BACKGROUND: The castor bean tick Ixodes ricinus is an important vector of several clinically important diseases, whose prevalence increases with accelerating global climate changes. Characterization of a tick life-cycle is thus of great importance. However, researchers mainly focus on specific organs of fed life stages, while early development of this tick species is largely neglected. METHODS: In an attempt to better understand the life-cycle of this widespread arthropod parasite, we sequenced the transcriptomes of four life stages (egg, larva, nymph and adult female), including unfed and partially blood-fed individuals. To enable a more reliable identification of transcripts and their comparison in all five transcriptome libraries, we validated an improved-fit set of five I. ricinus-specific reference genes for internal standard normalization of our transcriptomes. Then, we mapped biological functions to transcripts identified in different life stages (clusters) to elucidate life stage-specific processes. Finally, we drew conclusions from the functional enrichment of these clusters specifically assigned to each transcriptome, also in the context of recently published transcriptomic studies in ticks. RESULTS: We found that reproduction-related transcripts are present in both fed nymphs and fed females, underlining the poorly documented importance of ovaries as moulting regulators in ticks. Additionally, we identified transposase transcripts in tick eggs suggesting elevated transposition during embryogenesis, co-activated with factors driving developmental regulation of gene expression. Our findings also highlight the importance of the regulation of energetic metabolism in tick eggs during embryonic development and glutamate metabolism in nymphs. CONCLUSIONS: Our study presents novel insights into stage-specific transcriptomes of I. ricinus and extends the current knowledge of this medically important pathogen, especially in the early phases of its development.

BIOCEV Faculty of Science Charles University Prague Czech Republic

Biology Centre CAS Institute of Parasitology České Budějovice Czech Republic

Department of Plant Developmental Genetics Institute of Biophysics of the Czech Academy of Sciences Brno Czech Republic

Faculty of Science University of South Bohemia České Budějovice Czech Republic

GeneCore EMBL Heidelberg Germany

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Heyman P, Cochez C, Hofhuis A, Van Der Giessen J, Sprong H, Porter SR, et al. A clear and present danger: tick-borne diseases in Europe. Expert Rev Anti Infect Ther. 2010;8:33–50. PubMed

Michelet L, Delannoy S, Devillers E, Umhang G, Aspan A, Juremalm M, et al. High-throughput screening of tick-borne pathogens in Europe. Front Cell Infect Microbiol. 2014;4:103. PubMed PMC

Vayssier-Taussat M, Kazimirova M, Hubalek Z, Hornok S, Farkas R, Cosson JF, et al. Emerging horizons for tick-borne pathogens: From the “one pathogen-one disease” vision to the pathobiome paradigm. Future Microbiol. 2015;10:2033–2043. PubMed PMC

Honig V, Svec P, Halas P, Vavruskova Z, Tykalova H, Kilian P, et al. Ticks and tick-borne pathogens in South Bohemia (Czech Republic) - spatial variability in Ixodes ricinus abundance, Borrelia burgdorferi and tick-borne encephalitis virus prevalence. Ticks Tick Borne Dis. 2015;6:559–567. PubMed

Amicizia D, Domnich A, Panatto D, Lai PL, Cristina ML, Avio U, et al. Epidemiology of tick-borne encephalitis (TBE) in Europe and its prevention by available vaccines. Hum Vaccines Immunother. 2013;9:1163–1171. PubMed PMC

Lindgren E, Jaenson, Thomas GT. Lyme borreliosis in Europe: influences of climate and climate change, epidemiology, ecology and adaptation measures. Copenhagen: WHO Regional Office for Europe Copenhagen; 2006.

