Neuropeptides are small signaling molecules expressed in the tick central nervous system, i.e., the synganglion. The neuronal-like Ixodes scapularis embryonic cell line, ISE6, is an effective tool frequently used for examining tick-pathogen interactions. We detected 37 neuropeptide transcripts in the I. scapularis ISE6 cell line using in silico methods, and six of these neuropeptide genes were used for experimental validation. Among these six neuropeptide genes, the tachykinin-related peptide (TRP) of ISE6 cells varied in transcript expression depending on the infection strain of the tick-borne pathogen, Anaplasma phagocytophilum. The immunocytochemistry of TRP revealed cytoplasmic expression in a prominent ISE6 cell subpopulation. The presence of TRP was also confirmed in A. phagocytophilum-infected ISE6 cells. The in situ hybridization and immunohistochemistry of TRP of I. scapularis synganglion revealed expression in distinct neuronal cells. In addition, TRP immunoreaction was detected in axons exiting the synganglion via peripheral nerves as well as in hemal nerve-associated lateral segmental organs. The characterization of a complete Ixodes neuropeptidome in ISE6 cells may serve as an effective in vitro tool to study how tick-borne pathogens interact with synganglion components that are vital to tick physiology. Therefore, our current study is a potential stepping stone for in vivo experiments to further examine the neuronal basis of tick-pathogen interactions.

AgroParisTech Micalis Institute Université Paris Saclay PAPPSO INRAE 78350 Jouy en Josas France

Center for Veterinary Health Sciences Department of Veterinary Pathobiology Oklahoma State University Stillwater OK 74078 USA

Department of Virology Veterinary Research Institute Hudcova 70 62100 Brno Czech Republic

Faculty of Science Pavol Jozef Šafarik University in Košice 04180 Košice Slovakia

Institute of Parasitology Biology Centre of the Czech Academy of Sciences Branisovska 31 37005 Ceske Budejovice Czech Republic

Neuroplasticity and Neurodegeneration Group Regional Centre for Biomedical Research Ciu dad Real Medical School University of Castilla La Mancha 13071 Ciudad Real Spain

SaBio Instituto de Investigación en Recursos Cinegéticos IREC CSIC UCLM JCCM Ronda de Toledo s n 13005 Ciudad Real Spain

UMR BIPAR Laboratoire de Santé Animale ANSES INRAE Ecole Nationale Vétérinaire d'Alfort Paris Est Sup 94700 Maisons Alfort France

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Hamer S.A., Tsao J.I., Walker E.D., Hickling G.J. Invasion of the lyme disease vector Ixodes scapularis: Implications for Borrelia burgdorferi endemicity. Ecohealth. 2010;7:47–63. doi: 10.1007/s10393-010-0287-0. PubMed DOI

Rizzoli A., Silaghi C., Obiegala A., Rudolf I., Hubálek Z., Földvári G., Plantard O., Vayssier-Taussat M., Bonnet S., Špitalská E., et al. Ixodes ricinus and Its Transmitted Pathogens in Urban and Peri-Urban Areas in Europe: New Hazards and Relevance for Public Health. Front. Public Health. 2014;2 doi: 10.3389/fpubh.2014.00251. PubMed DOI PMC

Kocan K.M., De la Fuente J., Cabezas-Cruz A. The genus Anaplasma: New challenges after reclassification. Rev. Off. Int. Epizoot. 2015;34:577–586. doi: 10.20506/rst.34.2.2381. PubMed DOI

Kocan K.M., De la Fuente J., Blouin E.F., Garcia-Garcia J.C. Anaplasma marginale (Rickettsiales: Anaplasmataceae): Recent advances in defining host-pathogen adaptations of a tick-borne rickettsia. Parasitology. 2004;129:S285–S300. doi: 10.1017/S0031182003004700. PubMed DOI

De la Fuente J., Antunes S., Bonnet S., Cabezas-Cruz A., Domingos A.G., Estrada-Peña A., Johnson N., Kocan K.M., Mansfield K.L., Nijhof A.M., et al. Tick-Pathogen Interactions and Vector Competence: Identification of Molecular Drivers for Tick-Borne Diseases. Front. Cell. Infect. Microbiol. 2017;7 doi: 10.3389/fcimb.2017.00114. PubMed DOI PMC

