16S rRNA Gene Sequencing of Six Psyllid Species of the Family Carsidaridae Identified Various Bacteria Including <i>Symbiopectobacterium</i>

Abstract

Psyllids (Hemiptera: Sternorrhyncha: Psylloidea) are plant sap-sucking insects that are closely associated with various microbes. To obtain a more detailed understanding of the ecological and evolutionary behaviors of microbes in Psylloidea, the bacterial populations of six psyllid species, belonging to the family Carsidaridae, were analyzed using high-throughput amplicon sequencing of the 16S rRNA gene. The majority of the secondary symbionts identified in the present study were gammaproteobacteria, particularly those of the order Enterobacterales , including Arsenophonus and Sodalis , which are lineages found in a wide variety of insect hosts. Additionally, Symbiopectobacterium , another Enterobacterales lineage, which has recently been recognized and increasingly shown to be vertically transmitted and mutualistic in various invertebrates, was identified for the first time in Psylloidea. This lineage is closely related to Pectobacterium spp., which are plant pathogens, but forms a distinct clade exhibiting no pathogenicity to plants. Non- Enterobacterales gammaproteobacteria found in the present study were Acinetobacter , Pseudomonas (both Pseudomonadales ), Delftia , Comamonas (both Burkholderiales ), and Xanthomonas ( Xanthomonadales ), a putative plant pathogen. Regarding alphaproteobacteria, three Wolbachia ( Rickettsiales ) lineages belonging to supergroup B, the major group in insect lineages, were detected in four psyllid species. In addition, a Wolbachia lineage of supergroup O, a minor group recently found for the first time in Psylloidea, was detected in one psyllid species. These results suggest the pervasive transfer of bacterial symbionts among animals and plants, providing deeper insights into the evolution of the interactions among these organisms.

Microbiome of psyllids of the family Aphalaridae, includingAphalara itadori, a potential biocontrol agent againstReynoutriaspp

“…SV7 was 98.1% identical to that of “ Candidatus Sodalis pierantonius” (AF548137), the primary symbiont of the rice weevil Sitophilus oryzae (Coleoptera: Curculionidae) (Dale et al ., 2003), and 97.9% identical to the sequence of the type species Sodalis glossinidius (AP008232), a secondary symbiont of the tsetse fly Glossina morsitans (Diptera: Glossinidae) (Dale and Maudlin, 1999). SV7 formed a clade with Sodalis symbionts found in various insects, including those of other psyllid species, Homotoma unifasciata (Carsidaridae: Homotominae; TAAE01000023, TAAE01000026, and TAAE01000031, 95.8% – 96.7% identical to SV7) (Maruyama et al ., 2023), Cacopsylla kiushuensis (Psyllidae: Psyllinae; TAAB01000030, 96.5% identical to SV7), Cacopsylla burckhardti (Psyllidae: Psyllinae; TAAB01000016, 96.7% identical to SV7) (Nakabachi et al ., 2022b), and C. miyatakeai (Aphalaridae: Aphalarinae; TAAR01000006=SV6, 96.7% identical to SV7, see below) (Fig. 3).…”

“…Bacterial populations in psyllids were analyzed using the MiSeq system (Illumina), as described previously (Nakabachi, Malenovský, et al ., 2020; Nakabachi et al ., 2022b, 2022a; Maruyama et al ., 2023). Amplicon polymerase chain reaction (PCR) was performed using DNA extracted from psyllids, KAPA HiFi HotStart ReadyMix (KAPA Biosystems), and the primer set 16S_341Fmod (5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGYYTAMGGRNGGCWGC AG-3′) and 16S_805R (5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATC TAATCC-3′), targeting the V3 and V4 regions of the 16S rRNA gene.…”

“…The output sequences were processed using the QIIME2 platform (version 2022.11) (Bolyen et al ., 2019) as described previously (Nakabachi, Malenovský, et al ., 2020; Nakabachi et al ., 2022b, 2022a; Maruyama et al ., 2023). Following primer removal, paired-end reads were denoised and joined, and low-quality or chimeric reads were discarded using the dada2 plugin (Callahan et al ., 2016).…”

“…Through this feeding habit, some psyllid lineages transmit plant pathogens, including “ Candidatus Liberibacter spp.” (Alphaproteobacteria: Rhizobiales: Rhizobiaceae) and “ Candidatus Phytoplasma spp.” (Bacilli: Acholeplasmatales: Acholeplasmataceae), causing severe problems for agriculture (Jarausch and Jarausch, 2010; Grafton-Cardwell et al ., 2013; Mora et al ., 2021). Psyllids depend on vertically transmitted bacterial mutualists to compensate for the nutritional deficiencies in the phloem sap (Profft, 1937; Buchner, 1965; Fukatsu and Nikoh, 1998; Subandiyah et al ., 2000; Thao, Clark, et al ., 2000; Thao, Moran, et al ., 2000; Spaulding and von Dohlen, 2001; Nakabachi et al ., 2006, 2010; Sloan and Moran, 2012; Nakabachi, Ueoka, et al ., 2013; Arp et al ., 2014; Sloan et al ., 2014; Hall et al ., 2016; Morrow et al ., 2017; Nakabachi, Malenovský, et al ., 2020; Nakabachi, Piel, et al ., 2020; Kwak et al ., 2021; Nakabachi et al ., 2022a, 2022b; Nakabachi and Moran, 2022; Dittmer et al ., 2023; Maruyama et al ., 2023; Nakabachi and Suzaki, 2023). They have a specialized organ called the bacteriome (Nakabachi et al ., 2010; Sloan et al ., 2014; Nakabachi and Suzaki, 2023), harboring typically two intracellular symbiont species transovarially transmitted through generations.…”

“…One of them is the primary symbiont “ Candidatus Carsonella ruddii” (Gammaproteobacteria: Oceanospirillales: Halomonadaceae) (Thao, Moran, et al ., 2000), providing the host with essential amino acids scarce in the phloem sap (Nakabachi et al ., 2006; Sloan and Moran, 2012; Nakabachi, Ueoka, et al ., 2013; Nakabachi, Piel, et al ., 2020; Dittmer et al ., 2023), and is thus considered essential for Psylloidea. Molecular phylogenetic analyses demonstrated a single acquisition of a Carsonella ancestor by a psyllid common ancestor and its subsequent stable vertical transmission (Thao, Moran, et al ., 2000; Spaulding and von Dohlen, 2001; Hall et al ., 2016; Nakabachi, Malenovský, et al ., 2020; Nakabachi et al ., 2022b, 2022a; Maruyama et al ., 2023). Another bacteriome-associated bacterium is classified as a ‘secondary symbiont,’ with wide phylogenetic variations among host lineages, suggesting its repeated replacements during the evolution of Psylloidea (Thao, Clark, et al ., 2000; Spaulding and von Dohlen, 2001; Sloan and Moran, 2012; Hall et al ., 2016; Morrow et al ., 2017; Nakabachi, Malenovský, et al ., 2020; Nakabachi et al ., 2022b, 2022a).…”