Heinz FX, Stiasny K, Holzmann H, Grgic-Vitek M, Kriz B, Essl A, et al. Vaccination and tick-borne encephalitis, central Europe. Emerg Infect Dis. 2013;19:69–76. PubMed PMC

Bogovic P. Tick-borne encephalitis: a review of epidemiology, clinical characteristics, and management. World J Clin Cases. 2015;3:430. PubMed PMC

Steffen R. Epidemiology of tick-borne encephalitis (TBE) in international travellers to western/central Europe and conclusions on vaccination recommendations. J Travel Med. 2016;23:taw018. PubMed

vvan den Wijngaard CC, Hofhuis A, Simões M, Rood E, van Pelt W, Zeller H, et al. Surveillance perspective on Lyme borreliosis across the European Union and European Economic Area. Euro Surveill. 2017;22:30569. PubMed PMC

Sykes RA, Makiello P. An estimate of Lyme borreliosis incidence in western Europe. J Public Health (Oxf). 2017;39:74–81. PubMed

Cerny V. Sezónní dynamika klíštěte Ixodes risinus v divokém místě zaklíštění. Československá Parazitol. 1957;4:57–58.

Roe RM, Donohue KV, Khalil SMS, Sonenshine DE. Hormonal regulation of metamorphosis and reproduction in ticks. Front Biosci. 2008;13:7250–7268. PubMed

Sonenshine DE, Roe RM, editors. Biology of Ticks, vol. 1, 2nd edn. New York: Oxford University Press (OUP); 2014.

Walker AR. Review of “Ticks: biology, disease and control” by Alan Bowman & Patricia Nuttall (eds.). Parasit Vectors. 2009;2:1.

Foldvari G. Life cycle and ecology of Ixodes ricinus: the roots of public health importance. In: Braks MAH, Van Wieren SE, Takken W, Sprong H, editors. Ecology and prevention of Lyme borreliosis. Ecology and control of vector-borne diseases, vol. 4. Wageningen: Wageningen Academic Publishers; 2016. p. 31–40.

Balashov YS. Ixodid ticks: parasites and vectors of infections. St. Petersburg: Nauka; 1998.

Balashov YS. Bloodsucking ticks (Ixodoidea) - vectors of disease in man and animals. Misc Publ Entomol Soc Am. 1972;8:163–376.

Honzakova E, Olejnicek J, Cerny V, Daniel M, Dusbabek F. Relationship between number of eggs deposited and body weight of engorged Ixodes ricinus female. Folia Parasitol. 1975;22:37–42.

Gray JS, Kahl O, Lane RS, Levin ML, Tsao JI. Diapause in ticks of the medically important Ixodes ricinus species complex. Ticks Tick Borne Dis. 2016;7:992–1003. PubMed PMC

Dana AN, Hong YS, Kern MK, Hillenmeyer ME, Harker BW, Lobo NF, et al. Gene expression patterns associated with blood-feeding in the malaria mosquito Anopheles gambiae. BMC Genomics. 2005;6:5. PubMed PMC

Reid WR, Zhang L, Liu N. Temporal gene expression profiles of pre blood-fed adult females immediately following eclosion in the southern house mosquito Culex quinquefasciatus. Int J Biol Sci. 2015;11:1306–1313. PubMed PMC

Thangamani S, Wikel SK. Differential expression of Aedes aegypti salivary transcriptome upon blood feeding. Parasit Vectors. 2009;2:34. PubMed PMC

Karim S, Singh P, Ribeiro JMC. A deep insight into the sialotranscriptome of the gulf coast tick. Amblyomma maculatum. PLoS One. 2011;6:e28525. PubMed PMC

Schwarz A, Medrano-Mercado N, Schaub GA, Struchiner CJ, Bargues MD, Levy MZ, et al. An updated insight into the sialotranscriptome of Triatoma infestans: developmental stage and geographic variations. PLoS Negl Trop Dis. 2014;8:e3372. PubMed PMC

Bensaoud C, Nishiyama MY, Ben Hamda C, Lichtenstein F, Castro De Oliveira U, Faria F, et al. De novo assembly and annotation of Hyalomma dromedarii tick (Acari: Ixodidae) sialotranscriptome with regard to gender differences in gene expression. Parasit Vectors. 2018;11:314. PubMed PMC

Perner J, Provaznik J, Schrenkova J, Urbanova V, Ribeiro JMC, Kopacek P. RNA-seq analyses of the midgut from blood- and serum-fed Ixodes ricinus ticks. Sci Rep. 2016;6:36695. PubMed PMC

