist within
diverse proteins across Spiroplasma, but many VT Spiroplasma
species are equipped with several structural variants of these
domains on the same protein. In one notable example, our analysis
revealed a Spiroplasma genome within the WGS assembly of the
South American buttery J. suekentonmiller that encodes a protein
with RIP domains, an OTU domain, an ankyrin domain and a
C-terminal transmembrane domain, i.e., a RIP-Spaid fusion protein.
0.06
Spiroplasma
s
s
p. Ch
i
iS
s
Van
(
Myr
m
m
ica vand
e
e
l
i
i)
sPharaoh (Monomorium pharaonis)
sCroceus (Colias croceus)
S
p
iro
p
lasma
m
m
ellife
r
r
um
S
piroplasma
s
s
p. NB
R
R
C
s
Neo
(
Dro
s
s
ophila ne
o
o
t
e
e
stacea
)
sMer (Microceris merops)
M
S
R
O
(
Dr
o
o
sophila
m
me
e
lanogaste
r
r)
s
Moj
(
Dros
p
p
hila mojav
e
e
nsis
)
S
Sp
ir
o
op
lasma sa
b
b
audiens
e
sS
cab
(
M
yr
r
mica sca
b
b
r
i
i
nodis
)
Sp
iro
p
lasma
m
m
irum
s
Riversi
(
Nebria riversi
)
Mycoplasma mycoides
S
piroplasma p
l
l
atyheli
x
sPol (Parelbella polyzona)
sSue (Jemadia suekentonmiller)
sLong (Pogonognathellus longicornis)
sD
D
is
(
Lariophagus
d
d
istinguendus
)
sTox (Cecropterus toxeus)
sSelysi (Formica Selysi)
sC
C
inc
(
Cephus cin
c
c
tus
)
sC
C
hrys
(
Danaus c
h
h
rysippus
)
sThe (Elbella theseus)
sC
C
occus
(
Dactylop
i
i
us coccus
)
sKolod (Zaprionus kolodkinae)
S
S
pir
o
o
plasma cu
l
l
icicola
sHad (Pyrrhopyge hadassa)
sIph (Microceris iphinous)
s
H
y
d
(
Dro
s
s
phila h
y
d
e
e
i
)
)
s
Nigra
(
Me
g
g
aselia ni
g
gr
r
a
)
S
p
iro
p
lasma
e
e
rioch
e
e
iris
sA
A
tri
(
Drosophila a
t
t
ripex
)
S
piroplasma
c
c
itr
i
sTeh (Cecropterus tehuacana)
RIP OTU Ank ETX/MTX2
FIGURE6
Cryptic Spiroplasma carry domains associated with a heritable lifestyle. FastTree phylogeny built from MAFFT nucleotide alignment of concatenated
Spiroplasma ftsZ, rpoB, and gyrB and rooted to M. mycoides. This phylogram shows the placement of novel Spiroplasma strains (black bold) extracted
from insect WGS sequences. Many of these Spiroplasma strains carry RIPs, OTUs, and/or ankyrin domains that are strongly associated with a heritable
lifestyle. Circles indicate the presence of a given domain and dashes indicate their absence. Red shading indicates Apis clade, orange shading indicates
Citri clade and yellow shading indicates Ixodetis clade. WGS-extracted taxa are shown in bold typeface. Black dots adjacent to nodes indicate a
FastTree support value greater than 0.75.

Moore and Ballinger 10.3389/fmicb.2023.1148263
Frontiers in Microbiology 10 frontiersin.org
ese VT-associated domains appear to frequently recombine to
create novel protein congurations. For instance, Spaid and Spaid-
like proteins encode multiple ankyrin domains each and these
ankyrin domains vary greatly in copy number and protein family
(Figure 5; Supplementary Figure S7B), suggesting that ankyrin-
spanning region is especially prone to domain losses and gains. e
close association of RIPs, OTUs, and ankyrins on the same proteins
across Citri and Ixodetis Spiroplasma suggests functional
evolutionary ties between these domains.
