BACKGROUND: Parasitism as a life strategy has independently evolved multiple times within the eukaryotic tree of life. Each lineage has developed mechanisms to invade hosts, exploit resources, and ensure replication, but our knowledge of survival mechanisms in many parasitic taxa remain extremely limited. One such group is the Myxozoa, which are obligate, dixenous cnidarians. Evidence suggests that myxozoans evolved from free-living ancestors to endoparasites around 600 million years ago and are likely one of the first metazoan parasites on Earth. Some myxozoans pose significant threats to farmed and wild fish populations, negatively impacting aquaculture and fish stocks; one such example is Sphaerospora molnari, which forms spores in the gills of common carp (Cyprinus carpio), disrupting gill epithelia and causing somatic and respiratory failure. Sphaerospora molnari undergoes sequential development in different organs of its host, with large numbers of morphologically distinct stages occurring in the blood, liver, and gills of carp. We hypothesize that these parasite life-stages differ in regards to their host exploitation, pathogenicity, and host immune evasion strategies and mechanisms. We performed stage-specific transcriptomic profiling to identify differentially expressed key functional gene groups that relate to these functions and provide a fundamental understanding of the mechanisms S. molnari uses to optimize its parasitic lifestyle. We aimed to identify genes that are likely related to parasite pathogenicity and host cell exploitation mechanisms, and we hypothesize that genes unique to S. molnari might be indicative of evolutionary innovations and specific adaptations to host environments. RESULTS: We used parasite isolation protocols and comparative transcriptomics to study early proliferative and spore-forming stages of S. molnari, unveiling variation in gene expression between each stage. We discovered several apparent innovations in the S. molnari transcriptome, including proteins that are likely to function in the uptake of previously unknown key nutrients, immune evasion factors that may contribute to long-term survival in hosts, and proteins that likely improve adhesion to host cells that may have arisen from horizontal gene transfer. Notably, we identified genes that are similar to known virulence factors in other parasitic organisms, particularly blood and intestinal parasites like Plasmodium, Trypanosoma, and Giardia. Many of these genes are absent in published cnidarian and myxozoan datasets and appear to be specific to S. molnari; they may therefore represent potential innovations enabling Sphaerospora to exploit the host's blood system. CONCLUSIONS: In order to address the threat posed by myxozoans to both cultured fish species and wild stocks, it is imperative to deepen our understanding of their genetics. Sphaerospora molnari offers an appealing model for stage-specific transcriptomic profiling and for identifying differentially expressed key functional gene groups related to parasite development. We identified genes that are thus far unique to S. molnari, which reveal their evolutionary novelty and likely role as adaptations to specific host niches. In addition, we describe the pathogenicity-associated genetic toolbox of S. molnari and discuss the implications of our discoveries for disease control by shedding light on specific targets for potential intervention strategies.

Department of Life Sciences University of Modena and Reggio Emilia Modena Italy

Faculty of Science University of South Bohemia České Budějovice Czech Republic

Fish Health Division University of Veterinary Medicine Vienna Austria

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

National Biodiversity Future Center Palermo Italy

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Windsor DA. Controversies in parasitology, most of the species on earth are parasites. Int J Parasitol. 1998;28:1939–41. PubMed

de Meeûs T, Renaud F. Parasites within the new phylogeny of eukaryotes. Trends Parasitol. 2002;18:247–51. PubMed

Okamura B, Hartigan A, Naldoni J. Extensive uncharted biodiversity: the parasite dimension. Integr Comp Biol. 2018;58:1132–45. PubMed

Parker GA, Chubb JC, Ball MA, Roberts GN. Evolution of complex life cycles in helminth parasites. Nature. 2003;425:480–4. PubMed

Schultz DT, Haddock SHD, Bredeson JV, Green RE, Simakov O, Rokhsar DS. Ancient gene linkages support ctenophores as sister to other animals. Nature. 2023;618:110–7. PubMed PMC

Holzer AS, Bartosová-Sojková P, Born-Torrijos A, Lövy A, Hartigan A, Fiala I. The joint evolution of the Myxozoa and their alternate hosts: a cnidarian recipe for success and vast biodiversity. Mol Ecol. 2018;27:1651–66. PubMed

Zhang Z-Q. Animal biodiversity: an update of classification and diversity in 2013. In: Zhang, z.-q, editor animal biodiversity: an outline of higher-level classification and survey of taxonomic richness (addenda 2013). Zootaxa. 2013;3703:5–11. PubMed

Atkinson SD, Bartholomew JL, Lotan T, Myxozoans. Ancient metazoan parasites find a home in phylum Cnidaria. Zoology. 2018;129:66–8. PubMed