Baldridge G.D., Burkhardt N.Y., Labruna M.B., Pacheco R.C., Paddock C.D., Williamson P.C., Billingsley P.M., Felsheim R.F., Kurtti T.J., Munderloh U.G. Wide dispersal and possible multiple origins of low-copy-number plasmids in rickettsia species associated with blood-feeding arthropods. Appl. Environ. Microbiol. 2010;76:1718–1731. doi: 10.1128/AEM.02988-09. PubMed DOI PMC

Munderloh U.G., Blouin E.F., Kocan K.M., Ge N.L., Edwards W.L., Kurtti T.J. Establishment of the Tick (Acari: Ixodidae)-Borne Cattle Pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae) in Tick Cell Culture. J. Med. Entomol. 1996;33:656–664. doi: 10.1093/jmedent/33.4.656. PubMed DOI

Munderloh U.G., Silverman D.J., MacNamara K.C., Ahlstrand G.G., Chatterjee M., Winslow G.M. Ixodes ovatus Ehrlichia exhibits unique ultrastructural characteristics in mammalian endothelial and tick-derived cells. Ann. N. Y. Acad. Sci. 2009;1166:112–119. doi: 10.1111/j.1749-6632.2009.04520.x. PubMed DOI PMC

Munderloh U.G., Yabsley M.J., Murphy S.M., Luttrell M.P., Howerth E.W. Isolation and establishment of the raccoon Ehrlichia-like agent in tick cell culture. Vector Borne Zoonotic Dis. 2007;7:418–425. doi: 10.1089/vbz.2007.0640. PubMed DOI

Tate C.M., Howerth E.W., Mead D.G., Dugan V.G., Luttrell M.P., Sahora A.I., Munderloh U.G., Davidson W.R., Yabsley M.J. Anaplasma odocoilei sp. nov. (family Anaplasmataceae) from white-tailed deer (Odocoileus virginianus) Ticks Tick Borne Dis. 2013;4:110–119. doi: 10.1016/j.ttbdis.2012.09.005. PubMed DOI PMC

Pornwiroon W., Pourciau S.S., Foil L.D., Macaluso K.R. Rickettsia felis from Cat Fleas: Isolation and Culture in a Tick-Derived Cell Line. Appl. Environ. Microbiol. 2006;72:5589–5595. doi: 10.1128/AEM.00532-06. PubMed DOI PMC

Simser J.A., Palmer A.T., Fingerle V., Wilske B., Kurtti T.J., Munderloh U.G. Rickettsia monacensis sp. nov., a Spotted Fever Group Rickettsia, from Ticks (Ixodes ricinus) Collected in a European City Park. Appl. Environ. Microbiol. 2002;68:4559–4566. doi: 10.1128/AEM.68.9.4559-4566.2002. PubMed DOI PMC

Munderloh U.G., Tate C.M., Lynch M.J., Howerth E.W., Kurtti T.J., Davidson W.R. Isolation of an Anaplasma sp. Organism from White-Tailed Deer by Tick Cell Culture. J. Clin. Microbiol. 2003;41:4328–4335. doi: 10.1128/JCM.41.9.4328-4335.2003. PubMed DOI PMC

Obonyo M., Munderloh U.G., Fingerle V., Wilske B., Kurtti T.J. Borrelia burgdorferi in Tick Cell Culture Modulates Expression of Outer Surface Proteins A and C in Response to Temperature. J. Clin. Microbiol. 1999;37:2137–2141. doi: 10.1128/JCM.37.7.2137-2141.1999. PubMed DOI PMC

Garcia S., Billecocq A., Crance J.-M., Munderloh U., Garin D., Bouloy M. Nairovirus RNA Sequences Expressed by a Semliki Forest Virus Replicon Induce RNA Interference in Tick Cells. J. Virol. 2005;79:8942–8947. doi: 10.1128/JVI.79.14.8942-8947.2005. PubMed DOI PMC

