The closest relative of human pathogen Leishmania, the trypanosomatid Novymonas esmeraldas, harbors a bacterial endosymbiont "Candidatus Pandoraea novymonadis." Based on genomic data, we performed a detailed characterization of the metabolic interactions of both partners. While in many respects the metabolism of N. esmeraldas resembles that of other Leishmaniinae, the endosymbiont provides the trypanosomatid with heme, essential amino acids, purines, some coenzymes, and vitamins. In return, N. esmeraldas shares with the bacterium several nonessential amino acids and phospholipids. Moreover, it complements its carbohydrate metabolism and urea cycle with enzymes missing from the "Ca. Pandoraea novymonadis" genome. The removal of the endosymbiont from N. esmeraldas results in a significant reduction of the overall translation rate, reduced expression of genes involved in lipid metabolism and mitochondrial respiratory activity, and downregulation of several aminoacyl-tRNA synthetases, enzymes involved in the synthesis of some amino acids, as well as proteins associated with autophagy. At the same time, the genes responsible for protection against reactive oxygen species and DNA repair become significantly upregulated in the aposymbiotic strain of this trypanosomatid. By knocking out a component of its flagellum, we turned N. esmeraldas into a new model trypanosomatid that is amenable to genetic manipulation using both conventional and CRISPR-Cas9-mediated approaches. IMPORTANCENovymonas esmeraldas is a parasitic flagellate of the family Trypanosomatidae representing the closest insect-restricted relative of the human pathogen Leishmania. It bears symbiotic bacteria in its cytoplasm, the relationship with which has been established relatively recently and independently from other known endosymbioses in protists. Here, using the genome analysis and comparison of transcriptomic profiles of N. esmeraldas with and without the endosymbionts, we describe a uniquely complex cooperation between both partners on the biochemical level. We demonstrate that the removal of bacteria leads to a decelerated growth of N. esmeraldas, substantial suppression of many metabolic pathways, and increased oxidative stress. Our success with the genetic transformation of this flagellate makes it a new model trypanosomatid species that can be used for the dissection of mechanisms underlying the symbiotic relationships between protists and bacteria.

e Duve Institute Université Catholique de Louvain Brussels Belgium

Faculty of Sciences University of South Bohemia České Budějovice Czech Republic

Institute of Parasitology Biology Centre Czech Academy of Sciences České Budějovice Czech Republic

Life Science Research Centre Faculty of Science University of Ostrava Ostrava Czech Republic

Martsinovsky Institute of Medical Parasitology Tropical and Vector Borne Diseases Sechenov University Moscow Russia

Zoological Institute of the Russian Academy of Sciences St Petersburg Russia

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Maslov DA, Opperdoes FR, Kostygov AY, Hashimi H, Lukeš J, Yurchenko V. 2019. Recent advances in trypanosomatid research: genome organization, expression, metabolism, taxonomy and evolution. Parasitology 146:1–27. doi: 10.1017/S0031182018000951. PubMed DOI

Lukeš J, Butenko A, Hashimi H, Maslov DA, Votýpka J, Yurchenko V. 2018. Trypanosomatids are much more than just trypanosomes: clues from the expanded family tree. Trends Parasitol 34:466–480. doi: 10.1016/j.pt.2018.03.002. PubMed DOI

Maslov DA, Votýpka J, Yurchenko V, Lukeš J. 2013. Diversity and phylogeny of insect trypanosomatids: all that is hidden shall be revealed. Trends Parasitol 29:43–52. doi: 10.1016/j.pt.2012.11.001. PubMed DOI

Lukeš J, Skalický T, Týč J, Votýpka J, Yurchenko V. 2014. Evolution of parasitism in kinetoplastid flagellates. Mol Biochem Parasitol 195:115–122. doi: 10.1016/j.molbiopara.2014.05.007. PubMed DOI

