# From insect endosymbiont to phloem colonizer: comparative genomics unveils the lifestyle transition of phytopathogenic Arsenophonus strains

Mathieu Mahillon, Christophe Debonneville, Raphaël Groux, David Roquis, Justine Brodard, Franco Faoro, Xavier Foissac, Olivier Schumpp, Jessica Dittmer

<figure><img src="image_1.png" /></figure>

## To cite this version:

Mathieu Mahillon, Christophe Debonneville, Raphaël Groux, David Roquis, Justine Brodard, et al. From insect endosymbiont to phloem colonizer: comparative genomics unveils the lifestyle transition of phytopathogenic Arsenophonus strains. mSystems, 2025, pp.Early Access. 10.1128/msystems.01496-24. [https://doi.org/10.1128/msystems.01496-24](https://doi.org/10.1128/msystems.01496-24)

HAL Id: hal-05056226

https://hal.inrae.fr/hal-05056226v1

Submitted on 5 May 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_3.png" /></figure>

# From insect endosymbiont to phloem colonizer: comparative genomics unveils the lifestyle transition of phytopathogenic *Arsenophonus* strains

Mathieu Mahillon,¹ Christophe Debonneville,¹ Raphaël Groux,¹ David Roquis,² Justine Brodard,¹ Franco Faoro,³ Xavier Foissac,⁴ Olivier Schumpp,¹ Jessica Dittmer³,⁵

**AUTHOR AFFILIATIONS** See affiliation list on p. 16.

**ABSTRACT** Bacteria infecting the plant phloem represent a growing threat worldwide. While these organisms often resist *in vitro* culture, they multiply both in plant sieve elements and hemipteran vectors. Such cross-kingdom parasitic lifestyle has emerged in diverse taxa via distinct ecological routes. In the genus *Arsenophonus*, the phloem pathogens "Candidatus Arsenophonus phytopathogenicus" (Ap) and "Ca. Phlomobacter fragariae" (Pf) have evolved from insect endosymbionts, but the genetic mechanisms underlying this transition have not been explored. To fill this gap, we obtained the genomes of both strains from insect host metagenomes. The resulting assemblies are highly similar in size and functional repertoire, rich in viral sequences, and closely resemble the genomes of several facultative endosymbiotic *Arsenophonus* strains of sap-sucking hemipterans. However, a phylogenomic analysis demonstrated distinct origins, as Ap belongs to the "Triatominarum" clade, whereas Pf represents a distant species. We identified a set of orthologs encoded only by Ap and Pf in the genus, including hydrolytic enzymes likely targeting plant substrates. In particular, both bacteria encode putative plant cell wall-degrading enzymes and cysteine peptidases related to xylellain, a papain-like peptidase from *Xylella fastidiosa*, for which close homologs are found in diverse *Pseudomonadota* infecting the plant vasculature. *In silico* predictions and gene expression analyses further support a role during phloem colonization for several of the shared orthologs. We conclude that the double emergence of phytopathogenicity in *Arsenophonus* may have been mediated by a few horizontal gene transfer events, involving genes acquired from other *Pseudomonadota*, including phytopathogens.

**IMPORTANCE** We investigate the genetic mechanisms of a transition in bacterial lifestyle. We focus on two phloem pathogens belonging to the genus *Arsenophonus*: "Candidatus Arsenophonus phytopathogenicus" and "Ca. Phlomobacter fragariae." Both bacteria cause economically significant pathologies, and they have likely emerged among facultative insect endosymbionts. Our genomic analyses show that both strains are highly similar to other strains of the genus associated with sap-sucking hemipterans, suggesting a recent lifestyle shift. Importantly, although the phytopathogenic *Arsenophonus* strains belong to distant clades, they share a small set of orthologs unique in the genus pangenome. We provide evidence that several of these genes produce hydrolytic enzymes that are secreted and may target plant substrates. The acquisition and exchange of these genes may thus have played a pivotal role in the lifestyle transition of the phytopathogenic *Arsenophonus* strains.

**KEYWORDS** *Arsenophonus*, cixiid, endosymbiont, phloem, Phlomobacter, planthopper, proteobacteria, xylellain

**Editor** Juliana Almario, CNRS Delegation Alpes,
Villeurbanne, France

Address correspondence to Olivier Schumpp,
olivier.schumpp@agroscope.admin.ch, or Jessica
Dittmer, jessica.dittmer@inrae.fr.

The authors declare no conflict of interest.

See the funding table on p. 16.

**Received** 6 November 2024

**Accepted** 10 March 2025

**Published** 9 April 2025

Copyright © 2025 Mahillon et al. This is an open-
access article distributed under the terms of the
Creative Commons Attribution 4.0 International
license.
In vascular plants, the phloem ensures the translocation of photosynthesates from source to sink tissues. As a sugar-rich environment, the phloem also represents a niche for pathogenic microorganisms (1, 2). In particular, bacterial infections restricted to the sieve elements (SEs; i.e., the phloem conductive cells) are difficult to control and cause damaging diseases worldwide (3). The causal agents are generally non-culturable and "vector-borne" as they have evolved a biphasic lifestyle, alternating between plant SEs and sap-sucking hemipteran vectors (4). Notorious examples of these bacteria include *Candidatus (Ca.)* species of the genera *Phytoplasma* and *Liberibacter*. This lifestyle is shared by bacteria from distant taxa, and it can emerge through distinct evolutionary routes (3, 5). In the phylum *Pseudomonadota* (formerly *Proteobacteria*), vector-borne phloem pathogens have emerged either among plant-associated bacteria as in the genera *Liberibacter* and *Serratia* (1, 6, 7), or among insect endosymbionts as proposed for the genera *Rickettsia* and *Arsenophonus* (5, 8). In the latter, two phytopathogens have been identified, namely, "*Ca. Arsenophonus phytopathogenicus*" (Ap) and "*Ca. Phlomobacter fragariae*" (Pf) (9–11).

Ap is transmitted by the plantchoppers *Pentastiridius leporinus* and *Cixius wagneri*, and it also colonizes the SEs of sugar beet, thereby inducing the syndrome "basses richesses" (SBR). SBR was first described in France and now occurs in Germany and Switzerland as well (12–14). Recently, Ap has been detected in sugar beets across Central Europe as well as in potato and onion plants in Germany and Switzerland (15–19), indicating its growing distribution and host range and warning of potential novel epidemics. In contrast, Pf is transmitted by *C. wagneri* and causes strawberry marginal chlorosis (SMC) (20), first reported in France (21) and then in Japan (22). SMC was also documented in Italy, where it was associated with both Pf and Ap (23, 24).

While Ap and Pf are phytopathogens, other *Arsenophonus* strains are known as arthropod endosymbionts. *Arsenophonus* belongs to the family *Morganellaceae* in the order *Enterobacterales* (25). It represents one of the most widespread insect endosymbiotic genera (26), and its host range also comprises other arthropod groups such as arachnids (27). Host-symbiont interactions, tissue tropisms, and transmission routes of *Arsenophonus* strains are diverse (28, 29). Infections can be associated with reproductive parasitism (30), insecticide resistance (31), feeding behavior alteration (32, 33), and nutritional symbiosis (34). These bacteria can multiply extra- or intracellularly, and some are maternally inherited. The best-studied species is *Arsenophonus nasoniae*, an endosymbiont of parasitoid wasps sometimes associated with "male-killing" (35). Transmission of *A. nasoniae* can be vertical but also horizontal through multi-parasitism events (36). Horizontal transmission has also been documented for the bee endosymbiont *Arsenophonus apicola* (37). Unlike these two species, many members of the genus can only be grown in host cells or even completely resist *in vitro* culture (5). Some strains have become obligate "primary" endosymbionts (P-endosymbionts) (38, 39). This great lifestyle diversity is mirrored by various genomic architectures. While the complex genome of *A. nasoniae* consists of one large circular chromosome (>3.5 Mb) and 7–20 extrachromosomal elements (40, 41), the genomes of P-endosymbionts are highly eroded (<1.2 Mb), lack extrachromosomal elements, and display reduced metabolisms streamlined for the production of nutrients lacking from the host's diet (38, 39).

Several aspects support the idea of an ancestral insect-restricted lifestyle and the recent emergence of phytopathogenicity in *Arsenophonus*. (i) All known strains of the genus (including the phytopathogenic ones) are associated with arthropods. (ii) Some *Arsenophonus* strains are P-endosymbionts of insects with highly reduced genomes, indicating millions of years of co-evolution with certain insect species. In contrast, there is no evidence for similar long-lasting relationships between *Arsenophonus* and plants. (iii) The genetic diversity among phytopathogenic strains is very low, suggesting a recent emergence (10). (iv) Ap and Pf conserve traits typically associated with insect endosymbionts (e.g., high infection rate, vertical transmission, and presence in reproductive organs) (11). Furthermore, phylogenetic and ancestral state reconstruction analyses support this scenario (11). Accordingly, Ap and Pf represent ideal models to investigate
the genetic changes involved in the transition from insect endosymbiont to vector-borne phloem pathogen. Probable mechanisms underlying this transition would be the acquisition of new functions, such as virulence factors, through horizontal gene transfer (HGT). To investigate this possibility, we compiled the first genome assemblies for Pf and Ap. As both bacteria are currently non-culturable, we produced hybrid assemblies for Pf (Pf-FR) and Ap (Ap-CH) using metagenomes of insects recently collected in France and Switzerland, respectively. In addition, the assembly of a second strain of Ap (Ap-FR) was obtained using short reads generated from insects sampled during an earlier outbreak in France. Following a genus-wide comparative analysis, we identified orthologs exclusively shared by the phytopathogenic strains that may have enabled their lifestyle transition.

## RESULTS

### General features of the assemblies

The genome sizes of Ap and Pf (2.6–2.9 Mb) are much larger than those reported for other vector-borne phloem pathogens such as liberibacteria, phytoplasmas, and spiroplasmas (<1.7 Mb [42]). The main features of the three assemblies are summarized in Fig. 1A. The hybrid assembly approach produced 50 and 33 scaffolds for Pf-FR and Ap-CH, respectively. The best assembly was obtained for the latter, as 28% of the genome is contained in the longest scaffold (806 kb) and the genome N50 is 384 kb (97 kb for Pf-FR). The genome of Ap-FR is shorter and more discontinuous with 67 scaffolds and an N50 of 106 kb. The three assemblies exhibit a guanine-cytosine (GC) content of ca. 37%, which is similar to other facultative endosymbiotic *Arsenophonus* strains, whereas the genomes of P-endosymbionts are richer in adenine-thymine (AT) (Fig. 1B). The genome sizes, numbers of predicted proteins (2,100–2,500), and pseudogenization rates (15.7%–17.8 %) of the assemblies occupy an intermediate position within the genus and align with several strains of the *Triatominarum* clade (Fig. 1B) (43).

CheckM analyses confirmed a high level of genome completeness for the three assemblies: 96.76% for Ap-FR, 97.84% for Pf-FR, and 99.46% for Ap-CH. In the latter, seven complete rRNA operons were identified, while a previous study found only six copies of the 16S rRNA (44). Three complete rRNA operons were detected for Pf-FR, but this genome likely contains at least seven rRNA operons as additional rRNA genes occur at scaffold ends. Both Ap genomes show extensive similarities (Fig. S1); hence, some of the finer-scale analyses presented herein will focus only on Ap-CH.

<table><thead><tr><th></th><th>Pf-FR</th><th>Ap-CH</th><th>Ap-FR</th></tr></thead><tbody><tr><td>LR cov.</td><td>38</td><td>300</td><td>NA</td></tr><tr><td>SR cov.</td><td>632</td><td>121</td><td>1,632</td></tr><tr><td>Scaffolds</td><td>50</td><td>33</td><td>67</td></tr><tr><td>Size (bp)</td><td>2,797,651</td><td>2,875,309</td><td>2,583,717</td></tr><tr><td>GC (%)</td><td>36.71</td><td>37.68</td><td>37.08</td></tr><tr><td>Genes</td><td>3,209</td><td>3,213</td><td>2,710</td></tr><tr><td>Proteins</td><td>2,577</td><td>2,620</td><td>2,167</td></tr><tr><td>ψ</td><td>554</td><td>505</td><td>483</td></tr><tr><td>rRNA</td><td>21</td><td>21</td><td>7</td></tr><tr><td>tRNA</td><td>53</td><td>61</td><td>47</td></tr><tr><td>ncRNA</td><td>4</td><td>6</td><td>6</td></tr><tr><td>Phage (%)</td><td>20</td><td>17</td><td>NA</td></tr></tbody></table>

<figure><img src="image_4.png" /><figcaption>FIG 1 (A) Global genomic features of Pf-FR and Ap-CH/FR. LR: long reads; SR: short reads. (B) Distribution of GC content (in percent) and size (in megabases [Mb]) for the *Arsenophonus* genomes. Each point corresponds to a single genome. Strain names are provided for the largest and smallest genomes as well as the phytopathogens. Details on host species, transmission mode, and phenotype of each strain are provided in Table 1.</figcaption></figure>
## Phage and plasmid regions

Despite high sequencing coverages and multiple assembly approaches, the three assemblies could not be closed, consistent with previous *Arsenophonus* genome projects necessitating DNA from pure cultures (41, 45). The assemblies of Ap and Pf may thus contain extrachromosomal elements, as reported for other strains of the genus (Table 1) (43). Several *Arsenophonus* extrachromosomal elements were recently characterized as “phage-plasmids,” harboring both phage and plasmid modules (41, 46), and some correspond to helper prophages and their associated satellites (47).

