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Page 2 of 16 Stuehler et al. Microbiome Res Rep. 2025;4:19 https://dx.doi.org/10.20517/mrr.2024.84

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: Wolbachia, Leuronota fagarae, wild lime psyllid, Citrus greening, Huanglongbing, microbiome, Diaphorina
citri

INTRODUCTION
The Wild Lime Psyllid, Leuronota fagarae (L. fagarae) Burckhardt (Hemiptera: Psylloidea), is an invasive
[1]
species of Florida, USA, with origins traced to Paraguay, South America . The first report of L. fagarae in
the United States occurred in southern Florida when adults were discovered feeding and reproducing on
[1]
citrus relative Zanthoxylum fagara (L.) Sarg. (Sapindales: Rutaceae) . The 2001 discovery of L. fagarae in
southern Florida led to the first classification of the species since its original description in 1988 from two
[2]
locations in Paraguay . Rolled leaf edges that provide safeguards for developing nymphs indicate a typical
phenotype of L. fagarae feeding on citrus . In Florida, the habitat range of L. fagarae overlaps with the Asan
[1]
Citrus Psyllid (ACP, Diaphorina citri Kuwayama) in the transition zone from non-cultivated areas to areas
of cultivated citrus trees. This proximity, as well as taxonomic relationship and potentially shared food
source, provides an opportunity for the transfer of intracellular microbes through plant feeding , such as
[3]
Wolbachia spp. .
[4]
The parasitism-mutualism balance linking host and endosymbionts is a major influence on systemic
biological processes within an organism . Previous studies have determined that endosymbionts influence
[5]
fundamental aspects of host insect biology, including nutritional mechanisms , reproduction , tolerance of
[7]
[6]
[8,9]
abiotic conditions , and the reduction of an insect’s ability to vector pathogens . Molecular pathways
[10]
critical to the health of endosymbionts and insect hosts provide targets of interest for designing therapeutics
to induce phenotypic changes in the insect. Additionally, symbiotic relationships are of interest in
evolutionary genetics, as codependence can result in genetic markers in endosymbiont genomes, which
inform the evolutionary history and adaptation of the insect [11,12] . Insect diets deficient in vitamins, sterols,
[13]
and essential amino acids are supplemented by symbiotic microorganisms that produce the necessary
dietary nutrients . Sap ingesting psyllids, such as L. fagarae, are hosts to a variety of bacterial
[14]
endosymbionts, most notably Gram-negative Wolbachia of the order Rickettsiales, which are currently
found in 40%-60% of insect species [15,16] . Our characterization of Wolbachia in L. fagarae is specifically
[17]
noteworthy due to Wolbachia’s potential to modulate the fitness of disease-vectoring insects and its close
relationship to the Wolbachia strain found in the Huanglongbing (HLB) vector Diaphorina citri. Previous
analysis of Wolbachia genome assemblies further determined that these microorganisms are without
complete sets of metabolic pathways present in closely related, nonsymbiotic bacteria of the order
Rickettsiales , suggesting that Wolbachia can manipulate host metabolic pathways [19,20] . This dynamic
[18]
influence, along with the exclusive vertical transmission through maternal hosts, contributes to the
exploitation of major reproductive influences through Wolbachia-host relationships .
[21]