Butenko A, Hammond M, Field MC, Ginger ML, Yurchenko V, Lukeš J. 2021. Reductionist pathways for parasitism in euglenozoans? Expanded datasets provide new insights. Trends Parasitol 37:100–116. doi: 10.1016/j.pt.2020.10.001. PubMed DOI

Kostygov AY, Karnkowska A, Votýpka J, Tashyreva D, Maciszewski K, Yurchenko V, Lukeš J. 2021. Euglenozoa: taxonomy, diversity and ecology, symbioses and viruses. Open Biol 11:200407. doi: 10.1098/rsob.200407. PubMed DOI PMC

de Souza W, Motta MC. 1999. Endosymbiosis in protozoa of the Trypanosomatidae family. FEMS Microbiol Lett 173:1–8. doi: 10.1111/j.1574-6968.1999.tb13477.x. PubMed DOI

Votýpka J, Kostygov AY, Kraeva N, Grybchuk-Ieremenko A, Tesařová M, Grybchuk D, Lukeš J, Yurchenko V. 2014. PubMed DOI

Kostygov A, Dobáková E, Grybchuk-Ieremenko A, Váhala D, Maslov DA, Votýpka J, Lukeš J, Yurchenko V. 2016. Novel trypanosomatid-bacterium association: evolution of endosymbiosis in action. mBio 7:e01985-15. doi: 10.1128/mBio.01985-15. PubMed DOI PMC

Ganyukova AI, Frolov AO, Malysheva MN, Spodareva VV, Yurchenko V, Kostygov AY. 2020. A novel endosymbiont-containing trypanosomatid PubMed DOI

Catta-Preta CM, Nascimento MT, Garcia MC, Saraiva EM, Motta MC, Meyer-Fernandes JR. 2013. The presence of a symbiotic bacterium in PubMed DOI

Motta MC, Catta-Preta CM, Schenkman S, de Azevedo Martins AC, Miranda K, de Souza W, Elias MC. 2010. The bacterium endosymbiont of PubMed DOI PMC

Freymuller E, Camargo EP. 1981. Ultrastructural differences between species of trypanosomatids with and without endosymbionts. J Protozool 28:175–182. doi: 10.1111/j.1550-7408.1981.tb02829.x. PubMed DOI

Chang KP. 1974. Ultrastructure of symbiotic bacteria in normal and antibiotic-treated PubMed DOI

Motta MC, Soares MJ, Attias M, Morgado J, Lemos AP, Saad-Nehme J, Meyer-Fernandes JR, De Souza W. 1997. Ultrastructural and biochemical analysis of the relationship of PubMed

Klein CC, Alves JM, Serrano MG, Buck GA, Vasconcelos AT, Sagot MF, Teixeira MM, Camargo EP, Motta MC. 2013. Biosynthesis of vitamins and cofactors in bacterium-harbouring trypanosomatids depends on the symbiotic association as revealed by genomic analyses. PLoS One 8:e79786. doi: 10.1371/journal.pone.0079786. PubMed DOI PMC

Alves JM, Serrano MG, Maia da Silva F, Voegtly LJ, Matveyev AV, Teixeira MM, Camargo EP, Buck GA. 2013. Genome evolution and phylogenomic analysis of PubMed DOI PMC

Alves JM, Klein CC, da Silva FM, Costa-Martins AG, Serrano MG, Buck GA, Vasconcelos AT, Sagot MF, Teixeira MM, Motta MC, Camargo EP. 2013. Endosymbiosis in trypanosomatids: the genomic cooperation between bacterium and host in the synthesis of essential amino acids is heavily influenced by multiple horizontal gene transfers. BMC Evol Biol 13:190. doi: 10.1186/1471-2148-13-190. PubMed DOI PMC

Alves JMP. 2017. Working together: amino acid biosynthesis in endosymbiont-harbouring Trypanosomatidae, p 371–383. In D'Mello JPF (ed), The handbook of microbial metabolism of amino acids. CAB International, Wallingford, UK Boston, USA.