Four regions in Ap-CH and one in Pf-FR exhibit synteny with sequences from known *Arsenophonus* extrachromosomal elements (Fig. 2A, black blocks, and Table S2). Plasmid genes are present both inside and outside of these regions in both assemblies (Table S3). Phaster predicted 18 and 26 phage regions for Ap-CH and Pf-FR, respectively, representing 17%–20% of the genomes (Fig. 2A, green, blue, and red blocks). These repetitive regions likely contributed to the assembly breaks, as indicated by their locations at scaffold ends (Fig. 2A, inner ribbons). Most of these regions are short and predicted as “incomplete,” harboring only a few viral genes (Fig. 2B). Their status should thus be taken with caution since they may represent phage relics or partial sequences. Conversely, eight regions for each strain are classified as “questionable” and even “intact” and could represent functional phages.

Importantly, one intact phage DNA polymerase was detected in both hybrid assemblies (ApCH_3206 and PfFR_1270). They represent homologs of p45 (>87% aa id.), the polymerase of *Acyrtosiphon pisum* secondary endosymbiont (APSE) phages that infect the pea aphid endosymbiont *Hamiltonella defensa* (*Gammaproteobacteria*) (54). Similar p45 homologs are found in other *Arsenophonus* strains (Fig. 2C) which also harbor APSE-like phages that can be part of modules forming mosaics with unrelated phages (54). Likewise, in Ap-CH and Pf-FR, numerous APSE genes are found throughout the assemblies (Fig. 2B, column “APSE”), and the p45 homologs are located within clusters syntenic to APSE replicative modules 1 and 2 (Fig. S2). In the case of Ap-CH, these modules are part of the intact phage region 11 on scaffold 9, which also contains the incomplete phage region 10 and several plasmid genes (Table S3). This scaffold exhibits a high GC content of 43% and a high sequencing coverage (Fig. 2D), indicating that it may be a multi-copy phage-plasmid element. In Pf-FR, the APSE replicative modules are located within the incomplete phage region 21 on scaffold 24, which shows no discrepancy in coverage and GC content compared to other scaffolds (Fig. 2D), and might hence represent a single-copy region. These modules could be associated with an integrated prophage since the rest of the scaffold consists of non-viral genes. Unfortunately, the APSE replicative modules of both assemblies are located at scaffold ends (Fig. S2), limiting further characterization.

## Phylogenomic relationships

Both maximum-likelihood and Bayesian phylogenomic analyses combining Ap, Pf, and other facultative *Arsenophonus* symbionts produced congruent tree topologies (Fig. 3; Fig. S3, respectively). Specifically, they revealed two basal clades within the genus (“I” and “II”), consistent with previous studies (48, 49). Clade I accommodates the hymenoptera-infecting parasitic species *Nasoniae* and *Apicola* as well as the butterfly-infecting strain aPb. Clade II, or “*Triatominarum*” clade (43), comprises hemipteran endosymbionts, some of which (strains ARAF, Asia-II-3, Hangzhou) are vertically transmitted and likely provide their hosts with B vitamins, similar to P-endosymbionts (Table 1) (39, 51, 52).

In both trees, Ap and Pf unambiguously represent distinct species. The Ap strains are deeply embedded within the *Triatominarum* clade (bootstrap support: 100%, posterior probability: 1), whose members can be considered as strains of the same “species complex” since they share very high ANI levels (>95% [55]). In contrast, Pf is an early-branching member of clade I (bootstrap support: 98%, posterior probability: 1) and shares low ANI levels (<91%) with the other strains, indicating that it represents a distinct species within the genus. Additional analyses including the P-endosymbionts
<table><caption>TABLE 1 Genomes of *Arsenophonus* strains used in this study</caption><thead><tr><th>Strain</th><th>Host species</th><th>Transmission mode<sup>a</sup></th><th>Phenotype</th><th>Genome size (Mb)</th><th>Assembly status<sup>b</sup></th><th>Plasmid number</th><th>Accession</th><th>Reference</th></tr></thead><tbody><tr><td colspan="9"><strong>Vector-borne phytopathogens</strong></td></tr><tr><td>Ca. Ap-CH</td><td>P. leporinus</td><td>M</td><td>Plant pathogen</td><td>2.9</td><td>S</td><td>NA</td><td>GCA_047291415.1</td><td>This study</td></tr><tr><td>Ca. Ap-FR</td><td>P. leporinus</td><td>M</td><td>Plant pathogen</td><td>2.6</td><td>S</td><td>NA</td><td>GCA_047291325.1</td><td>This study</td></tr><tr><td>Ca. Pf-FR</td><td>C. wagneri</td><td>H</td><td>Plant pathogen</td><td>2.8</td><td>S</td><td>NA</td><td>GCA_047291315.1</td><td>This study</td></tr><tr><td colspan="9"><strong>Facultative endosymbionts</strong></td></tr><tr><td>A. nasoniae aNv-CAN</td><td>Nasonia vitripennis</td><td>M</td><td>Male-killing</td><td>5.1</td><td>C</td><td>20</td><td>GCF_029873515.1</td><td>(43)</td></tr><tr><td>A. nasoniae aNv-CH</td><td>Nasonia vitripennis</td><td>M</td><td>Male-killing</td><td>4.7</td><td>C</td><td>16</td><td>GCF_029873535.1</td><td>(37)</td></tr><tr><td>A. nasoniae aNv-DSM</td><td>Nasonia vitripennis</td><td>M</td><td>Male-killing</td><td>3.6</td><td>S</td><td>NA</td><td>GCF_000429565.1</td><td>(45)</td></tr><tr><td>A. nasoniae aNv-FIN</td><td>Nasonia vitripennis</td><td>M</td><td>Male-killing</td><td>5.0</td><td>C</td><td>17</td><td>GCF_004768525.1</td><td>(41)</td></tr><tr><td>A. nasoniae aNv-UK</td><td>Nasonia vitripennis</td><td>M</td><td>Male-killing</td><td>4.7</td><td>C</td><td>16</td><td>GCF_029873555.1</td><td>(43)</td></tr><tr><td>A. nasoniae aPv</td><td>Pachycrepoideus vindemmiae</td><td>V</td><td>Unknown</td><td>4.3</td><td>C</td><td>18</td><td>GCF_029873495.1</td><td>(43)</td></tr><tr><td>A. nasoniae alh</td><td>Ixodiphagus hookeri</td><td>V</td><td>Unknown</td><td>3.7</td><td>C</td><td>7</td><td>GCF_029873455.1</td><td>(43)</td></tr><tr><td>A. apicola aApi-US</td><td>Apis mellifera</td><td>H</td><td>Putative pathogen</td><td>3.6</td><td>C</td><td>6</td><td>GCF_020268605.1</td><td>(48)</td></tr><tr><td>A. apicola aApi-CH</td><td>Apis mellifera</td><td>H</td><td>Putative pathogen</td><td>3.3</td><td>S</td><td>NA</td><td>GCF_903968575.1</td><td>(37)</td></tr><tr><td>A. apicola aApi-AU</td><td>Apis mellifera</td><td>H</td><td>Putative pathogen</td><td>3.2</td><td>C</td><td>5</td><td>GCF_029906405.1</td><td>(43)</td></tr><tr><td>A. triatominarum ATi</td><td>Triatoma infestans</td><td>V</td><td>Unknown</td><td>3.9</td><td>S</td><td>NA</td><td>GCF_001640365.1</td><td>(42)</td></tr><tr><td>Arsenophonus sp. aPb</td><td>Polyommatus bellargus</td><td>H</td><td>Unknown</td><td>3.5</td><td>C</td><td>4</td><td>GCF_029873475.1</td><td>(49)</td></tr><tr><td>Ca. Arsenophonus sp. ENCA</td><td>Entylia carinata</td><td>NA</td><td>Unknown</td><td>3.2</td><td>S</td><td>NA</td><td>GCF_002287155.1</td><td>(50)</td></tr><tr><td>Ca. Arsenophonus sp. ARAF</td><td>Aleurodicus floccissimus</td><td>V</td><td>Provides B vitamins</td><td>3.0</td><td>S</td><td>NA</td><td>GCF_900343025.1</td><td>(39)</td></tr><tr><td>Ca. Arsenophonus sp.</td><td>Nilaparvata lugens</td><td>V</td><td>Likely provides B vitamins</td><td>2.9</td><td>S</td><td>NA</td><td>GCF_000757905.1</td><td>(51)</td></tr><tr><td colspan="9"><strong>Hangzhou</strong></td></tr><tr><td>Ca. Arsenophonus sp. Ash</td><td>Aphis craccivora</td><td>V</td><td>Mediates plant host range</td><td>2.4</td><td>C</td><td>1</td><td>GCF_013460135.1</td><td><sup>d</sup></td></tr><tr><td>Ca. Arsenophonus sp. Asia-II-3</td><td>Bemisia tabaci</td><td>V</td><td>Provides B vitamins</td><td>2.3</td><td>S</td><td>NA</td><td>GCF_004118055.1</td><td>(52)</td></tr><tr><td>Ca. Arsenophonus sp. MEDQ21</td><td>Bemisia tabaci</td><td>V</td><td>Likely provides B vitamins</td><td>1.9</td><td>S</td><td>NA</td><td>GCF_902713415.1</td><td>–</td></tr><tr><td colspan="9"><strong>P-endosymbionts</strong></td></tr><tr><td>Ca. A. melophagi ArsM</td><td>Melophagus ovinus</td><td>V</td><td>Provides B vitamins</td><td>1.1</td><td>S</td><td>NA</td><td>NA<sup>c</sup></td><td>(34)</td></tr><tr><td>Ca. Arsenophonus sp. CjNOSY1</td><td>Ceratovacuna japonica</td><td>V</td><td>Provides riboflavin</td><td>0.9</td><td>C</td><td>0</td><td>GCA_024349725.1</td><td>(53)</td></tr><tr><td>Ca. A. lipoptenae CB</td><td>Lipoptena cervi</td><td>V</td><td>Provides B vitamins</td><td>0.8</td><td>C</td><td>0</td><td>GCF_001534665.1</td><td>(38)</td></tr><tr><td>Ca. Arsenophonus sp. ARAD</td><td>Aleurodicus dispersus</td><td>V</td><td>Provides B vitamins</td><td>0.6</td><td>C</td><td>0</td><td>GCF_900343015.1</td><td>(39)</td></tr></tbody></table>

produced similar tree topologies, albeit with reduced branch support due to long-branch attractions caused by the reduced genomes (except in the tree produced using MrBayes where almost all branches were fully supported (posterior probability: 1; Fig. S4).

