High-resolution Microbiome Analyses of Nine Psyllid Species of the Family Triozidae Identified Previously Unrecognized but Major Bacterial Populations, including Liberibacter and Wolbachia of Supergroup O

Psyllids (Hemiptera: Sternorrhyncha: Psylloidea) are plant sap-sucking insects that include important agricultural pests. To obtain insights into the ecological and evolutionary behaviors of microbes, including plant pathogens, in Psylloidea, high-resolution analyses of the microbiomes of nine psyllid species belonging to the family Triozidae were performed using high-throughput amplicon sequencing of the 16S rRNA gene. Analyses identified various bacterial populations, showing that all nine psyllids have at least one secondary symbiont, along with the primary symbiont “Candidatus Carsonella ruddii” (Gammaproteobacteria: Oceanospirillales: Halomonadaceae). The majority of the secondary symbionts were gammaproteobacteria, particularly those of the order Enterobacterales, which included Arsenophonus and Serratia symbiotica, a bacterium formerly recognized only as a secondary symbiont of aphids (Hemiptera: Sternorrhyncha: Aphidoidea). The non-Enterobacterales gammaproteobacteria identified in the present study were Diplorickettsia (Diplorickettsiales: Diplorickettsiaceae), a potential human pathogen, and Carnimonas (Oceanospirillales: Halomonadaceae), a lineage detected for the first time in Psylloidea. Regarding alphaproteobacteria, the potential plant pathogen “Ca. Liberibacter europaeus” (Rhizobiales: Rhizobiaceae) was detected for the first time in Epitrioza yasumatsui, which feeds on the Japanese silverberry Elaeagnus umbellata (Elaeagnaceae), an aggressive invasive plant in the United States and Europe. Besides the detection of Wolbachia (Rickettsiales: Anaplasmataceae) of supergroup B in three psyllid species, a lineage belonging to supergroup O was identified for the first time in Psylloidea. These results suggest the rampant transfer of bacterial symbionts among animals and plants, thereby providing deeper insights into the evolution of interkingdom interactions among multicellular organisms and bacteria, which will facilitate the control of pest psyllids.

In the present study, we performed amplicon analyses based on Illumina sequencing of 16S rRNA genes to assess the microbiomes of nine Triozidae species collected in Japan (Table 1) using 1) ten pooled individuals for each species, 2) primers to detect genomes with a wider variety of GC contents, and 3) a method to resolve sequence variants (SVs) down to the level of single-nucleotide differences. Nakabachi et al.

Insects and DNA extraction
The adults of nine psyllid species belonging to the family Triozidae were collected from their host plants at various locations in Japan (Table 1). Insect samples were stored in acetone (Epitrioza yasumatsui, Trioza cinnamomi, and Trioza machilicola) or 99.5% ethanol (the other species) at -20°C until DNA extraction. After surface sterilization with ethanol, DNA was extracted from the whole bodies of pooled individuals of five adult males and five adult females of each psyllid species using the DNeasy Blood & Tissue Kit (Qiagen). The quality of the extracted DNA was assessed using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific) and its quantity was evaluated using a Qubit 2.0 Fluorometer with the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific).

Construction and sequencing of amplicon libraries
Bacterial populations in psyllids were analyzed using the MiSeq system (Illumina), as previously described (Nakabachi et al., 2020a. In brief, amplicon PCR was performed using DNA extracted from psyllids, KAPA HiFi HotStart ReadyMix (KAPA Biosystems), and the primer set 16S_341Fmod (5′-TCGTCGGCA GCGTCAGATGTGTATAAGAGACAGYYTAMGGRNGGCWG CAG-3′) and 16S_805R (5′-GTCTCGTGGGCTCGGAGATGTGT ATAAGAGACAGGACTACHVGGGTATCTAATCC-3′) targeting the V3 and V4 regions of the 16S rRNA gene. Both primers were based on the instructions by Illumina (Illumina, 2013), whereas 16S_341F was modified (underlined) with original CC, C, and G being replaced by the mixed bases YY (C or T), M (A or C), and R (A or G). This modification has been shown to increase the sensitivity of detecting symbionts with AT-rich genomes, including Carsonella, without reducing sensitivity for those with GC-rich genomes . Dual indices and Illumina sequencing adapters were attached to the amplicons with index PCR using Nextera XT Index Kit v2 (Illumina). The libraries were combined with PhiX Control v3 (Illumina), and 250 bp of both ends were sequenced on the MiSeq platform (Illumina) with MiSeq Reagent Kit v2 (500 cycles; Illumina).