Santiago PB, De Araújo CN, Motta FN, Praça YR, Charneau S, Bastos IMD, et al. Proteases of haematophagous arthropod vectors are involved in blood-feeding, yolk formation and immunity - a review. Parasit Vectors. 2017;10:79. PubMed PMC

Kotsyfakis M, Kopáček P, Franta Z, Pedra JHF, Ribeiro JMC. Deep sequencing analysis of the Ixodes ricinus haemocytome. PLoS Negl Trop Dis. 2015;9:e0003754. PubMed PMC

Charrier NP, Couton M, Voordouw MJ, Rais O, Durand-Hermouet A, Hervet C, et al. Whole body transcriptomes and new insights into the biology of the tick Ixodes ricinus. Parasit Vectors. 11:364. PubMed PMC

Schwarz A, Von Reumont BM, Erhart J, Chagas AC, Ribeiro JMC, Kotsyfakis M. De novo Ixodes ricinus salivary gland transcriptome analysis using two next-generation sequencing methodologies. FASEB J. 2013;27:4745–4756. PubMed PMC

Schwarz A, Tenzer S, Hackenberg M, Erhart J, Gerhold-Ay A, Mazur J, et al. A systems level analysis reveals transcriptomic and proteomic complexity in Ixodes ricinus midgut and salivary glands during early attachment and feeding. Mol Cell Proteomics. 2014;13:2725–2735. PubMed PMC

Kotsyfakis M, Schwarz A, Erhart J, Ribeiro JMC. Tissue- and time-dependent transcription in Ixodes ricinus salivary glands and midguts when blood feeding on the vertebrate host. Sci Rep. 2015;5:9103. PubMed PMC

Mans BJ, Andersen JF, Francischetti IMB, Valenzuela JG, Schwan TG, Pham VM, et al. Comparative sialomics between hard and soft ticks: implications for the evolution of blood-feeding behavior. Insect Biochem Mol Biol. 2008;38:42–58. PubMed PMC

Esteves E, Maruyama SR, Kawahara R, Fujita A, Martins LA, Righi AA, et al. Analysis of the salivary gland transcriptome of unfed and partially fed Amblyomma sculptum ticks and descriptive proteome of the saliva. Front Cell Infect Microbiol. 2017;7:476. PubMed PMC

Landulfo GA, Patané JSL, da Silva DGN, Junqueira-de-Azevedo ILM, Mendonca RZ, Simons SM, et al. Gut transcriptome analysis on females of Ornithodoros mimon (Acari: Argasidae) and phylogenetic inference of ticks. Rev Bras Parasitol Vet. 2017;26:185–204. PubMed

Araujo RN, Silva NCS, Mendes-Sousa A, Paim R, Costa GCA, Dias LR, et al. RNA-seq analysis of the salivary glands and midgut of the Argasid tick Ornithodoros rostratus. Sci Rep. 2019;9:6764. PubMed PMC

Bell-Sakyi L, Zweygarth E, Blouin EF, Gould EA, Jongejan F. Tick cell lines: tools for tick and tick-borne disease research. Trends Parasitol. 2007;23:450–457. PubMed

Bolger AM, Lohse M, Usadel B. Trimmomatic: A flexible trimmer for Illumina Sequence Data. Bioinformatics. 2014;30:2114–2120. PubMed PMC

Haas BJ, Papanicolaou A, Yassour M, Grabherr M, Blood PD, Bowden J, et al. De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nat Protoc. 2013;8:1494–1512. PubMed PMC

Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al. The sequence alignment/map format and SAMtools. Bioinformatics. 2009;25:2078–2079. PubMed PMC

Simao FA, Waterhouse RM, Ioannidis P, Kriventseva EV, Zdobnov EM. BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics. 2015;31:3210–3212. PubMed

Laetsch DR, Blaxter ML. BlobTools: interrogation of genome assemblies [version 1; peer review: 2 approved with reservations]. F1000Research. 2017;6:1287.