## Basal biosynthetic capacities

The diversity of lifestyles and genome sizes among *Arsenophonus* strains is reflected by substantial variations in basal biosynthetic capacities (Fig. 4, predicted based on Kyoto Encyclopedia of Genes and Genomes [KEGG] pathways). There is a clear erosion gradient from the well-furnished genomes of the culturable strains from clade I to the reduced genomes of P-endosymbionts. Notably, the strain Hangzhou from the *Triatominarum* clade exhibits a basal biosynthetic repertoire almost equivalent to that of clade I strains, consistent with the idea that the genus ancestor was a free-living insect-associated bacterium (37). Ap and Pf have experienced an intermediate level of erosion and have lost many biosynthetic pathways. Nevertheless, these strains have retained more capacities than most members of the *Triatominarum* clade, showing a repertoire highly

<footnote><sup>a</sup>H: horizontal; V: vertical; M: mixed.</footnote>

<footnote><sup>b</sup>S: scaffolds; C: closed.</footnote>

<footnote><sup>c</sup>Genome available online: http://users.prf.jcu.cz/novake01.</footnote>

<footnote><sup>d</sup>–, no reference available for this genome.</footnote>
<figure><img src="image_5.png" /></figure>

**FIG 2** Viral content in Ap-CH and Pf-FR. (A) Schematic representation of the genome assemblies of Ap-CH (top) and Pf-FR (bottom). Each assembly is represented as a circle accommodating the scaffolds. Black blocks indicate regions syntenic to *Arsenophonus* extrachromosomal elements. Other colored blocks represent phage regions (>1 kb) identified by Phaster. Inner ribbons connect syntenic blocks (>5 kb). (B) Composition of the phage regions. VP: number of viral proteins; HP: number of hypothetical non-viral proteins; APSE: number of APSE proteins. The barplot represents the size of each region in kb. (C) Maximum-likelihood phylogenetic tree of the p45 homologs, built with the model JTT + F + I + G4. Black circles on branches indicate >50% bootstrap support. The scale is given in substitutions per site. Strains are color-coded as in Fig. 1B. Polymerases from *Citrobacter koseri* (WP_200053203.1) and *Salmonella enterica* (EF59905187.1) were used to root the initial tree. (D) Normalized long-read (LR) and short-read (SR) coverages for the scaffolds of Ap-CH (left) and Pf-FR (right). The color scale indicates the difference in GC content from the assembly average.

similar to the strain ENCA. However, considering that both genomes are incomplete, it is
possible that their metabolic repertoires were underestimated.

In terms of energy transfer, Ap and Pf have intact NADH quinone oxidoreductase and
cytochromes, but the F-type ATPase is incomplete in the Ap strains. For the carbohydrate
metabolism, both Ap and Pf can rely on the glycolysis and non-mevalonate pathway, and
the Ap strains also have a complete tricarboxylic acid cycle. Interestingly, the phytopa-
thogenic strains have retained the capacity to ferment sugars into acetate, while they
have lost other mixed acid pathways. Ap and Pf can produce most cofactors, and this
capacity could be involved in nutritional symbiosis with their cixiid hosts feeding on
nutrient-limited sap (39, 56). On the other hand, the phytopathogenic strains have lost
the ability to produce numerous amino acids, indicating that these compounds must be
obtained from the hosts. In particular, glutamate and aspartate might be easily obtained
from plants as they represent the most abundant amino acids in the phloem sap (57).
Ap and Pf encode several phosphotransferase systems, many ABC transporters, and a
siderophore (Fig. 4; Table S4). This capacity for nutrient acquisition could create phloem
imbalances and partially explain the plant symptoms associated with SBR and SMC, as
suggested for other phloem-infecting bacteria (58–60).

Genes for virulence and symbiosis

Previous studies have evidenced a complex genetic arsenal dedicated to the virulence
and symbiotic lifestyle of *Arsenophonus* strains in their insect hosts (43, 45). Like for the
basal capacities, a gradient in this gene arsenal is observed among the strains (Fig. 5). As
facultative endosymbionts, Ap and Pf have retained a large portion of this arsenal, which
<figure><img src="image_6.png" /><figcaption>FIG 3 Phylogenomic and taxonomic analyses of facultative endosymbiotic *Arsenophonus* strains. Left: ML phylogenomic tree based on 118 single copy protein-coding genes. All branches have >90% bootstrap support. Genes from *P. stuartii* and *P. mirabilis* were used to root the initial tree. The tree scale represents 0.01 substitution per site. Basal clades are indicated by Latin numbers. Right: matrix of pairwise average nucleotide identity (ANI; in percent). Strains are color-coded as in Fig. 1B. The host ranges are given in parentheses.</figcaption></figure>

is likely required during the infection of cixiids in order to survive in the gut, cross cellular barriers, evade immunity, and reach the eggs and salivary glands (45).

Ap and Pf possess the genetic capacities to produce lipopolysaccharides and peptidoglycan, consistent with studies reporting typical gram-negative cell walls (61–63). Both strains also harbor genes involved in adhesion but lack the capacity for flagellum, pilus, and chemotaxis, which may explain their fastidious *in planta* multiplication (14, 63). The pathways for general secretion and twin arginine translocation are both conserved in Ap and Pf. These bacteria also have a defensin ABC transporter, and the type 3 secretion system (T3SS) is complete for both Ap strains and almost complete for Pf-FR (lacking only *sctQ*). Conversely, other secretion systems appear absent or incomplete. A few toxins and effectors are predicted in Ap and Pf genomes, and those probably play a role during the insect stage since they are also encoded by non-phytopathogenic strains.

## Genes specific to Ap and Pf

The molecular mechanisms underlying the phytopathogenicity of Ap and Pf have not been studied, and it is unclear whether they use similar or distinct strategies. Nevertheless, considering the unique lifestyle of Ap and Pf in the genus, it is imaginable that the genes involved in phloem colonization are lacking in other *Arsenophonus* strains. To identify such "phytopathogen-specific" genes, an orthology clustering analysis was first conducted, resulting in 93% of the *Arsenophonus* pangenome clustered into 4,947 orthogroups (OGs) (Fig. 6A). The core-genome was relatively small, comprising only 3,337 genes in 119 OGs. Importantly, with the exception of ATi and to a lesser extent ENCA, the proportions of strain-specific genes (i.e., unassigned genes and genes in strain-specific OGs) were quite low in all strains (Fig. 6B). Only 159 genes were specific to Ap and/or Pf (Fig. 6C). Of these, some represent regulatory and mobile genetic elements, but the majority encode short hypothetical proteins (HPs) with no known homolog or distant homologs in diverse enterobacteria (Table S5).

Importantly, eight OGs containing 35 genes were exclusively shared by Pf-FR and Ap-CH/FR in the genus (Fig. 6C; Table 2). Proteins from OG3381 are short Zn metallopeptidases with no known homolog. OG2682 corresponds to phage lysis proteins with distant homologs in diverse enterobacteria (Fig. 7A). OG3842 are homologs of the transcriptional repressor *DicA*, with the closest homologs found in *Enterobacter*,
<figure><img src="image_7.png" /><figcaption>**FIG 4** Biosynthetic capacities and transmembrane transport systems among *Arsenophonus* strains. Rows indicate the pathway products or the transported substrates, while columns indicate the strains. Colors indicate the completeness according to the KEGG database. Only pathways complete in at least one strain are shown. Strains are color-coded as in Fig. 1B. B6: vitamin B6; Coa: coenzyme A; Cyto bd: cytochrome bd complex; Cyto O: cytochrome O ubiquinol oxidase; NaNQR: Na⁺-NADH-ubiquinone oxidoreductase; NAD: nicotinamide adenine dinucleotide; NQO: NADH quinone oxidoreductase; MA: mixed acid fermentations; GNG: gluconeogenesis GlcNAc; N-acetylglucosamine. GalNAc: N-acetylgalactosamine. N-acetylmuramic acid: MurNAc; LPS: lipopolysaccharide. MEX-DOXP: non-mevalonate pathway.</figcaption></figure>

the endophyte *Kosakonia pseudosacchari* and the two phytopathogens *Erwinia billingiae* and *Pectobacterium brasiliense* (Fig. 7B). Likewise, OG4231 are putative chaperones with homologs found in both phytopathogenic (*Erwinia tracheiphila* and *Pantoea stewartii*) and non-phytopathogenic enterobacteria (Fig. 7C). Proteins from OG3860 contain the Domain of Unknown Function 3757 (DUF3757) with distant homologs in
the phytopathogenic genera *Ralstonia*, *Pseudomonas*, *Lonsdalea*, and *Burkholderia* (Fig. 7D). Similar homologs were also identified among unassigned genes (ApCH_1314 and PfFR_1763, Table S5).

Curiously, OG3615, OG3866, and two unassigned genes from Pf show homology to genes from the psyllid endosymbiont *Ca. Kirkpatrickella diaphorinae* (64). OG3615 encodes O-glycosyl hydrolases of the CAZyme family GH30. The closest homologs are found in the poorly characterized subfamily GH30-6 (65), with the sole characterized member belonging to *Parabacteroides gordonii* (WP_028726386.1). This enzyme is active on pNP-β-D-cellobioside (66), indicating that OG3615 could represent cell wall-degrading enzymes (CWDEs) targeting plant polysaccharides. Intriguingly, homologs are encoded by arthropod endosymbionts (*Yokenella* and *Rickettsiella*), gut commensals (*Parabacteroides* and *Bacteroides*), and also a fungus gnat (*Bradysia odoriphaga*) and several mites of the genera *Leptotrombidium* and *Dinothrombium* (Fig. 7E). OG3866 represents hydrolases of unknown function distantly related to proteins from diverse non-phytopathogenic enterobacteria (Fig. 7F). An unassigned gene from Pf-FR (PfFR_3139) is also closely related to this OG. These hydrolases putatively represent lipases/esterases as they carry the motif "GxSxG" (67).

Strikingly, OG3863 represents a set of C1A cysteine peptidases closely related to xylellain (AE003869, 65% aa id.), a papain-like peptidase described in *Xylella fastidiosa* (68). Hence, OG3863 and related proteins are hereafter dubbed "xylellain-like peptidases" (XLPs). Ap-CH harbors one version of XLP (ApCH_300, "XLP1" in Fig. 7G) that is highly similar to two copies present in Pf-FR (PfFR_1221 and PfFR_2961). A second, distinct XLP was found among the unassigned genes of Ap-CH (ApCH_1315, "XLP2"), and two additional XLP pseudogenes were found in Pf-FR (PfFR_663 and PfFR_664). Importantly, we detected XLPs in distantly related phytopathogenic *Pseudomonadota: Erwinia psidii*, *X. fastidiosa* and *X. taiwanensis*, *Xanthomonas albilineans* and "pseudalbilineans," and *Ca. Liberibacter africanus* (Fig. 7G). A homolog is also encoded by the leafhopper endosymbiont *Symbiopectobacterium purcellii*. The XLPs are closely related to enzymes from the phytopathogenic genera *Dickeya* and *Musicola* and from pectinolytic bacteria of the genus *Prodigiosinella* (69). Distant homologs are found in the endophyte *Liberibacter crescens* and nodule-forming *Mesorhizobium* (Fig. 7G).

Given their possible roles during the plant stage of Ap and Pf, the genomic context of the CWDE and XLP genes of Ap and Pf was further analyzed, revealing their presence inside or in close proximity of phage regions (Table S6; Fig. S5 and S6). Interestingly, a CWDE is found on Ap-CH scaffold 9, the putative phage-plasmid element harboring APSE replicative modules. Strikingly, a cluster of nine genes is shared between Ap-CH scaffold 14 and Pf-FR scaffold 18 in proximity of XLP genes. This cluster comprises a phage antirepressor, several HPs, and genes involved in phage integration/excision (excisionase, recombinase, and integrase; Fig. S6). Additional BLAST searches on this cluster indicated similar genes on chromosomes and plasmids in multiple strains of *A. nasoniae* and *A. apicola* (data not shown). Hence, genes from this cluster seem to have propagated across multiple *Arsenophonus* species, and it is tempting to speculate that they mediated the exchange of XLP between Ap and Pf.

## In silico predictions and gene expression analyses

Several *in silico* prediction tools were used to assess potential secretion and subcellular localization for the longest representative of each OG exclusively shared by Ap and Pf (Fig. 7H). A non-classical secretion was predicted by SecretomeP for the chaperone, CWDE, XLPs, and DUF3757. SignalP detected a signal peptide for the DUF3757 product, indicating a putative Sec-dependent secretion. Transmembrane regions were evidenced by ModHMM in the Zn metallopeptidase and phage lysis protein, and PSORTb predicted a localization in the outer membrane for the lipase/esterase.