Morales J, Kokkori S, Weidauer D, Chapman J, Goltsman E, Rokhsar D, Grossman AR, Nowack EC. 2016. Development of a toolbox to dissect host-endosymbiont interactions and protein trafficking in the trypanosomatid PubMed DOI PMC

Embley TM, Martin W. 2006. Eukaryotic evolution, changes and challenges. Nature 440:623–630. doi: 10.1038/nature04546. PubMed DOI

Keeling PJ, Koonin EV. 2014. The origin and evolution of eukaryotes: a subject collection from Cold Spring Harbor Perspectives in Biology. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

Moran NA, McCutcheon JP, Nakabachi A. 2008. Genomics and evolution of heritable bacterial symbionts. Annu Rev Genet 42:165–190. doi: 10.1146/annurev.genet.41.110306.130119. PubMed DOI

McFall-Ngai M, Hadfield MG, Bosch TC, Carey HV, Domazet-Loso T, Douglas AE, Dubilier N, Eberl G, Fukami T, Gilbert SF, Hentschel U, King N, Kjelleberg S, Knoll AH, Kremer N, Mazmanian SK, Metcalf JL, Nealson K, Pierce NE, Rawls JF, Reid A, Ruby EG, Rumpho M, Sanders JG, Tautz D, Wernegreen JJ. 2013. Animals in a bacterial world, a new imperative for the life sciences. Proc Natl Acad Sci USA 110:3229–3236. doi: 10.1073/pnas.1218525110. PubMed DOI PMC

Dubilier N, Bergin C, Lott C. 2008. Symbiotic diversity in marine animals: the art of harnessing chemosynthesis. Nat Rev Microbiol 6:725–740. doi: 10.1038/nrmicro1992. PubMed DOI

McCutcheon JP, Moran NA. 2011. Extreme genome reduction in symbiotic bacteria. Nat Rev Microbiol 10:13–26. doi: 10.1038/nrmicro2670. PubMed DOI

Müller DB, Vogel C, Bai Y, Vorholt JA. 2016. The plant microbiota: systems-level insights and perspectives. Annu Rev Genet 50:211–234. doi: 10.1146/annurev-genet-120215-034952. PubMed DOI

Adl SM, Bass D, Lane CE, Lukeš J, Schoch CL, Smirnov A, Agatha S, Berney C, Brown MW, Burki F, Cárdenas P, Čepička I, Chistyakova L, Del Campo J, Dunthorn M, Edvardsen B, Eglit Y, Guillou L, Hampl V, Heiss AA, Hoppenrath M, James TY, Karnkowska A, Karpov S, Kim E, Kolisko M, Kudryavtsev A, Lahr DJG, Lara E, Le Gall L, Lynn DH, Mann DG, Massana R, Mitchell EAD, Morrow C, Park JS, Pawlowski JW, Powell MJ, Richter DJ, Rueckert S, Shadwick L, Shimano S, Spiegel FW, Torruella G, Youssef N, Zlatogursky V, Zhang Q. 2019. Revisions to the classification, nomenclature, and diversity of eukaryotes. J Eukaryot Microbiol 66:4–119. doi: 10.1111/jeu.12691. PubMed DOI PMC

Burki F, Roger AJ, Brown MW, Simpson AGB. 2020. The new tree of eukaryotes. Trends Ecol Evol 35:43–55. doi: 10.1016/j.tree.2019.08.008. PubMed DOI

Nowack EC, Price DC, Bhattacharya D, Singer A, Melkonian M, Grossman AR. 2016. Gene transfers from diverse bacteria compensate for reductive genome evolution in the chromatophore of PubMed DOI PMC

Singer A, Poschmann G, Muhlich C, Valadez-Cano C, Hansch S, Huren V, Rensing SA, Stuhler K, Nowack ECM. 2017. Massive protein import into the early-evolutionary-stage photosynthetic organelle of the amoeba PubMed DOI