Jones P, Binns D, Chang HY, Fraser M, Li W, McAnulla C, et al. InterProScan 5: genome-scale protein function classification. Bioinformatics. 2014;30:1236–1240. PubMed PMC

Rozewicki J, Li S, Amada KM, Standley DM, Katoh K. MAFFT-DASH: integrated protein sequence and structural alignment. Nucleic Acids Res. 2019;47:W5–W10. PubMed PMC

Glaser RW. “Ringer” solutions and some notes on the physiological basis of their ionic composition. Comp Biochem Physiol. 1917;2:241–289. PubMed

Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper - Excel-based tool using pair-wise correlations. Biotechnol Lett. 2004;26:509–515. PubMed

Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002;3(research0034):1. PubMed PMC

Li B, Dewey CN. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinf. 2011;12:323. PubMed PMC

Evans C, Hardin J, Stoebel DM. Selecting between-sample RNA-Seq normalization methods from the perspective of their assumptions. Brief Bioinform. 2018;19:776–792. PubMed PMC

Maza E, Frasse P, Senin P, Bouzayen M, Zouine M. Comparison of normalization methods for differential gene expression analysis in RNA-seq experiments: a matter of relative size of studied transcriptomes. Commun Integr Biol. 2013;6:e25849. PubMed PMC

Klopfenstein DV, Zhang L, Pedersen BS, Ramírez F, Vesztrocy AW, Naldi A, et al. GOATOOLS: a python library for gene ontology analyses. Sci Rep. 2018;8:10872. PubMed PMC

Pagel Van Zee J, Geraci NS, Guerrero FD, Wikel SK, Stuart JJ, Nene VM, et al. Tick genomics: the Ixodes genome project and beyond. Int J Parasitol. 2007;37:1297–305. PubMed

Sojka D, Franta Z, Frantová H, Bartošová P, Horn M, Váchová J, et al. Characterization of gut-associated cathepsin D hemoglobinase from tick Ixodes ricinus (IrCD1) J Biol Chem. 2012;287:21152–21163. PubMed PMC

Leboulle G, Crippa M, Decrem Y, Mejri N, Brossard M, Bollen A, et al. Characterization of a novel salivary immunosuppressive protein from Ixodes ricinus ticks. J Biol Chem. 2002;277:10083–10089. PubMed

Rego ROM, Hajdusek O, Kovar V, Kopacek P, Grubhoffer L, Hypsa V. Molecular cloning and comparative analysis of fibrinogen-related proteins from the soft tick Ornithodoros moubata and the hard tick Ixodes ricinus. Insect Biochem Mol Biol. 2005;35:991–1004. PubMed

Yang C, Pan H, Liu Y, Zhou X. Stably expressed housekeeping genes across developmental stages in the two-spotted spider mite, Tetranychus urticae. PLoS One. 2015;10:e0120833. PubMed PMC

Nijhof AM, Balk JA, Postigo M, Jongejan F. Selection of reference genes for quantitative RT-PCR studies in Rhipicephalus (Boophilus) microplus and Rhipicephalus appendiculatus ticks and determination of the expression profile of Bm86. BMC Mol Biol. 2009;10:112. PubMed PMC

Koci J, Simo 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. PubMed PMC

Bionaz M, Loor JJ. Identification of reference genes for quantitative real-time PCR in the bovine mammary gland during the lactation cycle. Physiol Genomics. 2007;29:312–319. PubMed

Kaur R, Sodhi M, Sharma A, Sharma VL, Verma P, Swami SK, et al. Selection of suitable reference genes for normalization of quantitative RT-PCR (RT-qPCR) expression data across twelve tissues of riverine buffaloes (Bubalus bubalis) PLoS One. 2018;13:e0191558. PubMed PMC

Razavi SA, Afsharpad M, Modarressi MH, Zarkesh M, Yaghmaei P, Nasiri S, et al. Validation of reference genes for normalization of relative qRT-PCR studies in papillary thyroid carcinoma. Sci Rep. 2019;9:15241. PubMed PMC