The expression levels of several OGs, in particular the hydrolytic enzymes and DUF3757, were determined by real-time quantitative PCR (RT-qPCR) analyses on RNA samples extracted from Ap-infected stems of periwinkle (*Catharanthus roseus*) and
<figure><img src="image_8.png" /><figcaption>FIG 5 Gene arsenal involved in virulence/symbiosis in the *Arsenophonus* strains. Rows indicate the pathway/protein products, and columns represent the strains. Colors indicate the pathway completeness according to the KEGG database. Only pathways complete or near-complete in at least one strain are shown. Strains are color-coded as in Fig. 1B. TA: tight adherence; TAT: twin arginine translocation; Sec: general secretion; SS: secretion system.</figcaption></figure>

female *P. leporinus* bodies (Fig. 7I). Importantly, expression profiles for all tested genes were similar regardless of the house-keeping gene used as reference (*gyrA* or *rpoB*). Significantly higher expressions *in planta* were detected for the DUF3757, lipase/esterase and CWDE (Wilcoxon rank sum test, all *P* < 0.05), and to a lesser extent XLP1 (Wilcoxon rank sum test, *P* < 0.01). In contrast, XLP2 expression was much higher in the insects than *in planta* (Wilcoxon rank sum test, *P* < 0.05).

DISCUSSION

For this first genomic analysis of Ap and Pf, three assemblies were obtained from
planthopper metagenomes. Despite the use of long-read sequencing, the genomes
remain at the scaffold level, partly due to the presence of repetitive viral sequences.
Axenic cultures of Ap and Pf will probably be required to obtain longer sequences and
<figure><img src="image_9.png" /><figcaption>**FIG 6** Identification of genes specific to Ap and Pf in the genus. (A) Orthology clustering of the *Arsenophonus* pangenome. (B) Distribution of genes in each *Arsenophonus* strain. Strains are color-coded as in Fig. 1B. (C) Venn diagram of genes only found in Ap and Pf.</figcaption></figure>

achieve gap closure, and this will be valuable to further explore the putative phage-plasmids. Specifically, the APSE modules deserve more scrutiny as they seem widespread in *Arsenophonus* and are known to play important biological roles in other insect endosymbionts (70, 71).

Comparisons of global genomic features and biosynthetic capacities indicate that Ap and Pf are highly similar to several other *Arsenophonus* strains, especially facultative endosymbionts of sap-sucking hemipterans. Our results show that Ap and Pf harbor very low fractions of strain-specific genes, suggesting that the shift from an insect-only to a dual insect-phytopathogenic lifestyle occurred with only a few gene changes. Phylogenomic and ANI analyses further support a recent shift at least for Ap, as evidenced by its very close relationship with other strains from the *Triatominarum* clade. The situation may be more complex for Pf, which represents a distinct species basal to clade I with no other closely related strains known to date. Therefore, it will be interesting to see whether Pf-like strains will be detected in other insect or plant hosts in future research. Importantly, Ap and Pf share a small set of eight OGs unique in the *Arsenophonus* pangenome. Of these OGs, little can be speculated about the putative chaperones, phage lysis proteins, metallopeptidases, DUF3757, and lipases/esterases, although similar proteins have been previously characterized as virulence factors in bacterial phytopathogens (72–74). In contrast, a role in the plant stage of Ap and Pf can be more confidently assumed for the putative CWDEs and XLPs.

The shared CWDEs belong to the large glycoside hydrolase family GH30, which includes enzymes used by phytopathogens to degrade cellulose, hemicellulose, and pectin (75–77). Both Ap and Pf cause physiological alterations in plants including SE

<table><caption>**TABLE 2** Description of the OGs shared exclusively by Ap and Pf among *Arsenophonus* strains<sup>a</sup></caption><thead><tr><th rowspan="2">OG</th><th rowspan="2">Size (aa)</th><th rowspan="2">Domain<br>(E value)</th><th rowspan="2">Annotation</th><th colspan="4">Best BlastP hit</th></tr><tr><th>Accession</th><th>Size (aa)</th><th>ID<br>(%)</th><th>Species/strain</th></tr></thead><tbody><tr><td>2682</td><td>150</td><td>PF03245 (6<sup>e-27</sup>)</td><td>Phage Rz lysis protein</td><td>WP_249540969.1</td><td>154</td><td>41.2</td><td>Escherichia coli E15</td></tr><tr><td>3381</td><td>109</td><td>COG2321 (2<sup>e-3</sup>)</td><td>Zn metallopeptidase</td><td>No hit</td><td></td><td></td><td></td></tr><tr><td>3615</td><td>533</td><td>PF14587 (1.2<sup>e-11</sup>)</td><td>O-glycosyl hydrolase</td><td>WP_319806414.1</td><td>532</td><td>86.5</td><td>Ca. K. diaphorinae</td></tr><tr><td>3842</td><td>132</td><td>PRK09706 (6<sup>e-8</sup>)</td><td>Repressor DicA</td><td>WP_265910051.1</td><td>240</td><td>60.0</td><td>E. billingiae LS-1</td></tr><tr><td>3860</td><td>142</td><td>DUF3757 (3<sup>e-17</sup>)</td><td>Unknown</td><td>CBJ39888.1</td><td>140</td><td>53.0</td><td>Ralstonia solanacearum CMR15</td></tr><tr><td>3863</td><td>271</td><td>COG4870 (2<sup>e-45</sup>)</td><td>C1A cysteine peptidase</td><td>WP_233592545.1</td><td>271</td><td>76.7</td><td>E. psidii LPF 681</td></tr><tr><td>3866</td><td>504</td><td>PF09994 (3<sup>e-18</sup>)</td><td>Alpha/beta hydrolase</td><td>WP_319806123.1</td><td>590</td><td>40.4</td><td>Ca. K. diaphorinae</td></tr><tr><td>4231</td><td>99</td><td>COG2833 (9.2<sup>e-1</sup>)</td><td>Molecular chaperone</td><td>WP_233480985.1</td><td>96</td><td>73.7</td><td>E. tracheiphila MDcuke</td></tr></tbody></table>

<footnote><sup>a</sup>Blastp results are shown for the longest gene of each OG.</footnote>
<figure><img src="image_10.png" /><figcaption>FIG 7 Characterization of a subset of OGs specific to Ap and Pf in the *Arsenophonus* pangenome. (A–G) Maximum-likelihood phylogenetic trees. Only the closest homologs are shown in the trees. Black circles on branches indicate >50% bootstrap support. The black scale bars represent one substitution per site. Numbers in brackets indicate the number of genes present in the collapsed clades. *Arsenophonus* genes are highlighted in green. Arthropod genes are highlighted in blue. Genes from *Ca. K. diaphorinae* are highlighted in yellow. The trees were built using the models WAG + I + G4, JTT + I + G4, JTT + G4, VT + I + G4, LG + F + I + G4, VT + I + G4, and WAG + I + G4, respectively. (H) *In silico* predictions for secretion and subcellular localization signals. (I) Box-plots of relative expression (in log2) for selected genes of Ap-CH in insect (red) and plant (green) tissues. Expression levels (n = 5) are given relative to the expression of gyrA (top) or rpoB (bottom). Single and double asterisks indicate *P* < 0.05 and < 0.01, respectively, based on Wilcoxon Rank Sum tests.</figcaption></figure>

obstruction (61, 63), which might be targeted by the CWDEs to facilitate systemic invasion. These enzymes might also have nutritional functions through the degradation of plant polysaccharides into consumable forms. This is supported by the presence of homologs in other arthropod endosymbionts and gut commensals. Surprisingly, homologs of these CWDEs are also found in several mites and a fungus gnat, indicating probable cross-kingdom HGT. Notably, HGT between bacteria and arthropods has been previously documented and includes genes crucial for plant adaptation (78, 79).

The occurrence of XLPs in diverse phytopathogenic *Pseudomonadota* is reported here for the first time and clearly suggests a role of these enzymes in plant tissue colonization. Although a xylellain from *X. fastidiosa* has been purified and its structure resolved, its substrate remains unknown (68). The XLPs represent a sister clade of enzymes found in soft rot agents, endophytes and rhizobia, suggesting that plant proteins represent targets of all these C1A cysteine peptidases. Importantly, all XLP-encoding phytopathogenic bacteria infect the xylem, phloem, or both tissues (80–83). Therefore, these enzymes may specifically act on vasculature proteins. The XLPs are hypothetically secreted, similar to several different cysteine peptidases characterized in other phytopathogens, which are T3SS effectors targeting the plant immunity (84). However, low homology between these effectors and XLPs precludes further comparisons. Intriguingly,
an XLP is found in *S. purcellii* SyEd1T, a leafhopper endosymbiont with no known plant stage (85). It should be noted that the closely related strain BEV is plant-transmitted (86), and similar strains have been recently detected in potato (87), indicating that SyEd1T might also have a plant stage. Interestingly, in contrast to Ap XLP1, Ap XLP2 appears to be more expressed in insect than plant tissues. This could be associated with a specific role in the plant adaptation of the cixiid vector through the digestion of phloem proteins, as hypothesized for peptidases encoded by sap-sucking hemipterans and their endosymbionts (88–90). Notably, most if not all the phytopathogenic bacteria encoding XLPs are insect-transmitted, suggesting that XLPs could have multiple roles, both for plant colonization and for the insect vector.

It is likely that some OGs found exclusively in Ap and Pf were transferred from one strain to the another by direct HGT, which is further supported for the XLPs given their shared genomic context. These HGT events might have taken place in a co-infected *C. wagneri* specimen as this planthopper can host both phytopathogens. Prior to these exchanges, some shared OGs were likely acquired from other *Pseudomonadota* (Fig. 7A through G). Importantly, Ap and Pf might have developed the ability to survive in the phloem before acquiring phytopathogenic traits. Indeed, diverse non-phytopathogenic endosymbionts use SEs for horizontal transmission between hemipterans (91–94), and such a plant-mediated transmission would explain the diversity of sap-sucking insects harboring closely related strains of the *Triatominarum* clade. Through this capacity to survive in the phloem, Ap and/or Pf might have been in contact with and acquired genes (e.g., XLP) from other phytopathogens. Noteworthy, several genes seem to have been exchanged with *Ca. K. diaphorinae*, an endosymbiont of the *Liberibacter* vector *Diaphorina citri*. HGT with this bacterium could have occurred in a co-infected hemipteran. HGT in planta is also imaginable, as *Ca. K. diaphorinae* is closely related to *Asaia*, a genus of arthropod endosymbionts horizontally transmitted via plants (95, 96). Importantly, to date, the functional roles of most *Arsenophonus* strains from sap-feed-ing Hemipterans remain unexplored, with only a few documented exceptions (31–33, 39). This suggests the possibility that other strains may have the potential to induce unidentified plant diseases. Therefore, further investigation into the biology and ecology of these poorly characterized strains from the *Triatominarum* clade is imperative.

In conclusion, our results strengthen the idea of an “insect first” scenario during the evolution of Ap and Pf toward becoming vector-borne phytopathogens. A limited number of HGT events probably acted as key mechanisms in this lifestyle transition, which aligns with recent studies associating HGT with the shift from a non-vascular to a vascular phytopathogen (97, 98). In particular, the acquisition and exchange of CWDEs and XLPs might underlie the double emergence of phytopathogenicity in *Arsenophonus*. Biochemical characterization of these enzymes and their substrates will be required to further support this hypothesis.

# MATERIALS AND METHODS

## DNA extraction for insect metagenomes

For Pf-FR, adult *C. wagneri* specimens were collected in a strawberry field in Dordogne (France) in June 2019. For Ap-FR, adult *P. leporinus* specimens were collected during the first SBR outbreak in Burgundy (France) in the early 2000s. For Ap-CH, adult *P. leporinus* specimens were collected in a sugar beet field in Gilly (Switzerland) in 2020. Insects from France were surface sterilized by serial washes in 20% bleach, sterile water, 70% ethanol, and sterile water. Individual insects were then ground in 400 µL 2% cetyltrimethylammonium bromide (CTAB) buffer (2% CTAB, 2% polyvinylpyrrolidone [PVP] K40, 1.4 M NaCl, 20 mM EDTA, 100 mM Tris-HCl, and 0.02% β-mercaptoethanol) and incubated at 65°C for 1.5 hours. The DNA was then extracted as previously described (63). No surface sterilization was performed for insects collected in Switzerland, and they were stored in 70% ethanol at -20°C until further use. For these specimens, DNA was extracted using a 3% CTAB protocol (14). All DNA samples were treated with 20 µg RNase A at 37°C for 30 min,
followed by chloroform/isoamyl alcohol extraction and isopropanol precipitation. DNA pellets were resuspended in 40 µL of 1x TE buffer for samples from France, whereas 100 µL of DNase-free water was used for Swiss samples.