Computational analysis of bacterial populations
Output sequences were imported into the QIIME2 platform (version 2022.2) (Bolyen et al., 2019) and processed as previously described (Nakabachi et al., 2020a. Briefly, after removing primer sequences, the denoising and joining of paired-end reads and the removal of low-quality or chimeric reads were performed using the dada2 plugin (Callahan et al., 2016). The q2-featureclassifier plugin (Bokulich et al., 2018) was trained using the V3 and V4 regions of the 16S rRNA gene sequences (Silva 138 SSURef NR99) (Glöckner et al., 2017). Dereplicated amplicon reads were then classified, to which taxonomic information was assigned using the trained q2-feature-classifier. The sequence variants (SVs) obtained were manually checked by performing BLASTN searches against the National Center for Biotechnology Information non-redundant database (Camacho et al., 2009).

Data availability
Nucleotide sequence data are available in the DDBJ/EMBL/ GenBank databases under the accession numbers DRR403084-DRR403092 (MiSeq output) and TAAC01000001-TAAC01000048 (dereplicated sequence variants).

Results and Discussion
All nine Triozidae species have Carsonella and at least one secondary symbiont MiSeq sequencing of amplicon libraries yielded 43,775-90,039 pairs of forward and reverse reads for the nine psyllid species. The denoising and joining of paired-end reads along with the removal of low-quality or chimeric reads resulted in 17,395-77,671 non-chimeric high-quality reads (Supplementary Table S1). The dereplication of these reads resulted in 206 independent sequence variants (SVs), among which 44 accounted for >1% of total reads (Supplementary Table S2). We focused on these 44 SVs because filtering with a threshold of 1% was shown to be among the most effective and accurate methods to remove potential contaminants (Karstens et al., 2019). SVs with a relative abundance of less than 1% were collectively categorized as 'others' in Fig. 1, which accounted for 0% (T. machilicola)-3.6% (E. yasumatsui) of total reads in each psyllid species (Supplementary Table S2). Extremely simple bacterial communities of this type have been reported for sternorrhynchan insects with a bacteriome, including aphids, whiteflies, and other psyllid species (Nachappa et al., 2011;Russell et al., 2013;Jing et al., 2014;Overholt et al., 2015;Morrow et al., 2017;Meng et al., 2019;Nakabachi et al., 2020aKwak et al., 2021). Taxonomic classification by QIIME2 (Supplementary Table S2) followed by independent BLAST searches and phylogenetic analyses showed that all nine psyllid species possess distinct lineages of Carsonella ( Fig.  1). In the maximum likelihood (ML) tree, the Carsonella sequences identified in the present study formed a moderately supported clade (bootstrap: 64%) with those of psyllids belonging to the family Psyllidae ( Fig. 2), which is consistent with the host psyllid phylogeny inferred by mitochondrial and nuclear data analyses (Burckhardt et al., 2021). Two types each of Carsonella sequences were observed in Baeoalitriozus swezeyi and Stenopsylla nigricornis ( Fig. 1 and 2 Table S2). In S. nigricornis, SV6 (43.8% of reads) and SV41 (1.3% of reads) were also 99.8% identical (Supplementary Table S2). Although the dada2 plugin corrects sequencing errors during the denoising process (Callahan et al., 2016), SV40 and SV41 may have been derived from PCR/sequencing errors because they accounted for only small percentages of the reads. Previous studies detected only a trace amount of Carsonella reads (Nachappa et al., 2011;Morrow et al., 2017;Kwak et al., 2021), whereas the present study, which used primers with increased sensitivity to AT-rich symbiont genomes, detected a large percentage of Carsonella reads ( Fig. 1 and Supplementary Table S2), more precisely reflecting actual populations (Nakabachi et al., 2020a.

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Article ME22078 Besides Carsonella, all nine psyllids analyzed in the present study possessed at least one other symbiont (Fig. 1).

Various bacteria of Enterobacterales reside in Triozidae
Among the 44 main SVs obtained in the present study, 37 corresponded to gammaproteobacteria, among which 23 belonged to the order Enterobacterales (Supplementary  Table S2). Enterobacterales is a group of bacteria that encompasses a large fraction of intimate insect symbionts, including those associated with the bacteriome (Moran et al., 2008). Enterobacterales bacteria identified in the present study included Arsenophonus, Serratia symbiotica, and several lineages with ambiguous phylogenetic placements ( Fig. 1 and 4, Supplementary Table S2).