Caracausi M, Piovesan A, Antonaros F, Strippoli P, Vitale L, Pelleri MC. Systematic identification of human housekeeping genes possibly useful as references in gene expression studies. Mol Med Rep. 2017;16:2397–2410. PubMed PMC

Chim SSC, Wong KKW, Chung CYL, Lam SKW, Kwok JSL, Lai CY, et al. Systematic selection of reference genes for the normalization of circulating RNA transcripts in pregnant women based on RNA-Seq data. Int J Mol Sci. 2017;18:E1709. PubMed PMC

Sojka D, Francischetti IMB, Calvo E, Kotsyfakis M. Cysteine proteases from bloodfeeding arthropod ectoparasites. Adv Exp Med Biol. 2011;712:177–191. PubMed PMC

Bork P, Beckmann G. The CUB domain: a widespread module in developmentally regulated proteins. J Mol Biol. 1993;231:539–545. PubMed

Francischetti IMB, Sa-Nunes A, Mans BJ, Santos IM, Ribeiro JMC. The role of saliva in tick feeding. Front Biosci. 2009;14:2051–2088. PubMed PMC

Suppan J, Engel B, Marchetti-Deschmann M, Nürnberger S. Tick attachment cement—reviewing the mysteries of a biological skin plug system. Biol Rev. 2018;93:1056–1076. PubMed PMC

Tirloni L, Kim TK, Berger M, Termignoni C, da Silva Vaz I, Mulenga A. Amblyomma americanum serpin 27 (AAS27) is a tick salivary anti-inflammatory protein secreted into the host during feeding. PLoS Negl Trop Dis. 2019;13:e0007660. PubMed PMC

Rosendale AJ, Dunlevy ME, McCue MD, Benoit JB. Progressive behavioural, physiological and transcriptomic shifts over the course of prolonged starvation in ticks. Mol Ecol. 2019;28:49–65. PubMed

Leboulle G, Rochez C, Louahed J, Rutti B, Brossard M, Bollen A, et al. Isolation of Ixodes ricinus salivary gland mRNA encoding factors induced during blood feeding. Am J Trop Med Hyg. 2002;66:225–233. PubMed

Ganfornina MD, Kayser H, Sanchez D. Lipocalins in Arthropoda: diversification and functional explorations. In: Akerstrom B, Borregaard N, Flower DR, Salier JP, editors. Lipocalins. Austin: Landes Bioscience; 2006. pp. 49–74.

Choy R, Thomas JH. Fluoxetine-resistant mutants in C. elegans define a novel family of transmembrane proteins. Mol Cell. 1999;4:143–52. PubMed

Choy RKM, Kemner JM, Thomas JH. Fluoxetine-resistance genes in Caenorhabditis elegans function in the intestine and may act in drug transport. Genetics. 2006;172:885–892. PubMed PMC

Chmelar J, Anderson JM, Mu J, Jochim RC, Valenzuela JG, Kopecky J. Insight into the sialome of the castor bean tick, Ixodes ricinus. BMC Genomics. 2008;9:233. PubMed PMC

Ogihara MH, Hikiba J, Suzuki Y, Taylor DM, Kataoka H. Ovarian ecdysteroidogenesis in both immature and mature stages of an acari, Ornithodoros moubata. PLoS One. 2015;10:e0124953. PubMed PMC

Richards MH. Vitellogenin and vitellogenin-like genes: not just for egg production. Insects Soc. 2019;66:505–506.