Grabowski J.M., Perera R., Roumani A.M., Hedrick V.E., Inerowicz H.D., Hill C.A., Kuhn R.J. Changes in the Proteome of Langat-Infected Ixodes scapularis ISE6 Cells: Metabolic Pathways Associated with Flavivirus Infection. PLoS Negl. Trop. Dis. 2016;10:e0004180. doi: 10.1371/journal.pntd.0004180. PubMed DOI PMC

Munderloh U.G., Liu Y., Wang M., Chen C., Kurtti T.J. Establishment, maintenance and description of cell lines from the tick Ixodes scapularis. J. Parasitol. 1994;80:533–543. doi: 10.2307/3283188. PubMed DOI

Oliver J.D., Chávez A.S.O., Felsheim R.F., Kurtti T.J., Munderloh U.G. An Ixodes scapularis cell line with a predominantly neuron-like phenotype. Exp. Appl. Acarol. 2015;66:427–442. doi: 10.1007/s10493-015-9908-1. PubMed DOI PMC

Alberdi P., Mansfield K.L., Manzano-Román R., Cook C., Ayllón N., Villar M., Johnson N., Fooks A.R., De la Fuente J. Tissue-Specific Signatures in the Transcriptional Response to Anaplasma phagocytophilum Infection of Ixodes scapularis and Ixodes ricinus Tick Cell Lines. Front. Cell Infect. Microbiol. 2016;6:20. doi: 10.3389/fcimb.2016.00020. PubMed DOI PMC

Artigas-Jerónimo S., Alberdi P., Rayo M.V., Cabezas-Cruz A., Prados P.J.E., Mateos-Hernández L., De la Fuente J. Anaplasma phagocytophilum modifies tick cell microRNA expression and upregulates isc-mir-79 to facilitate infection by targeting the Roundabout protein 2 pathway. Sci. Rep. 2019;9:1–15. doi: 10.1038/s41598-019-45658-2. PubMed DOI PMC

Cabezas-Cruz A., Espinosa P., Alberdi P., De la Fuente J. Tick–Pathogen Interactions: The Metabolic Perspective. Trends Parasitol. 2019;35:316–328. doi: 10.1016/j.pt.2019.01.006. PubMed DOI

Shaw D.K., Wang X., Brown L.J., Chávez A.S.O., Reif K.E., Smith A.A., Scott A.J., McClure E.E., Boradia V.M., Hammond H.L., et al. Infection-derived lipids elicit an immune deficiency circuit in arthropods. Nat. Commun. 2017;8:1–13. doi: 10.1038/ncomms14401. PubMed DOI PMC

De la Fuente J., Villar M., Cabezas-Cruz A., Estrada-Peña A., Ayllón N., Alberdi P. Tick–Host–Pathogen Interactions: Conflict and Cooperation. PLoS Pathog. 2016;12:e1005488. doi: 10.1371/journal.ppat.1005488. PubMed DOI PMC

Lin M., Kikuchi T., Brewer H.M., Norbeck A.D., Rikihisa Y. Global Proteomic Analysis of Two Tick-Borne Emerging Zoonotic Agents: Anaplasma Phagocytophilum and Ehrlichia Chaffeensis. Front. Microbiol. 2011;2 doi: 10.3389/fmicb.2011.00024. PubMed DOI PMC

Ayllón N., Villar M., Galindo R.C., Kocan K.M., Šíma R., López J.A., Vázquez J., Alberdi P., Cabezas-Cruz A., Kopáček P., et al. Systems Biology of Tissue-Specific Response to Anaplasma phagocytophilum Reveals Differentiated Apoptosis in the Tick Vector Ixodes scapularis. PLoS Genet. 2015;11:e1005120. doi: 10.1371/journal.pgen.1005120. PubMed DOI PMC

Cabezas-Cruz A., Alberdi P., Valdés J.J., Villar M., De la Fuente J. Anaplasma phagocytophilum Infection Subverts Carbohydrate Metabolic Pathways in the Tick Vector, Ixodes scapularis. Front. Cell Infect. Microbiol. 2017;7:23. doi: 10.3389/fcimb.2017.00023. PubMed DOI PMC