Kraeva N, Butenko A, Hlaváčová J, Kostygov A, Myškova J, Grybchuk D, Leštinová T, Votýpka J, Volf P, Opperdoes F, Flegontov P, Lukeš J, Yurchenko V. 2015. PubMed DOI PMC

Coburn CM, Otteman KM, McNeely T, Turco SJ, Beverley SM. 1991. Stable DNA transfection of a wide range of trypanosomatids. Mol Biochem Parasitol 46:169–179. doi: 10.1016/0166-6851(91)90210-w. PubMed DOI

Miranda MR, Saye M, Reigada C, Carrillo C, Pereira CA. 2015. PubMed DOI

Sloan MA, Ligoxygakis P. 2020. Tools for the genetic manipulation of PubMed DOI PMC

Liu Q, Lei J, Kadowaki T. 2019. Gene disruption of honey bee trypanosomatid parasite, PubMed DOI PMC

Jirků M, Yurchenko V, Lukeš J, Maslov DA. 2012. New species of insect trypanosomatids from Costa Rica and the proposal for a new subfamily within the Trypanosomatidae. J Eukaryot Microbiol 59:537–547. doi: 10.1111/j.1550-7408.2012.00636.x. PubMed DOI

Kostygov AY, Yurchenko V. 2017. Revised classification of the subfamily Leishmaniinae (Trypanosomatidae). Folia Parasitol 64:20. doi: 10.14411/fp.2017.020. PubMed DOI

Olivier M, Atayde VD, Isnard A, Hassani K, Shio MT. 2012. PubMed DOI

Jain M, Dole VS, Myler PJ, Stuart KD, Madhubala R. 2007. Role of DOI

Naderer T, Heng J, McConville MJ. 2010. Evidence that intracellular stages of PubMed DOI PMC

Kostygov AY, Butenko A, Nenarokova A, Tashyreva D, Flegontov P, Lukeš J, Yurchenko V. 2017. Genome of “Ca. Pandoraea novymonadis,” an endosymbiotic bacterium of the trypanosomatid Novymonas esmeraldas. Front Microbiol 8:1940. doi: 10.3389/fmicb.2017.01940. PubMed DOI PMC

Fish WR, Looker DL, Marr JJ, Berens RL. 1982. Purine metabolism in the bloodstream forms of PubMed DOI

Opperdoes FR, Butenko A, Flegontov P, Yurchenko V, Lukeš J. 2016. Comparative metabolism of free-living PubMed DOI

Nara T, Aoki T. 2002. The pyrimidine-biosynthetic (pyr) gene cluster in trypanosomes. Tanpakushitsu Kakusan Koso 47:13–20. PubMed

Opperdoes FR, Michels PA. 2007. Horizontal gene transfer in trypanosomatids. Trends Parasitol 23:470–476. doi: 10.1016/j.pt.2007.08.002. PubMed DOI

Čermáková P, Maďarová A, Baráth P, Bellová J, Yurchenko V, Horváth A. 2021. Differences in mitochondrial NADH dehydrogenase activities in trypanosomatids. Parasitology 148:1161–1170. doi: 10.1017/S0031182020002425. PubMed DOI PMC

Puustinen A, Finel M, Haltia T, Gennis RB, Wikström M. 1991. Properties of the two terminal oxidases of PubMed DOI

Motta MC, Martins AC, de Souza SS, Catta-Preta CM, Silva R, Klein CC, de Almeida LG, de Lima Cunha O, Ciapina LP, Brocchi M, Colabardini AC, de Araujo Lima B, Machado CR, de Almeida Soares CM, Probst CM, de Menezes CB, Thompson CE, Bartholomeu DC, Gradia DF, Pavoni DP, Grisard EC, Fantinatti-Garboggini F, Marchini FK, Rodrigues-Luiz GF, Wagner G, Goldman GH, Fietto JL, Elias MC, Goldman MH, Sagot MF, Pereira M, Stoco PH, de Mendonca-Neto RP, Teixeira SM, Maciel TE, de Oliveira Mendes TA, Urmenyi TP, de Souza W, Schenkman S, de Vasconcelos AT. 2013. Predicting the proteins of PubMed DOI PMC