Taylor D, Chinzei Y, Ito K, Higuchi N, Ando K. Stimulation of vitellogenesis by pyrethroids in mated and virgin female adults, male adults, and fourth instar females of Ornithodoros moubata (Acari: Argasidae) J Med Entomol. 1991;28:322–329. PubMed

Lumaret JP, Errouissi F, Floate K, Rombke J, Wardhaugh K. A review on the toxicity and non-target effects of macrocyclic lactones in terrestrial and aquatic environments. Curr Pharm Biotechnol. 2012;13:1004–1060. PubMed PMC

Hodzic E, Fish D, Maretzki CM, De Silva AM, Feng S, Barthold SW. Acquisition and transmission of the agent of human granulocytic ehrlichiosis by Ixodes scapularis ticks. J Clin Microbiol. 1998;36:3574–3578. PubMed PMC

Kocan KM, de la Fuente J, Coburn LA. Insights into the development of Ixodes scapularis: a resource for research on a medically important tick species. Parasit Vectors. 2015;8:592. PubMed PMC

Scott JD, Clark KL, Coble NM, Ballantyne TR. Detection and transstadial passage of Babesia species and Borrelia burgdorferi sensu lato in ticks collected from avian and mammalian hosts in Canada. Healthcare. 2019;7:155. PubMed PMC

Liu XY, Bonnet SI. Hard tick factors implicated in pathogen transmission. PLoS Negl Trop Dis. 2014;8:e2566. PubMed PMC

Villar M, Popara M, Ayllón N, De Fernández Mera IG, Mateos-Hernández L, Galindo RC, et al. A systems biology approach to the characterization of stress response in Dermacentor reticulatus tick unfed larvae. PLoS One. 2014;9:e89564. PubMed PMC

Ribeiro JMC, Alarcon-Chaidez F, Ivo IM, Mans BJ, Mather TN, Valenzuela JG, et al. An annotated catalog of salivary gland transcripts from Ixodes scapularis ticks. Insect Biochem Mol Biol. 2006;36:111–129. PubMed

Kim YH, Islam MS, You MJ. Proteomic screening of antigenic proteins from the hard tick, Haemaphysalis longicornis (Acari: Ixodidae) Korean J Parasitol. 2015;53:85–93. PubMed PMC

Nuttall PA, Trimnell AR, Kazimirova M, Labuda M. Exposed and concealed antigens as vaccine targets for controlling ticks and tick-borne diseases. Parasite Immunol. 2006;28:155–163. PubMed

Labuda M, Trimnell AR, Ličková M, Kazimírová M, Davies GM, Lissina O, et al. An antivector vaccine protects against a lethal vector-borne pathogen. PLoS Pathog. 2006;2:e27. PubMed PMC

Merino O, Alberdi P, Pérez De La Lastra JM, de la Fuente J. Tick vaccines and the control of tick-borne pathogens. Front Cell Infect Microbiol. 2013;3:30. PubMed PMC

Sprong H, Trentelman J, Seemann I, Grubhoffer L, Rego ROM, Hajdušek O, et al. ANTIDotE: anti-tick vaccines to prevent tick-borne diseases in Europe. Parasit Vectors. 2014;7:77. PubMed PMC

Rego ROM, Trentelman JJA, Anguita J, Nijhof AM, Sprong H, Klempa B, et al. Counterattacking the tick bite: towards a rational design of anti-tick vaccines targeting pathogen transmission. Parasit Vectors. 2019;12:229. PubMed PMC

Santos VT, Ribeiro L, Fraga A, de Barros CM, Campos E, Moraes J, et al. The embryogenesis of the tick Rhipicephalus (Boophilus) microplus: the establishment of a new chelicerate model system. Genesis. 2013;51:803–818. PubMed

Seixas A, Oliveira P, Termignoni C, Logullo C, Masuda A, da Silva Vaz I. Rhipicephalus (Boophilus) microplus embryo proteins as target for tick vaccine. Vet Immunol Immunopathol. 2012;148:149–156. PubMed

Hussein HE, Johnson WC, Taus NS, Suarez CE, Scoles GA, Ueti MW. Silencing expression of the Rhipicephalus microplus vitellogenin receptor gene blocks Babesia bovis transmission and interferes with oocyte maturation. Parasit Vectors. 2019;12:7. PubMed PMC

Artieri CG, Fraser HB. Transcript length mediates developmental timing of gene expression across drosophila. Mol Biol Evol. 2014;31:2879–2889. PubMed PMC