Cabezas-Cruz A., Espinosa P.J., Obregón D.A., Alberdi P., De la Fuente J. Ixodes scapularis Tick Cells Control Anaplasma phagocytophilum Infection by Increasing the Synthesis of Phosphoenolpyruvate from Tyrosine. Front. Cell Infect. Microbiol. 2017;7:375. doi: 10.3389/fcimb.2017.00375. PubMed DOI PMC

Sinclair S.H.G., Garcia-Garcia J.C., Dumler J.S. Bioinformatic and mass spectrometry identification of Anaplasma phagocytophilum proteins translocated into host cell nuclei. Front. Microbiol. 2015;6 doi: 10.3389/fmicb.2015.00055. PubMed DOI PMC

Sinclair S.H., Rennoll-Bankert K.E., Dumler J.S. Effector bottleneck: Microbial reprogramming of parasitized host cell transcription by epigenetic remodeling of chromatin structure. Front. Genet. 2014;5 doi: 10.3389/fgene.2014.00274. PubMed DOI PMC

Villar M., Ayllón N., Alberdi P., Moreno A., Moreno M., Tobes R., Mateos-Hernández L., Weisheit S., Bell-Sakyi L., De la Fuente J. Integrated Metabolomics, Transcriptomics and Proteomics Identifies Metabolic Pathways Affected by Anaplasma phagocytophilum Infection in Tick Cells. Mol. Cell Proteom. 2015;14:3154–3172. doi: 10.1074/mcp.M115.051938. PubMed DOI PMC

Cabezas-Cruz A., Alberdi P., Valdes J.J., Villar M., De la Fuente J. Remodeling of tick cytoskeleton in response to infection with Anaplasma phagocytophilum. Front. Biosci. 2017;22:1830–1844. doi: 10.2741/4574. PubMed DOI

Benelli G. Pathogens Manipulating Tick Behavior—Through a Glass, Darkly. Pathogens. 2020;9:664. doi: 10.3390/pathogens9080664. PubMed DOI PMC

Randolph S.E. Tick ecology: Processes and patterns behind the epidemiological risk posed by ixodid ticks as vectors. Parasitology. 2004;129:S37–S65. doi: 10.1017/S0031182004004925. PubMed DOI

Neelakanta G., Sultana H., Fish D., Anderson J.F., Fikrig E. Anaplasma phagocytophilum induces Ixodes scapularis ticks to express an antifreeze glycoprotein gene that enhances their survival in the cold. J. Clin. Investig. 2010;120:3179–3190. doi: 10.1172/JCI42868. PubMed DOI PMC

Busby A.T., Ayllón N., Kocan K.M., Blouin E.F., De la Fuente G., Galindo R.C., Villar M., De la Fuente J. Expression of heat shock proteins and subolesin affects stress responses, Anaplasma phagocytophilum infection and questing behaviour in the tick, Ixodes scapularis. Med. Vet. Entomol. 2012;26:92–102. doi: 10.1111/j.1365-2915.2011.00973.x. PubMed DOI

Lynn G.E., Oliver J.D., Nelson C.M., Felsheim R.F., Kurtti T.J., Munderloh U.G. Tissue Distribution of the Ehrlichia muris-Like Agent in a Tick Vector. PLoS ONE. 2015;10 doi: 10.1371/journal.pone.0122007. PubMed DOI PMC

Šimo L., Daniel S.E., Park Y., Žitňan D. The Nervous and Sensory Systems: Structure, Function, Proteomics and Genomics. In: Sonenshine D.E., Roe R.M., editors. Biology of Ticks. Volume 1. Oxford University Press; New York, NY, USA: 2014. pp. 309–367.