## Quantification of bacterial titre

Bacterial titers were estimated in DNA samples by qPCR. For Pf-FR and Ap-FR, samples were normalized to 50 ng/µL to compare Ct values between samples. All samples were tested in duplicates with the previously published primers and FAM-labeled TaqMan probes targeting the *spoT* gene of Pf (4). For Ap-FR, primers and probe (SBR-F/R/FAM) are listed in Table S1. Reactions were performed as previously described (63). For samples from Switzerland, a similar qPCR assay was performed using a different protocol (14). Samples with the highest bacterial titers were chosen for high-throughput sequencing.

## Genome sequencing and assembly

For Ap-CH and Pf-FR, long-read sequencing libraries were prepared using the Ligation Sequencing kits SQK-LSK 110 and 109 (Oxford Nanopore Technologies, UK), respectively. Each library was sequenced on an R9.4 flow cell on the MinION sequencer, producing 16 and 6 Gb of data, respectively. Base calling was done using Guppy v5.0.11 (in high-accuracy mode) and only ≥500 bp reads passing the Q7 quality filter were retained. In addition, 2 × 150 bp paired-end reads were obtained from an Illumina Novaseq platform (Macrogen), producing 50 and 476 million reads for Ap-CH and Pf-FR, respectively. The reads were quality-trimmed using Trimmomatic v0.38 (99), retaining only reads ≥Q30.

Reads belonging to *Arsenophonus* were extracted from the data sets via mapping (using Minimap2 v2.15 [100]) for the long reads and bwa mem v0.7.17 for the short reads, respectively) against a database of all published *Arsenophonus* genomes (as of September 2023; Table 1). Several hybrid assemblers were then tested: Masurca v4.0.7 (101), Spades v3.15.1 (102), and Unicycer v0.4.9 (103). For Pf-FR, the most contiguous assembly was obtained using Masurca and was further polished with short reads using Polca (part of the Masurca package) and scaffolded using two iterations of SSPACE v2.1.1 (104). For Ap-CH, the most contiguous assembly was obtained using Unicycer, and this assembly was also scaffolded using two iterations of SSPACE.

The genome of Ap-FR was assembled only from short reads since the samples had been stored for more than 15 years prior to DNA extraction, resulting in highly fragmented DNA precluding long-read sequencing. Illumina sequencing was performed as mentioned above, producing 486 million 150 bp paired-end reads. Following quality trimming, the reads were assembled using Megahit v1.2.9 (105) and contigs belonging to Ap were identified using BlobTools v1.1.1 (https://github.com/DRL/blobtools) and mapping against a database of all published *Arsenophonus* genomes using bwa mem. The contigs were scaffolded with Redundans (106) using the Ap-CH assembly as reference, followed by one iteration of SSPACE and Gapfiller v2.1.2 (107). Coverages were determined with Mosdepth v0.3.4 (108). CheckM v1.1.6 (109) was used to assess genome completeness. Synteny blocks were identified with Sibelia v3.0.7 (110).

## Functional annotation

The three assemblies were annotated using the National Center for Biotechnology Information (NCBI) PGAP v2023-05-17.build6771. Clusters of Orthologous Groups (COG) categories and KEGG ontology terms were determined using eggNOG-mapper v2 (111). Phage regions were detected with Phaster (112). Schematic representations of genetic modules were obtained with Clinker (113). Circular representations of the scaffolds were produced with the R package Circlize v0.4.10 (114). Heatmaps were constructed using the ComplexHeatmap R package (115). Biosynthetic capacities were evaluated using KEGG Decoder v1.3 (116), TXSscan v1.1.0 (117), and antiSMASH v7.beta (118). Toxins and effectors were detected by Blastp searches in a local database of *Arsenophonus* proteomes.
## Phylogenomics and taxonomy

Orthofinder v2.5.4 (119) was used to identify single-copy orthologs shared between Pf-FR, Ap-CH, Ap-FR, and 22 *Arsenophonus* genomes published as of September 2023 (Table 1). *Proteus mirabilis* HI4320 (GCF_000069965.1) and *Providencia stuartii* MRSN 2154 (GCF_000259175.1) were used as outgroups. Amino acid sequences of each gene were aligned using Muscle v3.1.31 (120), followed by concatenation with geneStitcher.py (https://github.com/ballesterus/Utensils/blob/master/geneStitcher.py). The resulting multi-gene matrix was analyzed using three different phylogenetic methods: (i) IQ-TREE v1.6.12 (121) was used to predict the best substitution model for each gene partition (122, 123) and to produce a maximum likelihood phylogenetic tree with 1,000 bootstrap iterations. The tree was edited in iTOL (124). (ii) A Bayesian phylogenetic analysis was performed using MrBayes v3.2.7 (125). The consensus tree was established from two independent runs, each with four chains (three hot and one cold chain) run for 3,000,000 generations. Trees were sampled every 1,000 generations with 25% burn-in. The best substitution model was determined by MrBayes (aamodelpr = mixed), with a gamma-distributed rate variation across sites (four discrete gamma categories) and a proportion of invariable sites (invgamma). (iii) Considering that some *Arsenophonus* strains are known to create extremely long branches (126), we performed a second Bayesian analysis using PhyloBayes-MPI v1.9 (127) with the CAT-GTR model to minimize the impact of long-branch attractions. Two separate chains were run for >10,000 generations, and convergence was assessed using the bpcomp and tracecomp functions (maxdiff < 0.3 and effect size > 50). The same phylogenetic analyses were performed including also "Candidatus Riesia" as well as several other insect endosymbionts from the Enterobacteriaceae family (*Buchnera aphidicola*, *H. defensa*, and *Wigglesworthia glossinidia*), in line with *Riesia's* current taxonomic assignment and the phylogenetic analyses by Boyd et al. (128). These analyses suggested that the frequently observed clustering of *Riesia* with some *Arsenophonus* P-endosymbionts might be an artifact caused by long-branch attractions (Fig. S7 to S9). Therefore, *Riesia* was not included in the comparative analyses presented herein.

ANI values were obtained using the enveomics toolbox collection (129).

## Pangenome analysis

An orthology clustering analysis was conducted using Orthofinder v2.5.4 (119) on all *Arsenophonus* genomes used for the phylogenomic analyses. Shared OGs were identified using the R package UpSetR v1.4.0 (130). To further analyze the genes present only in Ap and/or Pf, Blastp searches were conducted on the longest sequence for each OG. These searches excluded additional undetected pseudogenes and orthologs shared with recently published *Arsenophonus* genomes reconstructed from Sequence Read Archive (SRA) data sets and endosymbionts of louse flies (43, 126). For the OGs shared by Ap and Pf, functional domains were identified with MotifFinder (https://www.genome.jp/tools/motif/). In silico predictions were conducted using SignalP v6.0 (131), SecretomeP v2.0 (132), and PSORTb v3.0.3 (133). Phylogenetic analyses were performed as previously described (134).

## Gene expression analyses

RT-qPCR analyses were conducted for several Ap-CH genes in *P. leporinus* females and periwinkle stems. Insects were collected at the adult stage, after a rearing period of c. 5 months in controlled conditions (135). Periwinkle seedlings were inoculated with field-collected insects as previously described (14) and then maintained in insect-proof cages in greenhouse conditions for 3 months. Whole insect bodies and plant stem samples were freeze-dried in liquid nitrogen and stored at -80°C until further use. Total RNA was extracted using a 3% CTAB protocol (14), treated with DNase, and quantified using a Qubit fluorometer. Complementary DNA (cDNA) was synthetized for 1 µg of each RNA sample using random hexamers (Invitrogen) in combination with the Superscript
reverse transcriptase (Promega). qPCR assays were conducted as previously described (14), using primers and probes listed in Table S1. Expressions were obtained for five biological replicates and were normalized using the Ap-CH *gyrase* or *rpoB* genes. Mean relative expressions were compared by Wilcoxon Rank Sum tests in R. qPCR efficiencies were assessed based on five to six 10-fold serial dilutions of an infected sample in healthy RNA extracts. Specificity was checked by gel electrophoresis.

## ACKNOWLEDGMENTS

We would like to thank Floriane Bussereau (Agroscope, Switzerland) and Frédéric Gatineau (Cirad, France) for providing insects.

This research was funded by the European Union's Horizon 2020 research and innovation programme Marie Sklodowska-Curie, grant agreement No. 792813 to J.D., and the Swiss Federal Office for Agriculture (grant 2020/33/LES-Z II to O.S.).

M.M., C.D., J.B., D.R., X.F., and J.D. prepared samples and conducted H.T.S. analyses. M.M. conducted the gene expression analyses. M.M. and J.D. performed the bioinformatics analyses and wrote the initial draft. C.D., D.R., and R.G. provided technical assistance. J.D., F.F., X.F., and O.S. obtained funding. All authors reviewed the manuscript and agreed to publication.

## AUTHOR AFFILIATIONS

¹Research group Virology, Bacteriology and Phytoplasmology, Agroscope, Nyon, Switzerland

²Haute école du paysage, d'ingénierie et d'architecture de Genève, Geneva, Switzerland

³Dipartimento di Scienze agrarie e ambientali, Università degli Studi di Milano, Milano, Italy

⁴UMR 1332 Biologie du Fruit et Pathologie, INRAE, Université de Bordeaux, Bordeaux, France

⁵UMR 1345, Université d'Angers, Institut Agro, INRAE, IRHS, SFR Quasav, Angers, France

## PRESENT ADDRESS

Mathieu Mahillon, Department of Plants and Crops, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium

## AUTHOR ORCIDs

Mathieu Mahillon http://orcid.org/0000-0002-1974-7863

Olivier Schumpp http://orcid.org/0000-0002-2070-2144

Jessica Dittmer http://orcid.org/0000-0002-2600-9201

## FUNDING

<table><thead><tr><td>Funder</td><td>Grant(s)</td><td>Author(s)</td></tr></thead><tbody><tr><td>Swiss Federal Office for Agriculture</td><td>2020/33/LES-Z II</td><td>Olivier Schumpp</td></tr><tr><td>EU Horizon 2020 Marie Sklodowska Curie</td><td>792813</td><td>Jessica Dittmer</td></tr></tbody></table>

## AUTHOR CONTRIBUTIONS

Mathieu Mahillon, Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Resources, Software, Visualization, Writing – original draft, Writing – review and editing | Christophe Debonneville, Investigation, Supervision, Writing – review and editing | Raphaël Groux, Investigation, Resources, Software, Writing – review and editing | David Roquis, Methodology, Software, Writing – review and editing | Justine Brodard, Resources | Franco Faoro, Writing – review and editing | Xavier Foissac, Writing – review and editing | Olivier Schumpp, Funding acquisition, Project administration,
Supervision, Writing – review and editing | Jessica Dittmer, Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Visualization, Writing – original draft, Writing – review and editing

## DATA AVAILABILITY

The genomes produced in this work are accessible in the NCBI database under Gen-Bank accessions GCA_047291315.1 (Ca. Phlomobacter fragariae), GCA_047291415.1 (Ca. Arsenophonus pathogenicus Ap-CH) and GCA_047291325.1 (Ca. Arsenophonus pathogenicus Ap-FR).

## ADDITIONAL FILES

The following material is available online.

## Supplemental Material

Supplemental figures and tables (mSystems01496-24-s0001.pdf). Tables S1 to S6 and Fig. S1 to S9.