Muñoz-Gómez SA, Slamovits CH, Dacks JB, Baier KA, Spencer KD, Wideman JG. 2015. Ancient homology of the mitochondrial contact site and cristae organizing system points to an endosymbiotic origin of mitochondrial cristae. Curr Biol 25:1489–1495. doi: 10.1016/j.cub.2015.04.006. PubMed DOI

Kaurov I, Vancová M, Schimanski B, Cadena LR, Heller J, Bíly T, Potěšíl D, Eichenberger C, Bruce H, Oeljeklaus S, Warscheid B, Zdráhal Z, Schneider A, Lukeš J, Hashimi H. 2018. The diverged trypanosome MICOS complex as a hub for mitochondrial cristae shaping and protein import. Curr Biol 28:3393–3407.e5. doi: 10.1016/j.cub.2018.09.008. PubMed DOI

Flegontov P, Butenko A, Firsov S, Kraeva N, Eliáš M, Field MC, Filatov D, Flegontova O, Gerasimov ES, Hlaváčová J, Ishemgulova A, Jackson AP, Kelly S, Kostygov A, Logacheva MD, Maslov DA, Opperdoes FR, O'Reilly A, Sádlová J, Ševčíková T, Venkatesh D, Vlček Č, Volf P, Votýpka J, Záhonová K, Yurchenko V, Lukeš J. 2016. Genome of PubMed DOI PMC

Cunningham ML, Beverley SM. 2001. Pteridine salvage throughout the PubMed DOI

Opperdoes FR, Coombs GH. 2007. Metabolism of PubMed DOI

Kořený L, Lukeš J, Oborník M. 2010. Evolution of the haem synthetic pathway in kinetoplastid flagellates: an essential pathway that is not essential after all? Int J Parasitol 40:149–156. doi: 10.1016/j.ijpara.2009.11.007. PubMed DOI

Lee SH, Stephens JL, Englund PT. 2007. A fatty-acid synthesis mechanism specialized for parasitism. Nat Rev Microbiol 5:287–297. doi: 10.1038/nrmicro1617. PubMed DOI

Uttaro AD. 2014. Acquisition and biosynthesis of saturated and unsaturated fatty acids by trypanosomatids. Mol Biochem Parasitol 196:61–70. doi: 10.1016/j.molbiopara.2014.04.001. PubMed DOI

Kraeva N, Horáková E, Kostygov A, Kořený L, Butenko A, Yurchenko V, Lukeš J. 2017. Catalase in Leishmaniinae: with me or against me? Infect Genet Evol 50:121–127. doi: 10.1016/j.meegid.2016.06.054. PubMed DOI

Škodová-Sveráková I, Záhonová K, Bučková B, Füssy Z, Yurchenko V, Lukeš J. 2020. Catalase and ascorbate peroxidase in euglenozoan protists. Pathogens 9:317. doi: 10.3390/pathogens9040317. PubMed DOI PMC

Bombaça ACS, Brunoro GVF, Dias-Lopes G, Ennes-Vidal V, Carvalho PC, Perales J, d'Avila-Levy CM, Valente RH, Menna-Barreto RFS. 2020. Glycolytic profile shift and antioxidant triggering in symbiont-free and H PubMed DOI

Chevalier N, Bertrand L, Rider MH, Opperdoes FR, Rigden DJ, Michels PA. 2005. 6-Phosphofructo-2-kinase and fructose-2,6-bisphosphatase in Trypanosomatidae. Molecular characterization, database searches, modelling studies and evolutionary analysis. FEBS J 272:3542–3560. doi: 10.1111/j.1742-4658.2005.04774.x. PubMed DOI