Weinstein SB, Kuris AM. Independent origins of parasitism in Animalia. Biol Lett. 2016;12:20160324. PubMed PMC

Rodríguez E, Barbeitos MS, Brugler MR, Crowley LM, Grajales A, Gusmao L, et al. Hidden among sea anemones: the first comprehensive phylogenetic reconstruction of the order Actiniaria (Cnidaria, Anthozoa, Hexacorallia) reveals a novel group of xexacorals. PLoS ONE. 2014;9:e96998. PubMed PMC

Bentlage B, Osborn KJ, Lindsay DJ, Hopcroft RR, Raskoff KA, Collins AG. Loss of metagenesis and evolution of a parasitic life style in a group of open-ocean jellyfish. Mol Phylogenet Evol. 2018;124:50–9. PubMed

Okamura B, Gruhl A. Evolution, origins and diversification of parasitic cnidarians. The evolution and fossil record of parasitism: identification and macroevolution of parasites. Springer; 2021. pp. 109–52.

Wolf K, Markiw ME. Biology contravenes taxonomy in the Myxozoa - New discoveries show alternation of invertibrate and vertibrate hosts. Science. 1984;225:1449–52. PubMed

Feist SW, Morris DJ, Alama-Bermejo G, Holzer AS. Cellular processes in myxozoans. In: Okamura B, Gruhl A, Bartholomew J, editors. Myxozoan evolution, ecology and development. Cham: Springer; 2015.

Ben-David J, Atkinson SD, Pollak Y, Yossifon G, Shavit U, Bartholomew JL, et al. Myxozoan polar tubules display structural and functional variation. Parasites Vectors. 2016;9:549. PubMed PMC

Guo QX, Whipps CM, Zhai YH, Li D, Gu ZM. Quantitative insights into the contribution of nematocysts to the adaptive success of cnidarians based on proteomic analysis. Biology. 2022;11:91. PubMed PMC

El-Matbouli M, McDowell TS, Antonio DB, Andree KB, Hedrick RP. Effect of water temperature on the development, release and survival of the triactinomyxon stage of Myxobolus cerebralis in its oligochaete host. Int J Parasitol. 1999;29:627–41. PubMed

Lom J, Dyková I. Myxozoan genera:: definition and notes on taxonomy, life-cycle terminology and pathogenic species. Folia Parasitol. 2006;53:1–36. PubMed

Eszterbauer E, Atkinson S, Diamant A, Morris D, El-Matbouli M, Hartikainen H. Myxozoan life cycles: practical approaches and insights. In: Okamura B, Gruhl A, Bartholomew J, editors. Myxozoan evolution, ecology and development. Cham: Springer; 2015. pp. 175–98.

Alvarez-Pellitero P, Sitjá-Bobadilla A. Pathology of Myxosporea in marine fish culture. Dis Aquat Organ. 1993;17:229–38.

Kent ML, Margolis L, Corliss JO. The demise of a class of protists - taxonomic and nomenclatural revisions proposed for the protist phylum Myxozoa Grasse, 1970. Can J Zool. 1994. pp. 932–7.

Nehring RB, Walker PG. Whirling disease in the wild: the new reality in the intermountain west. Fisheries. 1996;21:28–30.

Pote LM, Hanson LA, Shivaji R. Small subunit ribosomal RNA sequences link the cause of proliferative gill disease in channel catfish to Henneguya n. Sp (Myxozoa: Myxosporea). J Aquat Anim Health. 2000;12:230–40.

Moran JDW, Whitaker DJ, Kent ML. A review of the myxosporean genus Kudoa Meglitsch, 1947, and its impact on the international aquaculture industry and commercial fisheries. Aquaculture. 1999. pp. 163–96.

Palenzuela O, Redondo MJ, Alvarez-Pellitero P. Description of Enteromyxum Scophthalmi gen. nov., sp. nov. (Myxozoa), an intestinal parasite of turbot (Scophthalmus maximus L.) using morphological and ribosomal RNA sequence data. Parasitology. 2002;124:369–79. PubMed

Jones SR, Bartholomew JL, Zhang JY. Mitigating myxozoan disease impacts on wild fish populations. In: Okamura B, Gruhl A, Bartholomew J, editors. Myxozoan evolution, ecology and development. Cham: Springer; 2015.

Hutchins PR, Sepulveda AJ, Hartikainen H, Staigmiller KD, Opitz ST, Yamamoto RM, et al. Exploration of the 2016 Yellowstone river fish kill and proliferative kidney disease in wild fish populations. Ecosphere. 2021;12:e03436.

