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Stuehler et al. Microbiome Res Rep. 2025;4:19 https://dx.doi.org/10.20517/mrr.2024.84 Page 3 of 16

[22]
Wolbachia are currently classified into separate monophyletic lineages, or supergroups, denoted A to S .
Here, we looked to classify the evolutionary placement of Wolbachia endosymbiont of Leuronota fagarae
(wLfag-FL) in the supergroup classification system and to examine the transcriptome of wLfag-FL to
uncover conserved or unique transcripts informing the symbiotic relationships of Wolbachia and psyllid
hosts. Our study uses an multilocus sequence typing (MLST) of nine loci to classify wLfag-FL phylogeny
and leverages published Wolbachia genomes from D. citri to perform a comparative analysis of Wolbachia
loci found in two psyllid species invasive to Florida, USA. L. fagarae presents an important organism to
study in parallel to D. citri, as recent research on the Australian finger lime produced a novel line of stable
antimicrobial peptides (SAMPs) to bolster citrus immunity against CLas infection. Since L. fagarae feeds on
the citrus relative wild lime, is closely related to D. citri, and is invasive to Florida, the biology of L. fagarae
and its endosymbionts informs HLB research and may reveal more naturally occurring therapeutic
molecules to combat HLB. A thorough comparative investigation of the wLfag-FL transcriptome was not
possible due to the low number of transcripts recovered from wLfag-FL, although transcripts encoding
proteins essential for nutrient provisioning, proteins with secretory domains, and proteins with ankyrin
repeat (ANK) domains characteristic to Wolbachia were identified to inform host-microbe interactions.

METHODS
L. fagarae collection
Colonies of wild-caught L. fagarae found on Zanthoxylum fagara were maintained at the University of
Florida Southwest Florida Research & Education Center in Immokalee, FL, U.S.A., for 5 years. L. fagarae
colonies were maintained on citrus relative Zanthoxylum fagara (L.) Sarg (Sapindales: Rutaceae) by the
Qureshi lab and sample collection from the colony occurred in 2017. After collection, psyllids were
immediately placed into TRIzol -LS Reagent (Invitrogen #10296028, 200 mL) for RNA purification .
TM
TM
[23]
RNA extraction, cDNA preparation, and library sequencing
Four cDNA libraries were made from RNA extracted from salivary glands, heads, or whole-body tissue of L.
fagarae. Both cDNA libraries from the salivary gland and head tissues were obtained from combined male
and female samples, while both whole-body cDNA libraries were made from either male or female psyllids.
In total, 900 psyllids were collected to build cDNA datasets where 500 psyllids were dissected for the mixed
salivary gland dataset, 380 psyllids were dissected for the mixed heads dataset, and 10 psyllids each, male
and female, were used for the whole-body dataset RNA extractions. RNA was purified using the Direct-zol
RNA MicroPrep method for the adult whole body and mixed heads extractions, and TRIzol LS, Na-Acetate/
ethanol precipitation was used to purify the mixed salivary glands extractions. Total RNA concentration
dissolved in nuclease-free water was assessed with the Qubit BR assay. The results showed that the mixed
salivary gland extractions yielded 2.8 ng/μL of RNA, the mixed head extractions yielded 4.4 ng/μL of RNA,
adult males yielded 30.8 ng/μL of RNA, and adult females yielded 134 ng/μL of RNA. To sequence the
transcriptome of each tissue or whole psyllid, we used the NEB Ultra II Directional RNAseq Library Prep
Kit from Illumina paired with the Illumina NextSeq 500 Mid mode throughput 2X150 method. Adapters
and low-quality reads (Q < 20) were removed at the University of Florida Interdisciplinary Center for
Biotechnology Research (UF|ICBR). Raw reads have been deposited into the Sequence Read Archive under
BioProject PRJNA789049 and assembled transcripts are found in BioSample SAMN46999246.

Data processing and sequence identification
De novo assemblies of four resulting cDNA libraries (salivary glands, heads, whole-body males, and whole-
body females) were performed using the TRINITY RNA-seq assembler v1.8.0 with default parameters.
[24]
For the initial characterization of Wolbachia sequences, nine local databases containing conserved
Wolbachia housekeeping genes: 16S rRNA, wsp, coxA, fbpA, ftsZ, gatB, gltA, groEL, and hcpA were built
from transcripts available on NCBI. The Trinity assembled transcripts were aligned to each of the nine local