Penha LL, Hoffmann L, Souza SS, Martins AC, Bottaro T, Prosdocimi F, Faffe DS, Motta MC, Ürményi TP, Silva R. 2016. Symbiont modulates expression of specific gene categories in PubMed DOI PMC

Randow F, Youle RJ. 2014. Self and nonself: how autophagy targets mitochondria and bacteria. Cell Host Microbe 15:403–411. doi: 10.1016/j.chom.2014.03.012. PubMed DOI PMC

Durante IM, Butenko A, Rašková V, Charyyeva A, Svobodová M, Yurchenko V, Hashimi H, Lukeš J. 2020. Large-scale phylogenetic analysis of trypanosomatid adenylate cyclases reveals associations with extracellular lifestyle and host-pathogen interplay. Genome Biol Evol 12:2403–2416. doi: 10.1093/gbe/evaa226. PubMed DOI PMC

Ishemgulova A, Hlaváčová J, Majerová K, Butenko A, Lukeš J, Votýpka J, Volf P, Yurchenko V. 2018. CRISPR/Cas9 in PubMed DOI PMC

Loreng TD, Smith EF. 2017. The central apparatus of cilia and eukaryotic flagella. Cold Spring Harb Perspect Biol 9:a028118. doi: 10.1101/cshperspect.a028118. PubMed DOI PMC

Ralston KS, Lerner AG, Diener DR, Hill KL. 2006. Flagellar motility contributes to cytokinesis in PubMed DOI PMC

Beneke T, Madden R, Makin L, Valli J, Sunter J, Gluenz E. 2017. A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids. R Soc Open Sci 4:170095. doi: 10.1098/rsos.170095. PubMed DOI PMC

Yurchenko V, Lukeš J. 2018. Parasites and their (endo)symbiotic microbes. Parasitology 145:1261–1264. doi: 10.1017/S0031182018001257. PubMed DOI

Catta-Preta CM, Brum FL, da Silva CC, Zuma AA, Elias MC, de Souza W, Schenkman S, Motta MC. 2015. Endosymbiosis in trypanosomatid protozoa: the bacterium division is controlled during the host cell cycle. Front Microbiol 6:520. doi: 10.3389/fmicb.2015.00520. PubMed DOI PMC

Loyola-Machado AC, Azevedo-Martins AC, Catta-Preta CMC, de Souza W, Galina A, Motta MCM. 2017. The symbiotic bacterium fuels the energy metabolism of the host trypanosomatid PubMed DOI

Hollar L, Lukeš J, Maslov DA. 1998. Monophyly of endosymbiont containing trypanosomatids: phylogeny versus taxonomy. J Eukaryot Microbiol 45:293–297. doi: 10.1111/j.1550-7408.1998.tb04539.x. PubMed DOI

Harmer J, Yurchenko V, Nenarokova A, Lukeš J, Ginger ML. 2018. Farming, slaving and enslavement: histories of endosymbiosis during kinetoplastid evolution. Parasitology 145:1311–1323. doi: 10.1017/S0031182018000781. PubMed DOI

Silva FM, Kostygov AY, Spodareva VV, Butenko A, Tossou R, Lukes J, Yurchenko V, Alves JMP. 2018. The reduced genome of PubMed DOI

Fampa P, Correa-da-Silva MS, Lima DC, Oliveira SM, Motta MC, Saraiva EM. 2003. Interaction of insect trypanosomatids with mosquitoes, sand fly and the respective insect cell lines. Int J Parasitol 33:1019–1026. doi: 10.1016/s0020-7519(03)00124-3. PubMed DOI

de Azevedo-Martins AC, Alves JM, de Mello FG, Vasconcelos AT, de Souza W, Einicker-Lamas M, Motta MC. 2015. Biochemical and phylogenetic analyses of phosphatidylinositol production in PubMed DOI PMC