Holland JW, Holzer AS. Myxozoan research forum 2021-the MyxoMixer: advances, methods, and problems yet to be solved in myxozoan research. Bull Eur Association Fish Pathologists. 2022;41:216–24.

Lom J, Dyková I, Pavlasková M, Grupcheva G. Sphaerospora molnari sp. nov. (Myxozoa, Myxosporea), an agent of gill, skin and blood sphaerosporosis of common carp in Europe. Parasitology. 1983;86:529–35.

Dykova I, Lom J. Review of pathogenetic myxosporeans in intensive culture of carp (Cyprinus carpio) in Europe. Folia Parasitol. 1988;35:289–307.

Korytář T, Wiegertjes GF, Zusková E, Tomanová A, Lisnerová M, Patra S, Sieranski V, Sima R, Born-Torrijos A, Wentzel AS, Blasco-Monleon S, Yanes-Roca C, Policar T, Holzer AS. The kinetics of cellular and humoral immune responses of common carp to presporogonic development of the myxozoan Sphaerospora Molnari. Parasites Vectors. 2019;12:208. PubMed PMC

Lu BX, Zeng ZB, Shi TL. Comparative study of de novo assembly and genome-guided assembly strategies for transcriptome reconstruction based on RNA-Seq. Sci China-Life Sci. 2013;56:143–55. PubMed

Lischer HEL, Shimizu KK. Reference-guided de novo assembly approach improves genome reconstruction for related species. BMC Bioinformatics. 2017;18:1–12. PubMed PMC

Liu Y, Zhou J, White KP. RNA-seq differential expression studies: more sequence or more replication? Bioinformatics. 2014;30:301–4. PubMed PMC

Wu T, Hu E, Xu S, Chen M, Guo P, Dai Z, et al. clusterProfiler 4.0: a universal enrichment tool for interpreting omics data. Innov. 2021;2:100141. PubMed PMC

Alexa A, Rahnenfuhrer J. topGO: enrichment analysis for gene ontology. R Package Version. 2010;2:2010.

Hartigan A, Estensoro I, Vancová M, Bíly T, Patra S, Eszterbauer E, et al. New cell motility model observed in parasitic cnidarian Sphaerospora Molnari (Myxozoa: Myxosporea) blood stages in fish. Sci Rep. 2016;6:39093. PubMed PMC

Chen Y-P, Riestra AM, Rai AK, Johnson PJ. A novel cadherin-like protein mediates adherence to and killing of Host Cells by the parasite trichomonas vaginalis. mBio. 2019;10:e00720–19. PubMed PMC

O’Connor RM, Wanyiri JW, Cevallos AM, Priest JW, Ward HD. Cryptosporidium parvum glycoprotein gp40 localizes to the sporozoite surface by association with gp15. Mol Biochem Parasitol. 2007;156:80–3. PubMed PMC

Dalton JP, Skelly P, Halton DW. Role of the tegument and gut in nutrient uptake by parasitic platyhelminths. Can J Zool. 2004;82:211–32.

Toh SQ, Glanfield A, Gobert GN, Jones MK. Heme and blood-feeding parasites: friends or foes? Parasites Vectors. 2010;3:108. PubMed PMC

Dean P, Major P, Nakjang S, Hirt RP, Embley TM. Transport proteins of parasitic protists and their role in nutrient salvage. Front Plant Sci. 2014;5:153. PubMed PMC

Yang YL, Xiong J, Zhou ZG, Huo FM, Miao W, Ran C, et al. The genome of the Myxosporean Thelohanellus kitauei shows adaptations to nutrient acquisition within its fish host. Genome Biol Evol. 2014;6:3182–98. PubMed PMC

Reyes-López M, Aguirre-Armenta B, Piña-Vázquez C, de la Garza M, Serrano-Luna J. Hemoglobin uptake and utilization by human protozoan parasites: a review. Front Cell Infect Microbiol. 2023;13:111150054. PubMed PMC

Shang F, Lan J, Wang L, Liu W, Chen Y, Chen J, et al. Crystal structure of the Siderophore-interacting protein SIP from Aeromonas hydrophila. Biochem Biophys Res Community. 2019;519:23–8. PubMed

Lombardo ME, Araujo LS, Batlle A. 5-aminolevulinic acid synthesis in epimastigotes of Trypanosoma Cruzi. Int J Biochem Cell Biol. 2003;35:1263–71. PubMed

Wiśniewska MM, Kyslík J, Alama-Bermejo G, Lövy A, Kolísko M, Holzer A et al. Comparative transcriptomics reveal stage-dependent parasitic adaptations in the myxozoan Sphaerospora Molnari, GenBank. 2024. https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1193866 PubMed PMC