S. symbiotica and its relatives
Four SVs found in Epitrioza mizuhonica and one in E. yasumatsui corresponded to the sequence of S. symbiotica, known as a prevalent secondary symbiont of aphids (Perreau et al., 2021) (Fig. 1). SV22, SV23, SV26, and SV37, which accounted for 13.3, 11.6, 10.1, and 2.2%, respectively, of E. mizuhonica reads, and SV14, accounting for 18.5% of E. yasumatsui reads, were 98.6-99.5% identical to a single sequence of S. symbiotica (e.g. AB522706) ( Fig. 4 and Supplementary Table S2). This reference sequence was derived from various aphid lineages, including Acyrthosiphon pisum, Aphis fabae, Aphis gossypii, Cinara pinikoraiensis, Cinara ponderosae, and Trama caudata (all Aphididae). The SVs described above and S. symbiotica sequences from aphids formed a robustly supported clade (bootstrap: 97%) together with SVs previously detected in another psyllid species Cacopsylla coccinea (Psyllidae: Psyllinae)   (Fig. 4). This result supports the recently proposed hypothesis that S. symbiotica is prevalent in psyllids , although it was formerly recognized only as a secondary symbiont of aphids. Since S. symbiotica was shown to enter plants and cause new infections in aphids, host plants are likely media for the intra-and inter-specific horizontal transmission of this bacterium (Perreau et al., 2021).
SV22, SV23, and SV26 were 98.6% (SV23 vs SV26)-99.3% (SV22 vs SV23) identical to one another. Similarities in nucleotide sequences and read frequencies ( Fig. 1 and Supplementary Table S2) implied that SVs corresponded to multiple copies of the 16S rRNA gene in a single S. symbiotica genome. This is consistent with a previous finding showing that the genomes of several S. symbiotica strains encoded more than a single copy of the 16S rRNA gene (Perreau et al., 2021), which is in contrast to primary symbionts with an extremely streamlined genome encoding only a single copy (Nakabachi et al., 2006(Nakabachi et al., , 2013b(Nakabachi et al., , 2020bMoran et al., 2008). A similar case was previously observed in C. coccinea described above . Although the ecological role of S. symbiotica widely varies depending on aphid lineages (Perreau et al., 2021), its role in psyllids is currently unknown and, thus, warrants further study.
Other Enterobacterales symbionts with uncertain identities SV34, which accounted for 2.3% of S. nigricornis reads (Supplementary Table S2), was closely related to Sodalis endosymbionts (Fig. 4). It was 93.7% identical to the
CLeu is a close relative of CLaf, "Ca. L. asiaticus", and "Ca. L. americanus", which are pathogens of the devastating greening disease in citrus (Rutaceae) (Grafton-Cardwell et al., 2013), and CLso, the pathogen causing serious diseases in solanaceous and apiaceous crops as described above (Mora et al., 2021). CLeu was detected in various psyllids from various locations: Cacopsylla spp. (Psyllidae: Psyllinae) in Italy and Hungary, Arytainilla spartiophila (Psyllidae: Psyllinae) in New Zealand and the U.K. (Tannières et al., 2020), D. cf. continua (Psyllidae: Diaphorininae) in Corsica island (Nakabachi et al., 2020a), and A. mori in Japan . CLeu was also detected from rosaceous plants and the Scotch broom Cytisus scoparius (Fabaceae), which are host plants of Cacopsylla spp. and Ar. spartiophila, respectively (Tannières et al., 2020). The presence of CLeu is associated with pathological symptoms in the Scotch broom (Tannières et al., 2020). The present study adds another example of CLeu from the psyllid species E. yasumatsui in Japan. Further studies are required to clarify whether the host plant E. umbellata, which is distantly related to previously known infected plants, is also infected with CLeu and if infection causes disease symptoms. Since E. umbellata is an aggressive invasive plant in the United States and Europe, if CLeu causes a disease in E. umbellata, CLeu transmitted by E. yasumatsui may potentially be exploited as a biological herbicide.