## REFERENCES

1. Bendix C, Lewis JD. 2018. The enemy within: phloem-limited pathogens. Mol Plant Pathol 19:238–254. https://doi.org/10.1111/mpp.12526

2. Lewis JD, Knoblauch M, Turgeon R. 2022. The phloem as an arena for plant pathogens. Annu Rev Phytopathol 60:77–96. https://doi.org/10.1146/annurev-phyto-020620-100946

3. Nadarasah G, Stavrinides J. 2011. Insects as alternative hosts for phytopathogenic bacteria. FEMS Microbiol Rev 35:555–575. https://doi.org/10.1111/j.1574-6976.2011.00264.x

4. Perilla-Henao LM, Casteel CL. 2016. Vector-borne bacterial plant pathogens: interactions with hemipteran insects and plants. Front Plant Sci 7:1163. https://doi.org/10.3389/fpls.2016.01163

5. Bressan A. 2014. Emergence and evolution of *Arsenophonus* bacteria as insect-vectored plant pathogens. Infect Genet Evol 22:81–90. https://doi.org/10.1016/j.meegid.2014.01.004

6. Petersen LM, Tisa LS. 2013. Friend or foe? A review of the mechanisms that drive *Serratia* towards diverse lifestyles. Can J Microbiol 59:627–640. https://doi.org/10.1139/cjm-2013-0343

7. Thapa SP, De Francesco A, Trinh J, Gurung FB, Pang Z, Vidalakis G, Wang N, Ancona V, Ma W, Coaker G. 2020. Genome-wide analyses of *Liberibacter* species provides insights into evolution, phylogenetic relationships, and virulence factors. Mol Plant Pathol 21:716–731. https://doi.org/10.1111/mpp.12925

8. Weinert LA, Werren JH, Aebi A, Stone GN, Jiggins FM. 2009. Evolution and diversity of *Rickettsia* bacteria. BMC Biol 7:6. https://doi.org/10.1186/1741-7007-7-6

9. Zreik L, Bové JM, Garnier M. 1998. Phylogenetic characterization of the bacterium-like organism associated with marginal chlorosis of strawberry and proposition of a *Candidates* taxon for the organism, *'Candidates Phlomobacter fragariae'* Int J Syst Bacteriol 48 Pt 1:257–261. https://doi.org/10.1099/00207713-48-1-257

10. Salar P, Sémétey O, Danet J-L, Boudon-Padieu E, Foissac X. 2010. *'Candidatus Phlomobacter fragariae'* and the proteobacterium associated with the low sugar content syndrome of sugar beet are related to bacteria of the arsenophonus clade detected in hemipteran insects. Eur J Plant Pathol 126:123–127. https://doi.org/10.1007/s10658-009-9520-5

11. Bressan A, Terlizzi F, Credi R. 2012. Independent origins of vectored plant pathogenic bacteria from arthropod-associated *Arsenophonus* endosymbionts. Microb Ecol 63:628–638. https://doi.org/10.1007/s0024-0811-9933-5

12. Bressan A, Sémétey O, Nusillard B, Clair D, Boudon-Padieu E. 2008. Insect vectors (Hemiptera: Cixiidae) and pathogens associated with the disease syndrome "basses richesses" of sugar beet in France. Plant Dis 92:113–119. https://doi.org/10.1094/PDIS-92-1-0113

13. Zübert C, Kube M. 2021. Application of TaqMan real-time pcr for detecting *'Candidatus Arsenophonus phytopathogenicus'* infection in

sugar beet. Pathogens 10:1466. https://doi.org/10.3390/pathogens1011466

14. Mahillon M, Groux R, Bussereau F, Brodard J, Debonneville C, Demal S, Kellenberger I, Peter M, Steinger T, Schumpp O. 2022. Virus yellows and syndrome "basses richesses" in western Switzerland: a dramatic 2020 season calls for urgent control measures. Pathogens 11:885. https://doi.org/10.3390/pathogens11080885

15. Behrmann SC, Rinklef A, Lang C, Vilcinskas A, Lee K-Z. 2023. Potato (*Solanum tuberosum*) as a new host for *Pentastiridius leporinus* (Hemiptera: Cixiidae) and *Candidatus Arsenophonus phytopathogenicus*. Insects 14:281. https://doi.org/10.3390/insects14030281

16. Rinklef A, Behrmann SC, Löffler D, Erner J, Meyer MV, Lang C, Vilcinskas A, Lee K-Z. 2024. Prevalence in potato of *'Candidatus Arsenophonus phytopathogenicus'* and *'Candidatus phytoplasma solani'* and their transmission via adult *Pentastiridius leporinus*. Insects 15:275. https://doi.org/10.3390/insects15040275

17. Therhaag E, Ulrich R, Gross J, Schneider B. 2024. Onion (*Allium cepa*) as a new host for *'Candidatus Arsenophonus phytopathogenicus'* in Germany. Plant Dis 108:2914. https://doi.org/10.1094/PDIS-03-24-0526-PDN

18. Duduk B, Stepanović J, Fránová J, Zwolińska A, Rekanović E, Stepanović M, Vučković N, Duduk N, Vico I. 2024. Geographical variations, prevalence, and molecular dynamics of fastidious phloem-limited pathogens infecting sugar beet across Central Europe. PLoS One 19:e0306136. https://doi.org/10.1371/journal.pone.0306136

19. Mahillon M, Bussereau F, Dubuis N, Brodard J, Debonneville C, Schumpp O. 2025. First detection of *Arsenophonus* in potato crop in switzerland: a threat for the processing industry? Potato Res. https://doi.org/10.1007/s11540-024-09840-y

20. Danet J-L, Foissac X, Zreik L, Salar P, Verdin E, Nourrisseau J-G, Garnier M. 2003. "Candidatus Phlomobacter fragariae" is the prevalent agent of marginal chlorosis of strawberry in French production fields and is transmitted by the planthopper *Cixius wagneri* (China). Phytopathology 93:644–649. https://doi.org/10.1094/PHYTO.2003.93.6.644

21. Nourrisseau J-G, Lansac M, Garnier M. 1993. Marginal chlorosis, a new disease of strawberries associated with a bacteriumlike organism. Plant Dis 77:1055. https://doi.org/10.1094/PD-77-1055

22. Tanaka M, Nao M, Usugi T. 2006. Occurrence of strawberry marginal chlorosis caused by "Candidatus Phlomobacter fragariae" in Japan. J Gen Plant Pathol 72:374–377. https://doi.org/10.1007/s10327-006-0308-6

23. Terlizzi F, Babini AR, Lanzoni C, Pisi A, Credi R, Foissac X, Salar P. 2007. First report of a γ 3-proteobacterium associated with diseased strawberries in Italy. Plant Dis 91:1688. https://doi.org/10.1094/PDIS-91-12-1688B

24. Characterization of two plant pathogenic *Arsenophonus* bacteria responsible for strawberry marginal chlorosis disease in Italy. Available
from: https://cris.unibo.it/handle/11585/548375. Retrieved 12 Dec 2023.

25. Nováková E, Hypsa V, Moran NA. 2009. *Arsenophonus*, an emerging clade of intracellular symbionts with a broad host distribution. BMC Microbiol 9:143. https://doi.org/10.1186/1471-2180-9-143

26. Duron O, Bouchon D, Boutin S, Bellamy L, Zhou L, Engelstädter J, Hurst GD. 2008. The diversity of reproductive parasites among arthropods: *Wolbachiado* not walk alone. BMC Biol 6:27. https://doi.org/10.1186/1741-7007-6-27

27. Jiang H, Ding Y, Zhao D, Liu X, Guo H. 2022. First discovery of *Arsenophonus* infection in spiders, predators of insect pests. J Applied Entomology 146:786-790. https://doi.org/10.1111/jen.13017

28. Zchori-Fein E, Bourtzis K, eds. 2012. The genus *Arsenophonus*, p 225-244. In Manipulative tenants. CRC Press.

29. Lo W-S, Huang Y-Y, Kuo C-H. 2016. Winding paths to simplicity: genome evolution in facultative insect symbionts. FEMS Microbiol Rev 40:855-874. https://doi.org/10.1093/femsre/fuw028

30. Skinner SW. 1985. Son-killer: a third extrachromosomal factor affecting the sex ratio in the parasitoid wasp, *Nasonia* (=*Mormoniella*) *vitripennis*. *Genetics* 109:745-759. https://doi.org/10.1093/genetics/109.4.745

31. Pang R, Chen M, Yue L, Xing K, Li T, Kang K, Liang Z, Yuan L, Zhang W. 2018. A distinct strain of *Arsenophonus* symbiont decreases insecticide resistance in its insect host. PLoS Genet 14:e1007725. https://doi.org/10.1371/journal.pgen.1007725

32. Wagner SM, Martinez AJ, Ruan Y-M, Kim KL, Lenhart PA, Dehnel AC, Oliver KM, White JA. 2015. Facultative endosymbionts mediate dietary breadth in a polyphagous herbivore. Funct Ecol 29:1402-1410. https://doi.org/10.1111/1365-2435.12459

33. Tian P-P, Chang C-Y, Miao N-H, Li M-Y, Liu X-D. 2019. Infections with *Arsenophonus* facultative endosymbionts alter performance of aphids (*Aphis gossypii*) on an amino-acid-deficient diet. Appl Environ Microbiol 85:e01407-19. https://doi.org/10.1128/AEM.01407-19

34. Nováková E, Husník F, Šochová E, Hypša V. 2015. *Arsenophonus* and *Sodalis* symbionts in louse flies: an analogy to the *Wigglesworthia* and *Sodalis* system in tsetse flies. Appl Environ Microbiol 81:6189-6199. https://doi.org/10.1128/AEM.01487-15

35. Ferree PM, Avery A, Azpurua J, Wilkes T, Werren JH. 2008. A bacterium targets maternally inherited centrosomes to kill males in *Nasonia*. Curr Biol 18:1409-1414. https://doi.org/10.1016/j.cub.2008.07.093

36. Duron O, Wilkes TE, Hurst GDD. 2010. Interspecific transmission of a male-killing bacterium on an ecological timescale. Ecol Lett 13:1139-1148. https://doi.org/10.1111/j.1461-0248.2010.01502.x

37. Drew GC, Budge GE, Frost CL, Neumann P, Siozios S, Yañez O, Hurst GDD. 2021. Transitions in symbiosis: evidence for environmental acquisition and social transmission within a clade of heritable symbionts. ISME J 15:2956-2968. https://doi.org/10.1038/s41396-021-0977-z

38. Nováková E, Hypša V, Nguyen P, Husník F, Darby AC. 2016. Genome sequence of *Candidatus* *Arsenophonus* lipopteni, the exclusive symbiont of a blood sucking fly *Lipoptena cervi* (Diptera: Hippobosciidae). Stand Genomic Sci 11:72. https://doi.org/10.1186/s40793-016-0195-1

39. Santos-Garcia D, Juravel K, Freilich S, Zchori-Fein E, Latorre A, Moya A, Morin S, Silva FJ. 2018. To B or Not to B: comparative genomics suggests *Arsenophonus* as a source of B vitamins in whiteflies. Front Microbiol 9:2254. https://doi.org/10.3389/fmicb.2018.02254

40. Darby AC, Choi J-H, Wilkes T, Hughes MA, Werren JH, Hurst GDD, Colbourne JK. 2010. Characteristics of the genome of *Arsenophonus nasoniae*, son-killer bacterium of the wasp *Nasonia*. Insect Mol Biol 19:75-89. https://doi.org/10.1111/j.1365-2583.2009.00950.x

41. Frost CL, Siozios S, Nadal-Jimenez P, Brockhurst MA, King KC, Darby AC, Hurst GDD. 2020. The hypercomplex genome of an insect reproductive parasite highlights the importance of lateral gene transfer in symbiont biology. mBio 11:e02590-19. https://doi.org/10.1128/mBio.02590-19

42. Huang W, Reyes-Caldas P, Mann M, Seifbarghi S, Kahn A, Almeida RPP, Béven L, Heck M, Hogenhout SA, Coaker G. 2020. Bacterial vector-borne plant diseases: unanswered questions and future directions. Mol Plant 13:1379-1393. https://doi.org/10.1016/j.molp.2020.08.010

43. Siozios S, Nadal-Jimenez P, Azagi T, Sprong H, Frost CL, Parratt SR, Taylor G, Brettell L, Liew KC, Croft L, King KC, Brockhurst MA, Hypša V, Novakova E, Darby AC, Hurst GDD. 2024. Genome dynamics across the evolutionary transition to endosymbiosis. Curr Biol 34:5659-5670. https://doi.org/10.1016/j.cub.2024.10.044

44. Sémétey O, Bressan A, Gatineau F, Boudon - Padieu E. 2007. Development of a specific assay using RISA for detection of the bacterial agent of 'basses richesses' syndrome of sugar beet and confirmation of a *Pentastiridius* sp. (Fulgoromopha, Cixiidae) as the economic vector. Plant Pathol 56:797-804. https://doi.org/10.1111/j.1365-3059.2007.01693.x

45. Wilkes TE, Darby AC, Choi J-H, Colbourne JK, Werren JH, Hurst GDD. 2010. The draft genome sequence of *Arsenophonus nasoniae*, son-killer bacterium of *Nasonia vitripennis*, reveals genes associated with virulence and symbiosis. Insect Mol Biol 19:59-73. https://doi.org/10.1111/j.1365-2583.2009.00963.x

46. Pfeifer E, Moura de Sousa JA, Touchon M, Rocha EPC. 2021. Bacteria have numerous distinctive groups of phage-plasmids with conserved phage and variable plasmid gene repertoires. Nucleic Acids Res 49:2655-2673. https://doi.org/10.1093/nar/gkab064