Faber M, Shaw S, Yoon S, Alves ED, Wang B, Qi ZT, et al. Comparative transcriptomics and host-specific parasite gene expression profiles inform on drivers of proliferative kidney disease. Sci Rep. 2021;11:2149. PubMed PMC

Brekhman V, Ofek-Lalzar M, Atkinson SD, Alama-Bermejo G, Maor-Landaw K, Malik A, et al. Proteomic analysis of the oarasitic cnidarian Ceratonova Shasta (Cnidaria: Myxozoa) reveals diverse roles of actin in motility and spore formation. Front Mar Sci. 2021;8:632700.

Leclère L, Horin C, Chevalier S, Lapébie P, Dru P, Peron S, et al. The genome of the jellyfish Clytia hemisphaerica and the evolution of the cnidarian life-cycle. Nat Ecol Evol. 2019;3:801–10. PubMed

Murphy JR. Corynebacterium diphtheriae: diphtheria toxin production. In: Baron S, et al. editors. Medical Microbiology. 4th ed. Galveston, Texas: University of Texas Medical Branch; 1996. PubMed

Shoop WL, Xiong Y, Wiltsie J, Woods A, Guo J, Pivnichny JV, et al. Anthrax lethal factor inhibition. Proc Natl Acad Sci USA. 2005;102:7958–63. PubMed PMC

Bowran K, Palmer T. Extreme genetic diversity in the type VII secretion system of Listeria monocytogenes suggests a role in bacterial antagonism. Microbiology. 2021;167:0001034. PubMed PMC

Hartigan A, Jaimes-Becerra A, Okamura B, Doonan LB, Ward M, Marques AC, et al. Recruitment of toxin-like proteins with ancestral venom function supports endoparasitic lifestyles of Myxozoa. PeerJ. 2021;9:e11208. PubMed PMC

Crauwels P, Bohn R, Thomas M, Gottwalt S, Jäckel F, Krämer S, et al. Apoptotic-like Leishmania exploit the host´s autophagy machinery to reduce T-cell-mediated parasite elimination. Autophagy. 2015;11:285–97. PubMed PMC

Americus B, Hams N, Klompen AML, Alama-Bermejo G, Lotan T, Bartholomew JL, et al. The cnidarian parasite Ceratonova Shasta utilizes inherited and recruited venom-like compounds during infection. PeerJ. 2021;9:e12606. PubMed PMC

Huene AL, Sanders SM, Ma Z, Nguyen AD, Koren S, Michaca MH, et al. A family of unusual immunoglobulin superfamily genes in an invertebrate histocompatibility complex. Proc Natl Acad Sci USA. 2022;119:e2207374119. PubMed PMC

Deitsch KW, Lukehart SA, Stringer JR. Common strategies for antigenic variation by bacterial, fungal and protozoan pathogens. Nat Rev Microbiol. 2009;7:493–503. PubMed PMC

Schmid-Hempel P. Parasite immune evasion: a momentous molecular war. Trends Ecol Evol. 2008;23:318–26. PubMed

Piriatinskiy G, Atkinson SD, Park S, Morgenstern D, Brekhman V, Yossifon G, et al. Functional and proteomic analysis of Ceratonova Shasta (Cnidaria: Myxozoa) polar capsules reveals adaptations to parasitism. Sci Rep. 2017;7:9010. PubMed PMC

Alama-Bermejo G, Bartošová-Sojková P, Atkinson SD, Holzer AS, Bartholomew JL. Proteases as therapeutic targets against the parasitic cnidarian Ceratonova Shasta: characterization of molecules key to parasite virulence in salmonid hosts. Front Cell Infect Microbiol. 2022;11:804864. PubMed PMC

Bartošová-Sojková P, Kyslík J, Alama-Bermejo G, Hartigan A, Atkinson SD, Bartholomew JL, et al. Evolutionary analysis of cystatins of early-emerging metazoans reveals a novel subtype in parasitic cnidarians. Biology. 2021;10:110. PubMed PMC

Hartigan A, Kosakyan A, Pecková H, Eszterbauer E, Holzer AS. Transcriptome of Sphaerospora molnari (Cnidaria, Myxosporea) blood stages provides proteolytic arsenal as potential therapeutic targets against sphaerosporosis in common carp. BMC Genomics. 2020;21:404. PubMed PMC

Nash TE. Surface antigenic variation in Giardia lamblia. Mol Microbiol. 2002;45:585–90. PubMed

