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

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

References

[1] Russell DN,Halbert SE.Host suitability of citrus and Zanthoxylum spp. for Leuronota fagarae burckhardt and Diaphorina citri kuwayama (Hemiptera: Psylloidea).Fla Entomol2014;97:1481-92

[2] Burckhardt D.Jumping plant lice (Homoptera: Psylloidea) of the temperate neotropical region. Part 3: calophyidae and triozidae.Zool J Linn Soc1988;92:115-91

[3] Chrostek E,Hurst GDD.Horizontal transmission of intracellular insect symbionts via plants.Front Microbiol2017;8:2237 PMCID:PMC5712413

[4] Sintupachee S,Poonchaisri S,Kittayapong P.Closely related Wolbachia strains within the pumpkin arthropod community and the potential for horizontal transmission via the plant.Microb Ecol2006;51:294-301

[5] Hoffmann AA.Describing endosymbiont-host interactions within the parasitism-mutualism continuum.Ecol Evol2024;14:e11705 PMCID:PMC11224498

[6] Baumann P.Biology bacteriocyte-associated endosymbionts of plant sap-sucking insects.Annu Rev Microbiol2005;59:155-89

[7] Benlarbi M.Host-specific Wolbachia strains in widespread populations of Phlebotomus perniciosus and P. papatasi (Diptera: Psychodidae), and prospects for driving genes into these vectors of Leishmania.Bull Entomol Res2003;93:383-91

[8] Charlesworth J,Araujo EV Jr.Wolbachia, Cardinium and climate: an analysis of global data.Biol Lett2019;15:20190273 PMCID:PMC6731486

[9] Hague MTJ,Cooper BS. Divergent effects of Wolbachia on host temperature preference. bioRxiv 2020. Available from: http://biorxiv.org/lookup/doi/10.1101/2020.06.11.146977. [Last accessed on 31 Mar 2025]

[10] Werren JH,Clark ME.Wolbachia: master manipulators of invertebrate biology.Nat Rev Microbiol2008;6:741-51

[11] Manthey JD,Hruska JP.Impact of host demography and evolutionary history on endosymbiont molecular evolution: a test in carpenter ants (genus Camponotus) and their Blochmannia endosymbionts.Ecol Evol2022;12:e9026 PMCID:PMC9251289

[12] Bontemps Z,Guy L.Host-bacteria interactions: ecological and evolutionary insights from ancient, professional endosymbionts.FEMS Microbiol Rev2024;48:fuae021 PMCID:PMC11338181

[13] Kraus S,Gomez-Moracho T.Insect diet. In: Vonk J, Shackelford T, editors. Encyclopedia of animal cognition and behavior. Cham: Springer International Publishing; 2019. p. 1-9.

[14] Douglas AE.Microbial nutrient factories in insects on extreme diets.Comptes Rendus Biol2019;342:260

[15] Fagen JR,Brown CT,Gano KA.Characterization of the relative abundance of the citrus pathogen Ca. Liberibacter asiaticus in the microbiome of its insect vector, Diaphorina citri, using high throughput 16S rRNA sequencing.Open Microbiol J2012;6:29-33 PMCID:PMC3330398

[16] Zug R.Still a host of hosts for Wolbachia: analysis of recent data suggests that 40% of terrestrial arthropod species are infected.PLoS One2012;7:e38544 PMCID:PMC3369835

[17] Roldán EL,Pelz-Stelinski KS.Reduction of Wolbachia in Diaphorina citri (Hemiptera: Liviidae) increases phytopathogen acquisition and decreases fitness.J Econ Entomol2024;117:733-49

[18] Bi J.The effect of the endosymbiont Wolbachia on the behavior of insect hosts.Insect Sci2020;27:846-58 PMCID:PMC7496987

[19] Saucereau Y,Dieryckx C.Comprehensive proteome profiling in Aedes albopictus to decipher Wolbachia-arbovirus interference phenomenon.BMC Genomics2017;18:635 PMCID:PMC5563009