Extracting symbiont genomes from publicly available nucleotide
databases is a powerful and convenient approach to studying symbiont
evolution (Salzberg etal., 2005; Gerth and Hurst, 2017; Pascar and
Chandler, 2018; Scholz etal., 2020; Pilgrim etal., 2021). Weexplored
the presence of RIP, OTU, ankyrin, and ETX/MTX2 domains in other
insect assemblies by mining partial to near complete genomes of both
Ixodetis and Citri clade Spiroplasma from a diverse array of insect
WGS assemblies. Within Citri, there is a large group of Spiroplasma
strains infecting various members of the South American buttery
family Hesperiidae, and are equipped with RIP, OTU, and ankyrin
domain-possessing proteins. Within Ixodetis, weidentied ve novel
Spiroplasma genomes extracted from a diverse variety of insect hosts
with a varied presence of RIP, OTU, ankyrin, and ETX/MTX2
domains. is analysis provides promising candidates for future
studies on heritable Spiroplasma and the identication of RIP, OTU,
ankyrin, and ETX/MTX2 domains within insect WGS assemblies may
be useful for developing early hypotheses on Spiroplasma-
host interactions.
Methods
Genome sequencing, assembly, and
annotation
Nucleic acids were extracted from ovaries of eight adult female
Drosophila atripex using the phenol-chloroform method. Paired end
150 bp Illumina genomicDNA reads were sequenced by Novogene
(CA, United States). Long reads were sequenced on an Oxford
Nanopore MinION device and R9.4.1 ow cell following library
preparation using the Ligation Sequencing Kit (SQK-LSK109). Guppy
6.1.5 implemented in MinKNOW 22.05.5 was used to generate high
accuracy basecalls. Only long reads above 10 kb were used in hybrid
assembly. Illumina reads were trimmed for adapter sequences and
quality (qtrim = r trimq = 10) with BBMap38.35 (Bushnell, 2014), and
a metagenome was hybrid assembled using Unicycler 0.5.0 (Wick
etal., 2017). Spiroplasma-derived contigs were retained through a
combination of metagenomic binning (Laczny et al., 2017) and
manual ltering. Manual ltering was performed through targeted
searches of sequences likely to contaminate the bin based on similar
nucleotide content and was guided by kmer abundance analyses
performed with KAT (Mapleson etal., 2016). e dra genome was
annotated through Prokka 1.14.5 (Seemann, 2014) and KEGG
numbers were assigned with BLASTKoala (Kanehisa etal., 2016).
Genome coverage and plasmid coverage were determined using
BBMAP (Bushnell, 2014). Genome completeness was estimated with
BUSCO v5 (Simão etal., 2015) on the gVolante webserver (Nishimura
etal., 2017). Spiroplasma homologs of interest, including those used
for phylogenetics, were identied using HMMER (Finn etal., 2011)
and PfamScan (Mistry etal., 2021) with a threshold of E-value = 0.01.
Proteins of interest that could not beannotated by the previously
mentioned approaches were investigated further with HHpred
(Zimmermann etal., 2018), BLASTp and alignments. Protein and
nucleotide sequences were aligned with MAFFT 7.388 (Katoh and
Standley, 2013) and phylogenies were built using FastTree 2.1.11
(Price etal., 2010) unless stated otherwise. BLASTp searches with
relaxed signicance thresholds (expect values ≤0.01) were also
performed to ensure detection of more distantly related protein
domains if present.
Characterizing distribution of heritability,
toxin, and virulence domains across
Spiroplasma genus
All Spiroplasma sp. genomes, plasmids, and reads were
downloaded from NCBI, and their accession numbers are listed in
Supplementary Table S5. Heritable Spiroplasma were identied based
on evidence provided from transgenerational screenings, ovarian
tissue and egg screens, high infection frequencies among populations,
multi-year screenings and systemic infections (Table2). Wesearched
for RIP, OTU, ankyrin, and ETX/MTX2 domains in all Spiroplasma
genomes using tBLASTn (threshold E-value = 0.01) with a curated list
of Spiroplasma domains that have been identied in this study and
extracted using HMMER domain annotations as a guide
(Supplementary Table S6). Protein domains were conrmed with
HMMER 3.3 and pfamscan with a.01 e-value threshold, and through
alignments to conrm presence of conserved residues. Domains
present on open reading frames and on pseudogenes are both included
in the total count for domains of interest. Phage regions were
determined using Phaster webserver (Zhou etal., 2011; Arndt etal.,
2016). Annotated phage regions were extracted from the genomes of
Spiroplasma strains known to contain RIP, ankyrin, OTU, or ETX/
MTX2 domains. ese phage regions were investigated to determine
if they included any of these domains.
We determined whether a correlation existed between heritability
and specic encoded domains using the soware BayesTraits V4.0.0
(Pagel and Meade, 2006). Heritability was treated a discrete binary
trait (heritable or non-heritable) and domains were also treated as a
binary trait (domain present domain absent in genome). Likelihood
scores were calculated for both a dependent model (i.e., assumes
heritability and domain acquisition/retention evolved dependently)
and an independent model (i.e., assumes heritability and domain
acquisition/retention evolved independently). Likelihood scores were
compared with a chi-square analysis as recommended by BayesTraits
documentation. We rejected the null hypothesis of independent
evolution at p< 0.001.