47. Tommasini D, Mageeney CM, Williams KP. 2023. Helper-embedded satellites from an integrase clade that repeatedly targets prophage late genes. NAR Genom Bioinform 5:lqad036. https://doi.org/10.1093/nargab/lqad036

48. Nadal-Jimenez P, Siozios S, Frost CL, Court R, Chrostek E, Drew GC, Evans JD, Hawthorne DJ, Burritt JB, Hurst G. 2022. *Arsenophonus apicola* sp. nov., isolated from the honeybee *Apis mellifera*. Int J Syst Evol Microbiol 72:005469. https://doi.org/10.1099/ijsem.0.005469

49. Nadal-Jimenez P, Parratt SR, Siozios S, Hurst GDD. 2023. Isolation, culture and characterization of *Arsenophonus* symbionts from two insect species reveal loss of infectious transmission and extended host range. Front Microbiol 14:1089143. https://doi.org/10.3389/fmicb.2023.1089143

50. Mao M, Yang X, Poff K, Bennett G. 2017. Comparative genomics of the dual-obligate symbionts from the treehopper, *Entylia carinata* (Hemiptera: Membracidae), provide insight into the origins and evolution of an ancient symbiosis. Genome Biol Evol 9:1803-1815. http://doi.org/10.1093/gbe/evx134

51. Fan H-W, Lu J-B, Ye Y-X, Yu X-P, Zhang C-X. 2016. Characteristics of the draft genome of "*Candidatus* *Arsenophonus* nilaparvatae", a facultative endosymbiont of *Nilaparvata lugens*. Insect Sci 23:478-486. https://doi.org/10.1111/1744-7917.12318

52. Zhu D-T, Rao Q, Zou C, Ban F-X, Zhao J-J, Liu S-S. 2022. Genomic and transcriptomic analyses reveal metabolic complementarity between whiteflies and their symbionts. Insect Sci 29:539-549. https://doi.org/10.1111/1744-7917.12943

53. Yorimoto S, Hattori M, Kondo M, Shigenobu S. 2022. Complex host/symbiont integration of a multi-partner symbiotic system in the eusocial aphid *Ceratovacuna japonica*. iScience 25:105478. https://doi.org/10.1016/j.isci.2022.105478

54. Boyd BM, Chevignon G, Patel V, Oliver KM, Strand MR. 2021. Evolutionary genomics of APSE: a tailed phage that lysogenically converts the bacterium Hamiltonella defensa into a heritable protective symbiont of aphids. Virol J 18:219. https://doi.org/10.1186/s12985-021-01685-y

55. Chun J, Oren A, Ventosa A, Christensen H, Arahal DR, da Costa MS, Rooney AP, Yi H, Xu X-W, De Meyer S, Trujillo ME. 2018. Proposed minimal standards for the use of genome data for the taxonomy of prokaryotes. Int J Syst Evol Microbiol 68:461-466. https://doi.org/10.1099/ijsem.0.002516

56. Douglas AE. 2006. Phloem-sap feeding by animals: problems and solutions. J Exp Bot 57:747-754. https://doi.org/10.1093/jxb/erj067

57. Broussard L, Abadie C, Lalande J, Limami AM, Lothier J, Tcherkez G. 2023. Phloem sap composition: what have we learnt from metabolomics?Int J Mol Sci 24:6917. https://doi.org/10.3390/ijms24086917

58. Bové JM, Garnier M. 2003. Phloem-and xylem-restricted plant pathogenic bacteria. Plant Sci 164:423-438. https://doi.org/10.1016/S0168-9452(03)00033-5

59. Buoso S, Pagliari L, Musetti R, Martini M, Marroni F, Schmidt W, Santi S. 2019. 'Candidatus Phytoplasma solani' interferes with the distribution and uptake of iron in tomato. BMC Genomics 20:703. https://doi.org/10.1186/s12864-019-6062-x

60. André M, Maucourt M, Moing A, Rolin D, Renaudin J. 2005. Sugar import and phytopathogenicity of *Spiroplasma citri*: glucose and fructose play distinct roles. Mol Plant Microbe Interact 18:33-42. https://doi.org/10.1094/MPMI-18-0033

61. Gatineau F, Jacob N, Vautrin S, Larrue J, Lherminier J, Richard-Molard M, Boudon-Padieu E. 2002. Association with the syndrome "basses richesses" of sugar beet of a phytoplasma and a bacterium-like
organism transmitted by a *Pentastiridius* sp. Phytopathology 92:384-392. https://doi.org/10.1094/PHYTO.2002.92.4.384

62. Bressan A, Sémétyot E, Arneodo J, Lherminier J, Boudon-Padieu E. 2009. Vector transmission of a plant-pathogenic bacterium in the *Arsenophonus* clade sharing ecological traits with facultative insect endosymbionts. Phytopathology 99:1289-1296. https://doi.org/10.1094/PHYTO-99-11-1289

63. Dittmer J, Lusseau T, Foissac X, Faoro F. 2021. Skipping the insect vector: plant stolon transmission of the phytopathogen *Ca. Phlomobacter fragariae* from the *Arsenophonus* clade of insect endosymbionts. Insects 12:93. https://doi.org/10.3390/insects12020093

64. Henry E, Carlson CR, Kuo Y-W. 2023. *Candidatus Kirkpatrickella diaphorinae* gen. nov., sp. nov., an uncultured endosymbiont identified in a population of *Diaphorina citri* from Hawaii. Int J Syst Evol Microbiol 73:006111. https://doi.org/10.1099/ijsem.0.006111

65. Crooks C, Bechle NJ, St John FJ. 2021. A new subfamily of glycoside hydrolase family 30 with strict xylobiohydrolase function. Front Mol Biosci 8:714238. https://doi.org/10.3389/fmolb.2021.714238

66. Helbert W, Poulet L, Drouillard S, Mathieu S, Loiodice M, Couturier M, Lombard V, Terrapon N, Turchetto J, Vincentelli R, Henrissat B. 2019. Discovery of novel carbohydrate-active enzymes through the rational exploration of the protein sequences space. Proc Natl Acad Sci USA 116:6063-6068. https://doi.org/10.1073/pnas.1815791116

67. Ma S, Dong Y, Wang N, Liu J, Lu C, Liu Y. 2020. Identification of a new effector-immunity pair of *Aeromonas hydrophila* type VI secretion system. Vet Res 51:71. https://doi.org/10.1186/s13567-020-00794-w

68. Nogaroto V, Tagliavini S, Gianotti A, Mikawa A, Barros NT, Puzer L, Carmona AK, Costa P, Henrique-Silva F. 2006. Recombinant expression and characterization of a *Xylella fastidiosa* cysteine protease differentially expressed in a nonpathogenic strain. FEMS Microbiol Lett 261:187-193. https://doi.org/10.1111/j.1574-6968.2006.00348.x

69. Hugouvieux-Cotte-Pattat N, Flandrois J-P, Briolay J, Reverchon S, Brochier-Armanet C. 2024. Description of a new genus of the *Pectobacteriaceae* family isolated from water in coastal brackish wetlands of the French Camargue region, *Prodigiosinella* gen. nov., including the new species *Prodigiosinella aquatilis* sp. nov. Syst Appl Microbiol 47:126497. https://doi.org/10.1016/j.syapm.2024.126497

70. Degnan PH, Moran NA. 2008. Diverse phage-encoded toxins in a protective insect endosymbiont. Appl Environ Microbiol 74:6782-6791. https://doi.org/10.1128/AEM.01285-08

71. Duron O. 2014. *Arsenophonus* insect symbionts are commonly infected with APSE, a bacteriophage involved in protective symbiosis. FEMS Microbiol Ecol 90:184-194. https://doi.org/10.1111/1574-6941.12381

72. Aparna G, Chatterjee A, Sonti RV, Sankaranarayanan R. 2009. A cell wall-degrading esterase of *Xanthomonas oryzae* requires a unique substrate recognition module for pathogenesis on rice. Plant Cell 21:1860-1873. https://doi.org/10.1105/tpc.109.066886

73. Seemüller E, Kampmann M, Kiss E, Schneider B. 2011. HflB gene-based phytopathogenic classification of *'Candidatus Phytoplasma mali'* strains and evidence that strain composition determines virulence in multiply infected apple trees. Mol Plant Microbe Interact 24:1258-1266. https://doi.org/10.1094/MPMI-05-11-0126

74. Nascimento R, Gouran H, Chakraborty S, Gillespie HW, Almeida-Souza HO, Tu A, Rao BJ, Feldstein PA, Bruening G, Goulart LR, Dandekar AM. 2016. The type II secreted lipase/esterase LesA is a key virulence factor required for *Xylella fastidiosa* pathogenesis in grapevines. Sci Rep 6:18598. https://doi.org/10.1038/srep18598

75. St John FJ, González JM, Pozharski E. 2010. Consolidation of glycosyl hydrolase family 30: a dual domain 4/7 hydrolase family consisting of two structurally distinct groups. FEBS Lett 584:4435-4441. https://doi.org/10.1016/j.fepslet.2010.09.051

76. Szczesny R, Jordan M, Schramm C, Schulz S, Cogez V, Bonas U, Büttner D. 2010. Functional characterization of the Xcs and Xps type II secretion systems from the plant pathogenic bacterium *Xanthomonas campestris* pv *vesicatoria*. New Phytol 187:983-1002. https://doi.org/10.1111/j.1469-8137.2010.03312.x

77. Blackman LM, Cullerne DP, Torreña P, Taylor J, Hardham AR. 2015. RNA-Seq analysis of the expression of genes encoding cell wall degrading enzymes during infection of lupin (*Lupinus angustifolius*) by *Phytophthora parasitica*. PLoS One 10:e0136899. https://doi.org/10.1371/journal.pone.0136899

78. Wybouw N, Dermauw W, Tirry L, Stevens C, Grbić M, Feyereisen R, Van Leeuwen T. 2014. A gene horizontally transferred from bacteria

protects arthropods from host plant cyanide poisoning. Elife 3:e02365. https://doi.org/10.7554/eLife.02365

79. Wybouw N, Pauchet Y, Heckel DG, Van Leeuwen T. 2016. Horizontal gene transfer contributes to the evolution of arthropod herbivory. Genome Biol Evol 8:1785-1801. https://doi.org/10.1093/gbe/evw119

80. Su C-C, Deng W-L, Jan F-J, Chang C-J, Huang H, Shih H-T, Chen J. 2016. *Xylella taiwanensis* sp. nov., causing pear leaf scorch disease. Int J Syst Evol Microbiol 66:4766-4771. https://doi.org/10.1099/ijsem.0.001426

81. Mensi I, Vernerey M-S, Gargani D, Nicole M, Rott P. 2014. Breaking dogmas: the plant vascular pathogen *Xanthomonas albilineans* is able to invade non-vascular tissues despite its reduced genome. Open Biol 4:130116. https://doi.org/10.1098/rsob.130116

82. Wang N, Pierson EA, Setubal JC, Xu J, Levy JG, Zhang Y, Li J, Rangel LT, Martins J. 2017. The *Candidatus* liberibacter-host interface: insights into pathogenesis mechanisms and disease control. Annu Rev Phytopathol 55:451-482. https://doi.org/10.1146/annurev-phyto-08051 6-035513

83. Caires NP, Guimarães LMS, Hermenegildo PS, Rodrigues FA, Badel JL, Alfenas AC. 2020. Bidirectional colonization and biofilm formation by *Erwinia psidii* in eucalypt plants. Plant Pathol 69:549-558. https://doi.org/10.1111/ppa.13136

84. Shindo T, Van der Hoorn RAL. 2008. Papain-like cysteine proteases: key players at molecular battlefields employed by both plants and their invaders. Mol Plant Pathol 9:119-125. https://doi.org/10.1111/j.1364-37 03.2007.00439.x

85. Nadal-Jimenez P, Siozios S, Halliday N, Câmera M, Hurst GDD. 2022. *Symbiopectobacterium purcellii*, gen. nov., sp. nov., isolated from the leafhopper *Empoasca decipiens*. Int J Syst Evol Microbiol 72. https://doi.org/10.1099/ijsem.0.005440

86. Purcell AH, Suslow KG, Klein M. 1994. Transmission via plants of an insect pathogenic bacterium that does not multiply or move in plants. Microb Ecol 27:19-26. https://doi.org/10.1007/BF00170111