Bombaça ACS, Dias FA, Ennes-Vidal V, Garcia-Gomes ADS, Sorgine MHF, d'Avila-Levy CM, Menna-Barreto RFS. 2017. Hydrogen peroxide resistance in PubMed DOI

Brunoro GVF, Menna-Barreto RFS, Garcia-Gomes AS, Boucinha C, Lima DB, Carvalho PC, Teixeira-Ferreira A, Trugilho MRO, Perales J, Schwammle V, Catanho M, de Vasconcelos ATR, Motta MCM, d'Avila-Levy CM, Valente RH. 2019. Quantitative proteomic map of the trypanosomatid PubMed DOI

d’Avila-Levy CM, Santos LO, Marinho FA, Matteoli FP, Lopes AHCS, Motta MCM, Santos ALS, Branquinha MH. 2008. PubMed DOI

d'Avila-Levy CM, Souza RF, Gomes RC, Vermelho AB, Branquinha MH. 2003. A metalloproteinase extracellularly released by PubMed DOI

Opperdoes FR, Borst P. 1977. Localization of nine glycolytic enzymes in a microbody-like organelle in PubMed DOI

Opperdoes F, Michels PA. 2008. The metabolic repertoire of

Ishemgulova A, Butenko A, Kortišová L, Boucinha C, Grybchuk-Ieremenko A, Morelli KA, Tesařová M, Kraeva N, Grybchuk D, Pánek T, Flegontov P, Lukeš J, Votýpka J, Pavan MG, Opperdoes FR, Spodareva V, d'Avila-Levy CM, Kostygov AY, Yurchenko V. 2017. Molecular mechanisms of thermal resistance of the insect trypanosomatid PubMed DOI PMC

Bushnell B, Rood J, Singer E. 2017. BBMerge—accurate paired shotgun read merging PubMed DOI PMC

Andrews S. 2019. FastQC: a quality control tool for high throughput sequence data. http://www.bioinformatics.babraham.ac.uk/projects/fastqc. Accessed 8 March 2021.

Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV, Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA. 2012. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19:455–477. doi: 10.1089/cmb.2012.0021. PubMed DOI PMC

Bolger AM, Lohse M, Usadel B. 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114–2120. doi: 10.1093/bioinformatics/btu170. PubMed DOI PMC

Langmead B, Salzberg SL. 2012. Fast gapped-read alignment with Bowtie 2. Nat Methods 9:357–359. doi: 10.1038/nmeth.1923. PubMed DOI PMC

Pertea M, Pertea GM, Antonescu CM, Chang TC, Mendell JT, Salzberg SL. 2015. StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nat Biotechnol 33:290–295. doi: 10.1038/nbt.3122. PubMed DOI PMC

Steinbiss S, Silva-Franco F, Brunk B, Foth B, Hertz-Fowler C, Berriman M, Otto TD. 2016. Companion: a web server for annotation and analysis of parasite genomes. Nucleic Acids Res 44:W29–W34. doi: 10.1093/nar/gkw292. PubMed DOI PMC

Waterhouse RM, Seppey M, Simão FA, Manni M, Ioannidis P, Klioutchnikov G, Kriventseva EV, Zdobnov EM. 2018. BUSCO applications from quality assessments to gene prediction and phylogenomics. Mol Biol Evol 35:543–548. doi: 10.1093/molbev/msx319. PubMed DOI PMC

Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R, Genome Project Data Processing S . 2009. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25:2078–2079. doi: 10.1093/bioinformatics/btp352. PubMed DOI PMC

Liao Y, Smyth GK, Shi W. 2014. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30:923–930. doi: 10.1093/bioinformatics/btt656. PubMed DOI

Love MI, Huber W, Anders S. 2014. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15:550. doi: 10.1186/s13059-014-0550-8. PubMed DOI PMC

Törönen P, Medlar A, Holm L. 2018. PANNZER2: a rapid functional annotation web server. Nucleic Acids Res 46:W84–W88. doi: 10.1093/nar/gky350. PubMed DOI PMC

Alexa A, Rahnenfuhrer J. 2020. topGO: enrichment analysis for Gene Ontology, v2.42.0.