Identification and extraction of
Spiroplasma genomes from insect whole
genome shotgun assemblies
Using Spaid toxin from MSRO, and sZ and rpoB from Ixodetis,
Citri, and Apis clade taxa as queries, we performed tBLASTn
searches of whole genome shotgun contigs (WGS) located on the
NCBI database. Default search parameters were used, and welimited

Moore and Ballinger 10.3389/fmicb.2023.1148263
Frontiers in Microbiology 11 frontiersin.org
our organism search to Insecta (taxid:50557). Assemblies with
matches to these genes were submitted to BusyBee web server for
genomic binning (Laczny etal., 2017). Bins outputted by BusyBee
were manually inspected for the presence of core Spiroplasma genes
including sZ, rpoB, and gyrB. ese core genes were also used to
construct the phylogeny in Figure6. Weperformed tBLASTn against
the extracted Spiroplasma genomes using a curated list of
Spiroplasma RIP, OTU and ankyrin peptide sequences. If a domain
was determined to be missing from an extracted Spiroplasma
genome, wesearched for it in the original insect assembly to ensure
it wasn’t present on a Spiroplasma contig missed in the binning
process. Genome completeness was determined using BUSCO
through gVolante webserver, and coding DNA sequences and tRNA
content was determined with Prokka 1.14.5. Genome statistics and
insect assembly accession numbers are available in
Supplementary Table S4.
Constructing phylogenies
MAFFT 7.388 was used to create all alignments and FastTree
2.1.11 was used to construct Spiroplasma phylogenies. Jukes-Cantor
model was used to build phylogenies from nucleotide sequence
alignments. RIP and OTU proteins are oen anked with diverse,
nonhomologous accessory domains. For this reason, RIP and OTU
regions immediately outside of the conserved active site residues
were trimmed out of alignments manually; conserved active site
residues span the majority of both domains. sSue RIP, sHyd RIP2,
and sChrys RIP3 all possess peptide insertion sequences ranging
from 54 to 157 aa that split their RIP domain. is appears to bea
conserved feature among this clade of RIPs. Due to their large size
and highly divergent sequence variation, we identied these
insertion sequence regions using alignments to other RIPs and
removed them manually. sHyd RIP2 and sRiversi RIP1 also group
among these RIPs, however they are small proteins encoding only a
partial RIP domain and therefore did not require additional
trimming. e trimmed RIP and OTU sequences were then aligned
with MAFFT 7.388 and submitted to MEGA soware (Kumar etal.,
2018) to determine the best substitution model for PhyML tree
building. WAG+G + I substitution model was used to construct a
RIP phylogeny and cpREV+G substitution model was used to
construct an OTU phylogeny. ETX/MTX2 are divergent toxins that
lack conserved active site residues to help guide trimming.
Wecreated an alignment of ETX/MTX2-possessing proteins with
MAFFT 7.388 and conrmed that the annotated ETX/MTX2
domains were aligned. e alignment was then trimmed using
ClipKIT soware (Steenwyk etal., 2020) in kpi-gappy mode. e
trimmed alignment output was submitted to MEGA soware to
determine the best substitution model for PhyML tree building.
WAG+G + F substitution model was used to construct an ETX/
MTX2 phylogeny.
Data availability statement
e datasets presented in this study can befound in online
repositories. e names of the repository/repositories and accession
number(s) can be found at: e Bioproject accession is
“PRJNA928682” and the Biosample accession is “SAMN32939082”
and the SRA accessions are “SRX19199603” and “SRX19199602.”
Author contributions
LM and MB conceived and designed the study, collected data and
performed analyses, and revised the manuscript and approved the
nal submission. LM prepared gures and wrote the rst dra of the
manuscript. All authors contributed to the article and approved the
submitted version.
Funding
is work was supported by National Science Foundation award
2144270 to MB.
Acknowledgments
We are grateful to Luciano Matzkin for sharing Drosophila
atripex lines.
Conflict of interest
e authors declare that the research was conducted in the
absence of any commercial or nancial relationships that could
beconstrued as a potential conict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their aliated organizations,
or those of the publisher, the editors and the reviewers. Any product
that may be evaluated in this article, or claim that may be made by its
manufacturer, is not guaranteed or endorsed by the publisher.
Supplementary material
e Supplementary material for this article can befound online
at: https://www.frontiersin.org/articles/10.3389/fmicb.2023.1148263/
full#supplementary-material
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