87. Nunes Leite L, Visnovsky SB, Wright PJ, Pitman AR. 2023. Draft genome sequences of three "*Candidatus Symbiopectobacterium" isolates collected from potato tubers grown in New Zealand. Microbiol Resour Announc 12:e01148-22. https://doi.org/10.1128/mra.01148-22

88. Foissac X, Edwards MG, Du JP, Gatehouse AMR, Gatehouse JA. 2002. Putative protein digestion in a sap-sucking homopteran plant pest (rice brown plant hopper; *Nilaparvata lugens*: Delphacidae)—identification of trypsin-like and cathepsin B-like proteases. Insect Biochem Mol Biol 32:967-978. https://doi.org/10.1016/s0965-1748(02)00033-4

89. Shao E, Song Y, Wang Y, Liao Y, Luo Y, Liu S, Guan X, Huang Z. 2021. Transcriptomic and proteomic analysis of putative digestive proteases in the salivary gland and gut of *Empoasca* (Matsumurasca) *onukii* Matsuda. BMC Genomics 22:271. https://doi.org/10.1186/s12864-021-0 7578-2

90. Skaljac M, Vogel H, Wielsch N, Mihajlovic S, Vilcinskas A. 2019. Transmission of a protease-secreting bacterial symbiont among pea aphids via host plants. Front Physiol 10:438. https://doi.org/10.3389/fphys.2019.00438

91. Caspi-Fluger A, Inbar M, Mozes-Daube N, Katzir N, Portnoy V, Belausov E, Hunter MS, Zchori-Fein E. 2012. Horizontal transmission of the insect symbiont *Rickettsia* is plant-mediated. Proc Biol Sci 279:1791-1796. http://doi.org/10.1098/rspb.2011.2095

92. Li S-J, Ahmed MZ, Lv N, Shi P-Q, Wang X-M, Huang J-L, Qiu B-L. 2017. Plant-mediated horizontal transmission of *Wolbachia* between whiteflies. ISME J 11:1019-1028. https://doi.org/10.1038/ismej.2016.164

93. Chrostek E, Pelz-Stelinski K, Hurst GDD, Hughes GL. 2017. Horizontal transmission of intracellular insect symbionts via plants. Front Microbiol 8:2237. https://doi.org/10.3389/fmicb.2017.02237

94. Pons I, Renoz F, Noël C, Hance T. 2019. Circulation of the cultivable symbiont *Serratia symbiotica* in aphids is mediated by plants. Front Microbiol 10:764. https://doi.org/10.3389/fmicb.2019.00764

95. Bassene H, Niang EHA, Fenollar F, Doucoure S, Faye O, Raoult D, Sokhna C, Mediannikov O. 2020. Role of plants in the transmission of *Asaia* sp., which potentially inhibit the *Plasmodium sporadic* cycle in *Anopheles* mosquitoes. Sci Rep 10:7144. https://doi.org/10.1038/s41598-020-6416-3

96. Li F, Hua H, Han Y, Hou M. 2020. Plant-mediated horizontal transmission of *Asaia* between white-backed planthoppers, *Sogatella furcifera*. Front Microbiol 11:593485. https://doi.org/10.3389/fmicb.2020.593485

97. Gluck-Thaler E, Cerutti A, Perez-Quintero AL, Butchacas J, Roman-Reyna V, Madhavan VN, Shantharaj D, Merfa MV, Pesce C, Jauneau A, et al.
2020. Repeated gain and loss of a single gene modulates the evolution of vascular plant pathogen lifestyles. Sci Adv 6:eabc4516. https://doi.org/g/10.1126/sciadv.abc4516

98. Rocha J, Shapiro LR, Kolter R. 2020. A horizontally acquired expansin gene increases virulence of the emerging plant pathogen *Erwinia tracheiphila*. Sci Rep 10:21743. https://doi.org/10.1038/s41598-020-78157-w

99. Bolger AM, Lohse M, Usadel B. 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114–2120. https://doi.org/10.1093/bioinformatics/btu170

100. Li H. 2018. Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics 34:3094–3100. https://doi.org/10.1093/bioinformatics/bty191

101. Zimin AV, Marçais G, Puiu D, Roberts M, Salzberg SL, Yorke JA. 2013. The MaSuRCA genome assembler. Bioinformatics 29:2669–2677. https://doi.org/10.1093/bioinformatics/btt476

102. Prjibelski A, Antipov D, Meleshko D, Lapidus A, Korobeynikov A. 2020. Using SPAdes of novo assembler. Curr Protoc Bioinformatics 70:e102. https://doi.org/10.1002/cpbi.102

103. Wick RR, Judd LM, Gorrie CL, Holt KE. 2017. Unicycler: resolving bacterial genome assemblies from short and long sequencing reads. PLOS Comput Biol 13:e1005595. https://doi.org/10.1371/journal.pcbi.1005595

104. Boetzer M, Henkel CV, Jansen HJ, Butler D, Pirovano W. 2011. Scaffolding pre-assembled contigs using SSPACE. Bioinformatics 27:578–579. https://doi.org/10.1093/bioinformatics/btq683

105. Li D, Luo R, Liu C-M, Leung C-M, Ting H-F, Sadakane K, Yamashita H, Lam T-W. 2016. MEGAHIT v1.0: a fast and scalable metagenome assembler driven by advanced methodologies and community practices. Methods 102:3–11. https://doi.org/10.1016/j.ymeth.2016.02.020

106. Pryszzc LP, Gabaldón T. 2016. Redundans: an assembly pipeline for highly heterozygous genomes. Nucleic Acids Res 44:e113. https://doi.org/g/10.1093/nar/gkw294

107. Nadalin F, Vezzi F, Policriti A. 2012. GapFiller: a de novo assembly approach to fill the gap within paired reads. BMC Bioinformatics 13:58. https://doi.org/10.1186/1471-2105-13-S14-S8

108. Pedersen BS, Quinlan AR. 2018. Mosdepth: quick coverage calculation for genomes and exomes. Bioinformatics 34:867–868. https://doi.org/10.1093/bioinformatics/btx699

109. Parks DH, Imelfort M, Skennerton CT, Hugenholtz P, Tyson GW. 2015. CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome Res 25:1043–1055. https://doi.org/10.1101/gr.186072.114

110. Minkin I, Patel A, Kolmogorov M, Vyahhi N, Pham S. 2013. Sibelia: a scalable and comprehensive synteny block generation tool for closely related microbial genomes, p 215–229. In Darling A, Stoye J (ed), Algorithms in bioinformatics. Springer, Berlin, Heidelberg.

111. Cantalapiedra CP, Hernández-Plaza A, Letunic I, Bork P, Huerta-Cepas J. 2021. eggNOG-mapper v2: functional annotation, orthology assignments, and domain prediction at the metagenomic scale. Mol Biol Evol 38:5825–5829. https://doi.org/10.1093/molbev/msab293

112. Arndt D, Grant JR, Marcu A, Sajed T, Pon A, Liang Y, Wishart DS. 2016. PHASTER: a better, faster version of the PHAST phage search tool. Nucleic Acids Res 44:W16–W21. https://doi.org/10.1093/nar/gkw387

113. Gilchrist CLM, Chooi Y-H. 2021. Clinker & clustermap.js: automatic generation of gene cluster comparison figures. Bioinformatics 37:2473–2475. https://doi.org/10.1093/bioinformatics/bta007

114. Gu Z, Gu L, Eils R, Schlesner M, Brors B. 2014. Cirlcize Implements and enhances circular visualization in R. Bioinformatics 30:2811–2812. https://doi.org/10.1093/bioinformatics/btu393

115. Gu Z. 2022. Complex heatmap visualization. iMeta 1:e43. https://doi.org/10.1002/imt2.43

116. Graham ED, Heidelberg JF, Tully BJ. 2018. Potential for primary productivity in a globally-distributed bacterial phototroph. ISME J 12:1861–1866. https://doi.org/10.1038/s41396-018-0091-3

117. Abby SS, Cury J, Guglielmini J, Néron B, Touchon M, Rocha EPC. 2016. Identification of protein secretion systems in bacterial genomes. Sci Rep 6:23080. https://doi.org/10.1038/srep23080

118. Blin K, Shaw S, Kloosterman AM, Charlop-Powers Z, van Wezel GP, Medema MH, Weber T. 2021. antiSMASH 6.0: improving cluster detection and comparison capabilities. Nucleic Acids Res 49:W29–W35. https://doi.org/10.1093/nar/gkab335

119. Emms DM, Kelly S. 2019. OrthoFinder: phylogenetic orthology inference for comparative genomics. Genome Biol 20:238. https://doi.org/g/10.1186/s13059-019-1832-y

120. Edgar RC. 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792–1797. https://doi.org/10.1093/nar/gkh340

121. Nguyen L-T, Schmidt HA, von Haeseler A, Minh BQ. 2015. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol 32:268–274. https://doi.org/10.1093/molbev/msu300

122. Kalyaanamoorthy S, Minh BQ, Wong TKF, von Haeseler A, Jermiin LS. 2017. ModelFinder: fast model selection for accurate phylogenetic estimates. Nat Methods 14:587–589. https://doi.org/10.1038/nmeth.4285

123. Chernomor O, von Haeseler A, Minh BQ. 2016. Terrace aware data structure for phylogenomic inference from supermatrices. Syst Biol 65:997–1008. https://doi.org/10.1093/sysbio/syw037

124. Letunic I, Bork P. 2019. Interactive Tree Of Life (iTOL) v4: recent updates and new developments. Nucleic Acids Res 47:W256–W259. https://doi.org/10.1093/nar/gkz239

125. Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP. 2012. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol 61:539–542. https://doi.org/10.1093/sysbio/sys029

126. Martin Ríhová J, Gupta S, Darby AC, Nováková E, Hypša V. 2023. *Arsenophonus* symbiosis with louse flies: multiple origins, coevolutionary dynamics, and metabolic significance. *mSystems* 8:e00706-23. https://doi.org/10.1128/msystems.00706-23

127. Tarllott N, Rodrigue N, Stubbs D, Richer J. 2013. PhyloBayes MPI: phylogenetic reconstruction with infinite mixtures of profiles in a parallel environment. Syst Biol 62:611–615. https://doi.org/10.1093/sysbio/syt022

128. Boyd BM, Allen JM, Nguyen N-P, Vachaspati P, Quicksall ZS, Warnow T, Mugisha L, Johnson KP, Reed DL. 2017. Primates, lice and bacteria: speciation and genome evolution in the symbionts of hominid lice. Mol Biol Evol 34:1743–1757. https://doi.org/10.1093/molbev/msx117

129. Rodriguez-R LM, Konstantinidis KT. 2016. The eneomics collection: a toolbox for specialized analyses of microbial genomes and metagenomes. PeerJ. https://doi.org/10.7287/peerj.preprints.1900v1

130. Conway JR, Lex A, Gehlenborg N. 2017. UpSetR: an R package for the visualization of intersecting sets and their properties. Bioinformatics 33:2938–2940. https://doi.org/10.1093/bioinformatics/btx364

131. Teufel F, Almagro Armenteros JJ, Johansen AR, Gíslason MH, Pihl SI, Tsirigos KD, Winther O, Brunak S, von Heijne G, Nielsen H. 2022. SignalP 6.0 predicts all five types of signal peptides using protein language models. Nat Biotechnol 40:1023–1025. https://doi.org/10.1038/s41587-021-01156-3

132. Bendtsen JD, Kiemer L, Fausbøll A, Brunak S. 2005. Non-classical protein secretion in bacteria. BMC Microbiol 5:58. https://doi.org/10.1186/1471-2180-5-58

133. Yu NY, Wagner JR, Laird MR, Melli G, Rey S, Lo R, Dao P, Sahinalp SC, Ester M, Foster LJ, Brinkman FSL. 2010. PSORTb 3.0: improved protein subcellular localization prediction with refined localization subcategories and predictive capabilities for all prokaryotes. Bioinformatics 26:1608–1615. https://doi.org/10.1093/bioinformatics/btq249

134. Mahillon M, Brodard J, Kellenberger I, Blouin AG, Schumm P. 2023. A novel weevil-transmitted tymovirus found in mixed infection on hollyhock. Virol J 20:17. https://doi.org/10.1186/s12985-023-01976-6

135. Pfitzer R, Varrelmann M, Schrameyer K, Rostás M. 2022. Life history traits and a method for continuous mass rearing of the planthopper *Pentastiridius leporinus*, a vector of the causal agent of syndrome 'basses richesses' in sugar beet. Pest Manag Sci 78:4700–4708. https://doi.org/10.1002/ps.7090