Šimo L., Slovák M., Park Y., Zitnan D. Identification of a complex peptidergic neuroendocrine network in the hard tick, Rhipicephalus appendiculatus. Cell Tissue Res. 2009;335:639–655. doi: 10.1007/s00441-008-0731-4. PubMed DOI PMC

Hewes R.S., Taghert P.H. Neuropeptides and Neuropeptide Receptors in the Drosophila melanogaster Genome. Genome Res. 2001;11:1126–1142. doi: 10.1101/gr.169901. PubMed DOI PMC

Caers J., Verlinden H., Zels S., Vandersmissen H.P., Vuerinckx K., Schoofs L. More than two decades of research on insect neuropeptide GPCRs: An overview. Front. Endocrinol. 2012;3 doi: 10.3389/fendo.2012.00151. PubMed DOI PMC

Yeoh J.G.C., Pandit A.A., Zandawala M., Nässel D.R., Davies S.-A., Dow J.A.T. DINeR: Database for Insect Neuropeptide Research. Insect Biochem. Mol. Biol. 2017;86:9–19. doi: 10.1016/j.ibmb.2017.05.001. PubMed DOI

Schoofs L., De Loof A., Van Hiel M.B. Neuropeptides as Regulators of Behavior in Insects. Annu. Rev. Entomol. 2017;62:35–52. doi: 10.1146/annurev-ento-031616-035500. PubMed DOI

Gulia-Nuss M., Nuss A.B., Meyer J.M., Sonenshine D.E., Roe R.M., Waterhouse R.M., Sattelle D.B., De la Fuente J., Ribeiro J.M., Megy K., et al. Genomic insights into the Ixodes scapularis tick vector of Lyme disease. Nat. Commun. 2016;7:1–13. doi: 10.1038/ncomms10507. PubMed DOI PMC

Neupert S., Russell W.K., Predel R., Russell D.H., Strey O.F., Teel P.D., Nachman R.J. The neuropeptidomics of Ixodes scapularis synganglion. J. Proteom. 2009;72:1040–1045. doi: 10.1016/j.jprot.2009.06.007. PubMed DOI

Šimo L., Žitňan D., Park Y. Two novel neuropeptides in innervation of the salivary glands of the black-legged tick, Ixodes scapularis: Myoinhibitory peptide and SIFamide. J. Comp. Neurol. 2009;517:551–563. doi: 10.1002/cne.22182. PubMed DOI PMC

Vancová M., Bílý T., Nebesářová J., Grubhoffer L., Bonnet S., Park Y., Šimo L. Ultrastructural mapping of salivary gland innervation in the tick Ixodes ricinus. Sci. Rep. 2019;9:1–13. doi: 10.1038/s41598-019-43284-6. PubMed DOI PMC

Šimo L., Koči J., Park Y. Receptors for the neuropeptides, myoinhibitory peptide and SIFamide, in control of the salivary glands of the blacklegged tick Ixodes scapularis. Insect. Biochem. Mol. Biol. 2013;43:376–387. doi: 10.1016/j.ibmb.2013.01.002. PubMed DOI PMC

Nässel D.R., Zandawala M., Kawada T., Satake H. Tachykinins: Neuropeptides That Are Ancient, Diverse, Widespread and Functionally Pleiotropic. Front. Neurosci. 2019;13 doi: 10.3389/fnins.2019.01262. PubMed DOI PMC

Jiang H., Kim D., Dobesh S., Evans J.D., Nachman R.J., Kaczmarek K., Zabrocki J., Park Y. Ligand selectivity in tachykinin and natalisin neuropeptidergic systems of the honey bee parasitic mite Varroa destructor. Sci. Rep. 2016;6:1–8. doi: 10.1038/srep19547. PubMed DOI PMC

Jiang H., Lkhagva A., Daubnerová I., Chae H.-S., Šimo L., Jung S.-H., Yoon Y.-K., Lee N.-R., Seong J.Y., Žitňan D., et al. Natalisin, a tachykinin-like signaling system, regulates sexual activity and fecundity in insects. Proc. Natl. Acad. Sci. USA. 2013;110:E3526–E3534. doi: 10.1073/pnas.1310676110. PubMed DOI PMC

Coast G.M., Schooley D.A. Toward a consensus nomenclature for insect neuropeptides and peptide hormones. Peptides. 2011;32:620–631. doi: 10.1016/j.peptides.2010.11.006. PubMed DOI

Miller J.R., Koren S., Dilley K.A., Harkins D.M., Stockwell T.B., Shabman R.S., Sutton G.G. A draft genome sequence for the Ixodes scapularis cell line, ISE6. F1000Res. 2018;7 doi: 10.12688/f1000research.13635.1. PubMed DOI PMC