Kanehisa M, Sato Y, Morishima K. 2016. BlastKOALA and GhostKOALA: KEGG tools for functional characterization of genome and metagenome sequences. J Mol Biol 428:726–731. doi: 10.1016/j.jmb.2015.11.006. PubMed DOI

Almagro Armenteros JJ, Salvatore M, Emanuelsson O, Winther O, von Heijne G, Elofsson A, Nielsen H. 2019. Detecting sequence signals in targeting peptides using deep learning. Life Sci Alliance 2:e201900429. doi: 10.26508/lsa.201900429. PubMed DOI PMC

Emms DM, Kelly S. 2019. OrthoFinder: phylogenetic orthology inference for comparative genomics. Genome Biol 20:238. doi: 10.1186/s13059-019-1832-y. PubMed DOI PMC

Katoh K, Standley DM. 2013. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol 30:772–780. doi: 10.1093/molbev/mst010. PubMed DOI PMC

Capella-Gutiérrez S, Silla-Martinez JM, Gabaldon T. 2009. trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics 25:1972–1973. doi: 10.1093/bioinformatics/btp348. PubMed DOI PMC

Nguyen LT, Schmidt HA, von Haeseler A, Minh BQ. 2015. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol 32:268–274. doi: 10.1093/molbev/msu300. PubMed DOI PMC

Lartillot N, Rodrigue N, Stubbs D, Richer J. 2013. PhyloBayes MPI: phylogenetic reconstruction with infinite mixtures of profiles in a parallel environment. Syst Biol 62:611–615. doi: 10.1093/sysbio/syt022. PubMed DOI

Kumar S, Stecher G, Li M, Knyaz C, Tamura K. 2018. MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms. Mol Biol Evol 35:1547–1549. doi: 10.1093/molbev/msy096. PubMed DOI PMC

Butenko A, Kostygov AY, Sádlová J, Kleschenko Y, Bečvář T, Podešvová L, Macedo DH, Žihala D, Lukeš J, Bates PA, Volf P, Opperdoes FR, Yurchenko V. 2019. Comparative genomics of PubMed DOI PMC

Robinson KA, Beverley SM. 2003. Improvements in transfection efficiency and tests of RNA interference (RNAi) approaches in the protozoan parasite PubMed DOI

Kelly S, Reed J, Kramer S, Ellis L, Webb H, Sunter J, Salje J, Marinsek N, Gull K, Wickstead B, Carrington M. 2007. Functional genomics in PubMed DOI PMC

Sollelis L, Ghorbal M, MacPherson CR, Martins RM, Kuk N, Crobu L, Bastien P, Scherf A, Lopez-Rubio JJ, Sterkers Y. 2015. First efficient CRISPR-Cas9-mediated genome editing in PubMed DOI

Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A. 2012. Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682. doi: 10.1038/nmeth.2019. PubMed DOI PMC

Záhonová K, Hadariová L, Vacula R, Yurchenko V, Eliáš M, Krajčovič J, Vesteg M. 2014. A small portion of plastid transcripts is polyadenylated in the flagellate PubMed DOI

Grybchuk D, Macedo DH, Kleschenko Y, Kraeva N, Lukashev AN, Bates PA, Kulich P, Leštinová T, Volf P, Kostygov AY, Yurchenko V. 2020. The first non-LRV RNA virus in PubMed DOI PMC

Ishemgulova A, Kraeva N, Hlaváčová J, Zimmer SL, Butenko A, Podešvová L, Leštinová T, Lukeš J, Kostygov A, Votýpka J, Volf P, Yurchenko V. 2017. A putative ATP/GTP binding protein affects PubMed DOI PMC

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