[20] Yuan LL,Zong Q.Quantitative proteomic analyses of molecular mechanisms associated with cytoplasmic incompatibility in Drosophila melanogaster induced by Wolbachia.J Proteome Res2015;14:3835-47

[21] Guo Y,Xu XQ.Vertical transmission of Wolbachia is associated with host vitellogenin in Laodelphax striatellus.Front Microbiol2018;9:2016 PMCID:PMC6127624

[22] Lefoulon E,Borveto F.Pseudoscorpion Wolbachia symbionts: diversity and evidence for a new supergroup S.BMC Microbiol2020;20:188 PMCID:PMC7325362

[23] Cicero JM.A stationary tweezer platform for high throughput dissections of minute arthropods and extirpation of their minute organs.MethodsX2021;8:101317 PMCID:PMC8374364

[24] Grabherr MG,Yassour M.Full-length transcriptome assembly from RNA-Seq data without a reference genome.Nat Biotechnol2011;29:644-52 PMCID:PMC3571712

[25] Kim D,Park C,Salzberg SL.Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype.Nat Biotechnol2019;37:907-15 PMCID:PMC7605509

[26] Manni M,Seppey M,Zdobnov EM.BUSCO update: novel and streamlined workflows along with broader and deeper phylogenetic coverage for scoring of eukaryotic, prokaryotic, and viral genomes.Mol Biol Evol2021;38:4647-54 PMCID:PMC8476166

[27] Okonechnikov K, Golosova O, Fursov M; UGENE team. Unipro UGENE: a unified bioinformatics toolkit. Bioinformatics. 2012;28:1166-7.

[28] Uday J.Comparative analysis of Wolbachia surface protein in D. melanoagster, A. tabida and B. malayi.Bioinformation2012;8:711-5 PMCID:PMC3449380

[29] Kanehisa M.Toward understanding the origin and evolution of cellular organisms.Protein Sci2019;28:1947-51 PMCID:PMC6798127

[30] Kanehisa M,Sato Y,Tanabe M.KEGG: integrating viruses and cellular organisms.Nucleic Acids Res2021;49:D545-51 PMCID:PMC7779016

[31] Kanehisa M.KEGG: kyoto encyclopedia of genes and genomes.Nucleic Acids Res2000;28:27-30 PMCID:PMC102409

[32] Gasteiger E,Hoogland C,Appel RD.ExPASy: the proteomics server for in-depth protein knowledge and analysis.Nucleic Acids Res2003;31:3784-8 PMCID:PMC168970

[33] Van Meer MM, Witteveldt J, Stouthamer R. Phylogeny of the arthropod endosymbiont Wolbachia based on the wsp gene.Insect Mol Biol1999;8:399-408

[34] Waterhouse AM,Martin DM,Barton GJ.Jalview Version 2 - a multiple sequence alignment editor and analysis workbench.Bioinformatics2009;25:1189-91 PMCID:PMC2672624

[35] Shen W,Li Y.SeqKit: a cross-platform and ultrafast toolkit for FASTA/Q file manipulation.PLoS One2016;11:e0163962 PMCID:PMC5051824

[36] Ankenbrand MJ,Hackl T.AliTV - interactive visualization of whole genome comparisons.PeerJ Computer Science2017;3:e116

[37] Alikhan NF,Ben Zakour NL.BLAST ring image generator (BRIG): simple prokaryote genome comparisons.BMC Genomics2011;12:402 PMCID:PMC3163573

[38] Correa CC.Wolbachia associations with insects: winning or losing against a master manipulator.Front Ecol Evol2016;3:153

[39] Stouthamer R,Hurst GD.Wolbachia pipientis: microbial manipulator of arthropod reproduction.Annu Rev Microbiol1999;53:71-102

[40] Singh ND.Wolbachia infection associated with increased recombination in drosophila.G32019;9:229-37 PMCID:PMC6325905

[41] Zhou W,O’Neil S.Phylogeny and PCR-based classification of Wolbachia strains using wsp gene sequences.Proc Biol Sci1998;265:509-15 PMCID:PMC1688917