Almazán C., Šimo L., Fourniol L., Rakotobe S., Borneres J., Cote M., Peltier S., Mayé J., Versillé N., Richardson J., et al. Multiple Antigenic Peptide-Based Vaccines Targeting Ixodes ricinus Neuropeptides Induce a Specific Antibody Response but Do Not Impact Tick Infestation. Pathogens. 2020;9:900. doi: 10.3390/pathogens9110900. PubMed DOI PMC

Brock C.M., Temeyer K.B., Tidwell J., Yang Y., Blandon M.A., Carreón-Camacho D., Longnecker M.T., Almazán C., Pérez de León A.A., Pietrantonio P.V. The leucokinin-like peptide receptor from the cattle fever tick, Rhipicephalus microplus, is localized in the midgut periphery and receptor silencing with validated double-stranded RNAs causes a reproductive fitness cost. Int. J. Parasitol. 2019;49:287–299. doi: 10.1016/j.ijpara.2018.11.006. PubMed DOI

Opdebeeck J.P., Wong J.Y., Jackson L.A., Dobson C. Vaccines to protect Hereford cattle against the cattle tick, Boophilus microplus. Immunology. 1988;63:363–367. PubMed PMC

Christie A.E. Neuropeptide discovery in Ixodoidea: An in silico investigation using publicly accessible expressed sequence tags. Gen. Comp. Endocrinol. 2008;157:174–185. doi: 10.1016/j.ygcen.2008.03.027. PubMed DOI

Park Y. Endocrine regulation of insect diuresis in the early postgenomic era1. Can. J. Zool. 2012 doi: 10.1139/z2012-013. DOI

Kim D., Šimo L., Park Y. Molecular characterization of neuropeptide elevenin and two elevenin receptors, IsElevR1 and IsElevR2, from the blacklegged tick, Ixodes scapularis. Insect. Biochem. Mol. Biol. 2018;101:66–75. doi: 10.1016/j.ibmb.2018.07.005. PubMed DOI

Livak K.J., Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25:402–408. doi: 10.1006/meth.2001.1262. PubMed DOI

Koči J., Šimo L., Park Y. Validation of Internal Reference Genes for Real-Time Quantitative Polymerase Chain Reaction Studies in the Tick, Ixodes scapularis (Acari: Ixodidae) J. Med. Entomol. 2013;50:79–84. doi: 10.1603/ME12034. PubMed DOI PMC

Emanuelsson O., Brunak S., Von Heijne G., Nielsen H. Locating proteins in the cell using TargetP, SignalP and related tools. Nat. Protoc. 2007;2:953–971. doi: 10.1038/nprot.2007.131. PubMed DOI

Silaghi C., Nieder M., Sauter-Louis C., Knubben-Schweizer G., Pfister K., Pfeffer M. Epidemiology, genetic variants and clinical course of natural infections with Anaplasma phagocytophilum in a dairy cattle herd. Parasites Vectors. 2018;11:20. doi: 10.1186/s13071-017-2570-1. PubMed DOI PMC

Stuen S., Van De Pol I., Bergström K., Schouls L.M. Identification of Anaplasma phagocytophila (Formerly Ehrlichia phagocytophila) Variants in Blood from Sheep in Norway. J. Clin. Microbiol. 2002;40:3192–3197. doi: 10.1128/JCM.40.9.3192-3197.2002. PubMed DOI PMC

Asanovich K.M., Bakken J.S., Madigan J.E., Rosenfeld M.A., Wormser G.P., Dumler J.S. Antigenic Diversity of Granulocytic Ehrlichia Isolates from Humans in Wisconsin and New York and a Horse in California. J. Infect. Dis. 1997;176:1029–1034. doi: 10.1086/516529. PubMed DOI

Mateos-Hernandéz L., Defaye B., Vancová M., Hajdusek O., Sima R., Park Y., Attoui H., Šimo L. Cholinergic axons regulate type I acini in salivary glands of Ixodes ricinus and Ixodes scapularis ticks. Sci. Rep. 2020;10:1–15. doi: 10.1038/s41598-020-73077-1. PubMed DOI PMC