Shawky JH, Davidson LA. Tissue mechanics and adhesion during embryo development. Dev Biol. 2015;401:152–164. PubMed PMC

Bourque G, Burns KH, Gehring M, Gorbunova V, Seluanov A, Hammell M, et al. Ten things you should know about transposable elements. Genome Biol. 2018;19:199. PubMed PMC

Estrela A, Seixas A, Termignoni C. A cysteine endopeptidase from tick (Rhipicephalus (Boophilus) microplus) larvae with vitellin digestion activity. Comp Biochem Physiol B Biochem Mol Biol. 2007;148:410–416. PubMed

Zhang TT, Qiu ZX, Li Y, Wang WY, Li MM, Guo P, et al. The mRNA expression and enzymatic activity of three enzymes during embryonic development of the hard tick Haemaphysalis longicornis. Parasit Vectors. 2019;12:96. PubMed PMC

Seixas A, Leal AT, Nascimento-Silva MCL, Masuda A, Termignoni C, da Silva Vaz I. Vaccine potential of a tick vitellin-degrading enzyme (VTDCE) Vet Immunol Immunopathol. 2008;124:332–340. PubMed

Martins R, Ruiz N, Fonseca RN da, Vaz Junior I da S, Logullo C. The dynamics of energy metabolism in the tick embryo. Rev Bras Parasitol Vet. 2018;27:259–66. PubMed

Song JL, Wong JL, Wessel GM. Oogenesis: single cell development and differentiation. Dev Biol. 2006;300:385–405. PubMed

Lee HX, Mendes FA, Plouhinec JL, De Robertis EM. Enzymatic regulation of pattern: BMP4 binds CUB domains of tolloids and inhibits proteinase activity. Genes Dev. 2009;23:2551–2562. PubMed PMC

Nunes da Fonseca R, van der Zee M, Roth S. Evolution of extracellular Dpp modulators in insects: the roles of tolloid and twisted-gastrulation in dorsoventral patterning of the Tribolium embryo. Dev Biol. 2010;345:80–93. PubMed

Radonić A, Thulke S, Mackay IM, Landt O, Siegert W, Nitsche A. Guideline to reference gene selection for quantitative real-time PCR. Biochem Biophys Res Commun. 2004;313:856–862. PubMed

Zyprych-Walczak J, Szabelska A, Handschuh L, Górczak K, Klamecka K, Figlerowicz M, et al. The impact of normalization methods on RNA-Seq data analysis. Biomed Res Int. 2015;2015:621690. PubMed PMC

Wang Z, Lyu Z, Pan L, Zeng G, Randhawa P. Defining housekeeping genes suitable for RNA-seq analysis of the human allograft kidney biopsy tissue. BMC Med Genomics. 2019;12:86. PubMed PMC

Paim RM, Pereira MH, Di Ponzio R, Rodrigues JO, Guarneri AA, Gontijo NF, et al. Validation of reference genes for expression analysis in the salivary gland and the intestine of Rhodnius prolixus (Hemiptera, Reduviidae) under different experimental conditions by quantitative real-time PCR. BMC Res Notes. 2012;5:128. PubMed PMC

Niu LL, Fallon AM. Differential regulation of ribosomal protein gene expression in Aedes aegypti mosquitoes before and after the blood meal. Insect Mol Biol. 2000;9:613–623. PubMed

Chadee DD, Beier JC, Mohammed RT. Fast and slow blood-feeding durations of Aedes aegypti mosquitoes in Trinidad. J Vector Ecol. 2002;27:172–177. PubMed

Valzania L, Mattee MT, Strand MR, Brown MR. Blood feeding activates the vitellogenic stage of oogenesis in the mosquito Aedes aegypti through inhibition of glycogen synthase kinase 3 by the insulin and TOR pathways. Dev Biol. 2019;454:85–95. PubMed PMC

Hajdusek O, Almazán C, Loosova G, Villar M, Canales M, Grubhoffer L, et al. Characterization of ferritin 2 for the control of tick infestations. Vaccine. 2010;28:2993–2998. PubMed

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