Transcriptomic characterization of Wolbachia endosymbiont from Leuronota fagarae (Hemiptera: Psylloidae)

Abstract

Aim: Wolbachia species are among the most abundant intracellular endosymbionts of insects worldwide. The extensive distribution of Gram-negative Wolbachia among insects highlights their evolutionary success and close relationship with many insect host species. This study aimed to characterize a novel Wolbachia strain from the Wild Lime Psyllid, Leuronota fagarae (L. fagarae), to understand its evolutionary relationship with Wolbachia from psyllid pests like Diaphorina citri, the vector of Huanglongbing (HLB). Methods: Wild-caught L. fagarae colonies from Florida, USA, were maintained on Zanthoxylum fagara. RNA was extracted from the salivary glands, heads, and whole bodies of male and female adult L. fagarae. Four cDNA libraries were sequenced using short read technology and de novo transcriptome assembly was performed. Multilocus sequence typing (MLST) of nine conserved loci and wsp gene analysis classified the strain's phylogeny, while sequence mapping and functional annotation provided insight into host-microbe interactions. Results: The new Wolbachia strain, designated Wolbachia endosymbiont of Leuronota fagarae (wLfag-FL), was assigned to supergroup B, showing relation to Wolbachia strains of other related psyllids. Transcriptome analysis identified 1,359 Wolbachia transcripts with 465 assigned functions encompassing metabolic and secretion system pathways. Ankyrin domain proteins and a partial bacterioferritin sequence were detected, suggesting nutritional provisioning roles. Conclusion: The characterization of wLfag-FL expands the known Wolbachia host range and informs HLB-related pest biology. Its phylogenetic placement and transcript annotations offer insights into symbiotic interactions, potentially guiding environmentally safe pest control strategies targeting psyllid fitness and pathogen transmission.

Keywords: Citrus greening; Diaphorina citri; Huanglongbing; Leuronota fagarae; Wolbachia; microbiome; wild lime psyllid.

Figure 1

Unrooted Maximum Likelihood tree for Wolbachia MLST concatenated sequences supporting placement of wLfag-FL in Supergroup B. MLST: Multilocus sequence typing; wLfag-FL: Wolbachia endosymbiont of Leuronota fagarae.

Figure 2

Unrooted Maximum Likelihood tree for Wolbachia wsp sequences highlighting the placement of wLfag-FL in Supergroup B with a distinct evolutionary lineage. wLfag-FL: Wolbachia endosymbiont of Leuronota fagarae.

Figure 3

AliTV linear alignment map of wLfag-FL concatenated transcripts mapped to closely related Wolbachia genomes of wDcit1 and wSpic. Accession numbers of full genome references are in Table 1. wLfag-FL: Wolbachia endosymbiont of Leuronota fagarae; wDcit1: Wolbachia endosymbiont of D. citri dawsonii; wSpic: Wolbachia endosymbiont of S. picta.

Figure 4

BRIG alignment of 11 Wolbachia endosymbiont genomes belonging to Supergroup B used in the MLST phylogenetic classification of wLfag-FL. Wolbachia endosymbiont of S. picta was set as the reference genome (red); the outermost ring (dark green) is made of wLfag-FL transcripts. Genome assembly lengths are in parenthesis next to the names of the Wolbachia strain. Genome accessions are in Table 1. BRIG: Blast Ring Image Generator; MLST: multilocus sequence typing; wLfag-FL: Wolbachia endosymbiont of Leuronota fagarae.

Figure 5

Bar graph containing 13 KEGG Orthology pathways having 10 or more sequence assignments from Wolbachia identified in L. fagarae FL-isolates. A full table of sequences assigned to KO identifiers is available in Supplementary Table 1.

Figure 6

MUSCLE alignment of 8 bacterioferritin sequences. Residues with a 100% identity between all 8 sequences are highlighted. Sequence accessions are available in Supplementary Table 2.