[42] Baldo L,Jolley KA.Multilocus sequence typing system for the endosymbiont Wolbachia pipientis.Appl Environ Microbiol2006;72:7098-110 PMCID:PMC1636189

[43] Chapman EG,Harwood JD.Determining the origin of the coffee berry borer invasion of Hawaii.Ann Entomol Soc Am2015;108:585-92

[44] Klasson L,Sebaihia M.Genome evolution of Wolbachia strain wPip from the Culex pipiens group.Mol Biol Evol2008;25:1877-87 PMCID:PMC2515876

[45] Saha S,Reese J.Survey of endosymbionts in the Diaphorina citri metagenome and assembly of a Wolbachia wDi draft genome.PLoS One2012;7:e50067 PMCID:PMC3500351

[46] Wu M,Vamathevan J.Phylogenomics of the reproductive parasite Wolbachia pipientis wMel: a streamlined genome overrun by mobile genetic elements.PLoS Biol2004;2:E69

[47] Al-Khodor S,Kalia A.Functional diversity of ankyrin repeats in microbial proteins.Trends Microbiol2010;18:132-9 PMCID:PMC2834824

[48] Hryniewicz-Jankowska A,Bok E.Ankyrins, multifunctional proteins involved in many cellular pathways.Folia Histochem Cytobiol2002;40:239-49

[49] Voronin DA.[Functional role of proteins containing ankyrin repeats].Tsitologiia2007;49:989-99

[50] Kremer N,Charif D,Mollereau B.Wolbachia interferes with ferritin expression and iron metabolism in insects.PLoS Pathog2009;5:e1000630 PMCID:PMC2759286

[51] Ebrahimi K, Hagedoorn PL, Hagen WR. Unity in the biochemistry of the iron-storage proteins ferritin and bacterioferritin.Chem Rev2015;115:295-326

[52] Foster J,Kamal I.The Wolbachia genome of Brugia malayi: endosymbiont evolution within a human pathogenic nematode.PLoS Biol2005;3:e121 PMCID:PMC1069646

[53] Nikoh N,Moriyama M,Hattori M.Evolutionary origin of insect-Wolbachia nutritional mutualism.Proc Natl Acad Sci U S A2014;111:10257-62 PMCID:PMC4104916

[54] Baldridge GD,Witthuhn BA,Baldridge AS.The Wolbachia WO bacteriophage proteome in the Aedes albopictus C/wStr1 cell line: evidence for lytic activity?.In Vitro Cell Dev Biol Anim2016;52:77-88 PMCID:PMC4701759

[55] Gan HM,Rahman AY,Savka MA.Comparative genomic analysis of six bacteria belonging to the genus Novosphingobium: insights into marine adaptation, cell-cell signaling and bioremediation.BMC Genomics2013;14:431 PMCID:PMC3704786

[56] Sullivan MB,Ignacio-Espinoza JC.Genomic analysis of oceanic cyanobacterial myoviruses compared with T4-like myoviruses from diverse hosts and environments.Environ Microbiol2010;12:3035-56 PMCID:PMC3037559

[57] Iyer LM,de Souza RF.Origin and evolution of peptide-modifying dioxygenases and identification of the wybutosine hydroxylase/hydroperoxidase.Nucleic Acids Res2010;38:5261-79 PMCID:PMC2938197

[58] Mistry J,Williams L.Pfam: the protein families database in 2021.Nucleic Acids Res2021;49:D412-9 PMCID:PMC7779014

[59] Beck K,Brunner J.Discrimination between SRP- and SecA/SecB-dependent substrates involves selective recognition of nascent chains by SRP and trigger factor.EMBO J2000;19:134-43 PMCID:PMC1171785

[60] Rancès E,Tran-Van V.Genetic and functional characterization of the type IV secretion system in Wolbachia.J Bacteriol2008;190:5020-30 PMCID:PMC2447017

[61] Higgins SA,Heck M.Strain tracking of ‘Candidatus Liberibacter asiaticus’, the citrus greening pathogen, by high-resolution microbiome analysis of Asian citrus psyllids.Phytopathology2022;112:2273-87