First detection of Wolbachia supergroup O in Psylloidea
Analyses identified five SVs corresponding to distinct lineages of Wolbachia (Alphaproteobacteria: Rickettsiales) ( Fig. 1 and 6, Supplementary Table S2). Wolbachia are rickettsial bacteria that are distributed in a wide variety of arthropods and nematodes (Werren et al., 2008), the strains of which are currently classified into supergroups A-Q (Lindsey et al., 2016). Supergroups A and B are the most common supergroups infecting arthropods, and all Wolbachia strains previously detected in psyllids belonged to supergroup B (Spaulding and von Dohlen, 2001;Sloan and Moran, 2012;Arp et al., 2014;Jain et al., 2017;Morrow et al., 2017;Chu et al., 2019;Nakabachi et al., 2020a. In contrast, SV10, which accounted for 25.9% of T. cinnamomi reads (Supplementary Table S2), was 100% identical to the sequence of Wolbachia belonging to supergroup O, which was detected in two aphid species, Kaburagia rhusicola (MT554837) and Schlechtendalia chinensis (MT554838) (Ren et al., 2020). The ML analysis placed the sequence within a robustly supported clade (bootstrap: 90%) of Wolbachia supergroup O (Fig. 6). To the best of our knowledge, this is the first detection of Wolbachia supergroup O in Psylloidea. All other Wolbachia strains found in the present study belonged to supergroup B (Fig.  6).
The majority of Wolbachia strains manipulate the reproduction of arthropod hosts to boost dissemination (Werren et al., 2008). Due to this ability, Wolbachia are recognized as promising agents to control insect pests by affecting their traits or microbiomes (Brinker et al., 2019). Based on the high infection rates of Wolbachia in pest psyllids worldwide (Spaulding and von Dohlen, 2001;Sloan and Moran, 2012;Arp et al., 2014;Chu et al., 2016Chu et al., , 2019Morrow et al., 2017;Nakabachi et al., 2020a and the suggested interactions between Wolbachia and other symbionts (Chu et al., 2016(Chu et al., , 2019Jain et al., 2017;Kruse et al., 2017;Killiny, 2022), the application of Wolbachia to control pest psyllids and/or plant pathogens is anticipated (Chu et al., 2016(Chu et al., , 2019Kruse et al., 2017). The present results suggest the rampant horizontal transmission of various Wolbachia strains among various insects, including pest psyllids (Fig.  6); therefore, the artificial infection of Wolbachia appears to be feasible in psyllids and will facilitate the exploitation of this bacterial group as a tool to control pest psyllids and/or the plant pathogens they carry. L. sulfurea reads, and 2) no other SVs in L. sulfurea were similar to the bacterial sequences found in the animal intestinal flora. Gram-positive E. faecalis (Firmicutes: Bacilli: Lactobacillales: Enterococcaceae), which is distantly related to all other Gram-negative proteobacterial symbionts identified in the present study, has only been recognized as a transient gut resident in insects (Akami et al., 2019). Therefore, the localization and functional role of this bacterium in L. sulfurea need to be examined in more detail.

Conclusions
The present study identified various bacterial symbionts in nine psyllid species of the family Triozidae. The majority of secondary symbionts were gammaproteobacteria, particularly those of the order Enterobacterales, including Arsenophonus and S. symbiotica. Regarding non-Enterobacterales gammaproteobacteria, Diplorickettsia (Diplorickettsiales: Diplorickettsiaceae), a potential human pathogen, and Carnimonas (Oceanospirillales: Halomonadaceae), a lineage independent of Carsonella within the family Halomonadaceae, were identified. As for alphaproteobacteria, the potential plant pathogen CLeu (Rhizobiales: Rhizobiaceae) was detected for the first time in E. yasumatsui. Since E. yasumatsui feeds on the Japanese silverberry E. umbellata (Elaeagnaceae), which is an aggressive invasive plant in the United States and Europe, the combination of E. yasumatsui and CLeu may potentially be exploited as a biological herbicide for E. umbellata. Wolbachia (Rickettsiales: Anaplasmataceae) strains of supergroup B were identified in three Triozidae species, whereas a lineage belonging to supergroup O was detected in T. cinnamomi, which is the first report of this supergroup in Psylloidea. Moreover, E. faecalis (Firmicutes: Bacilli: Lactobacillales: Enterococcaceae), which has only been recognized as a transient resident in insects, was suggested to constitute a large part of the microbiome in L. sulfurea, implying that this bacterium acquired the status of a stable symbiont. These results provide more detailed insights into the interactions among insects, bacteria, and plants, which may be exploited to facilitate the control of pest psyllids in the future.