Xu F, Jerlström-Hultqvist J, Einarsson E, Astvaldsson A, Svärd SG, Andersson JO. The genome of Spironucleus salmonicida highlights a fish pathogen adapted to fluctuating environments. PLoS Genet. 2014;10:e1004053. PubMed PMC

Wiśniewska MM, Kyslik J, Alama-Bermejo G, Lövy A, Kolísko M, Holzer AS et al. Comparative transcriptomics reveal stage-dependent parasitic adaptations in the myxozoan Sphaerospora Molnari. FigShare. 2024. 10.6084/m9.figshare.24639318 PubMed PMC

Spielmann T, Fergusen DJP, Beck H-P. Etramps, a new Plasmodium falciparum gene family coding for developmentally regulated and highly charged membrane proteins located at the parasite–host cell interface. Mol Biol Cell. 2003;14:1529–44. PubMed PMC

Kyes S, Horrocks P, Newbold C. Antigenic variation at the infected red cell surface in malaria. Annu Rev Microbiol. 2001;55:673–707. PubMed

Niang M, Yam XY, Preiser PR. The Plasmodium Falciparum STEVOR multigene family mediates antigenic variation of the infected erythrocyte. PLoS Pathog. 2009;5:e1000307. PubMed PMC

Bachmann A, Scholz JAM, Janssen M, Klinkert MQ, Tannich E, Bruchhaus I, et al. A comparative study of the localization and membrane topology of members of the RIFIN, STEVOR and PfMC-2TM protein families in Plasmodium falciparum-infected erythrocytes. Malar J. 2015;14:1–18. PubMed PMC

Andersson A, Kudva R, Magoulopoulou A, Lejqarre EQ, Lara P, Xu PB, et al. Membrane integration and topology of RIFIN and STEVOR proteins of the Plasmodium falciparum parasite. FEBS J. 2020;287:2744–62. PubMed

Huminiecki L, Wolfe KH. Divergence of spatial gene expression profiles following species-specific gene duplications in human and mouse. Genome Res. 2004;14:1870–9. PubMed PMC

Tautz D, Domazet-Loso T. The evolutionary origin of orphan genes. Nat Rev Genet. 2011;12:692–702. PubMed

Singh U, Wurtele ES. Genetic novelty: how new genes are born. eLife. 2020;9:e55136. PubMed PMC

Guo QX, Atkinson SD, Xiao B, Zhai YH, Bartholomew JL, Gu ZM. A myxozoan genome reveals mosaic evolution in a parasitic cnidarian. BMC Biol. 2022;20:51. PubMed PMC

McBride MJ, Braun TF, Brust JL. Flavobacterium johnsoniae GldH is a lipoprotein that is required for gliding motility and chitin utilization. J Bacteriol. 2003;185:6648–57. PubMed PMC

Hale CA, Shiomi D, Liu B, Bernhardt TG, Margolin W, Niki H, et al. Identification of Escherichia coli ZapC (YcbW) as a component of the division apparatus that binds and bundles FtsZ polymers. J Bacteriol. 2011;193:1393–404. PubMed PMC

Ravindran S, Boothroyd JC. Secretion of proteins into host cells by Apicomplexan parasites. Traffic. 2008;9:647–56. PubMed

Portes J, Barrias E, Travassos R, Attias M, de Souza W. ToxopGondiigondii mechanisms of entry into host cells. Front Cell Infect Microbiol. 2020;10:294. PubMed PMC

Patra S, Bartosová-Sojková P, Pecková H, Fiala I, Eszterbauer E, Holzer AS. Biodiversity and host-parasite cophylogeny of Sphaerospora (sensu stricto) (Cnidaria: Myxozoa). Parasites Vectors. 2018;11:1–24. PubMed PMC

Marsden RL, Ranea JAG, Sillero A, Redfern O, Yeats C, Maibaum M, et al. Exploiting protein structure data to explore the evolution of protein function and biological complexity. Philosophical Trans Royal Soc B-Biological Sci. 2006;361:425–40. PubMed PMC

Wilson GA, Feil EJ, Lilley AK, Field D. Large-scale comparative genomic ranking of taxonomically restricted genes (TRGs) in bacterial and archaeal genomes. PLoS ONE. 2007;2:e324. PubMed PMC

Weisman CM, Murray AW, Eddy SR. Many, but not all, lineage-specific genes can be explained by homology detection failure. PLoS Biol. 2020;18:e3000862. PubMed PMC

Wilson GA, Bertrand N, Patel Y, Hughes JB, Feil EJ, Field D. Orphans as taxonomically restricted and ecologically important genes. Microbiology. 2005;151:2499–501. PubMed

Ma DN, Lai ZF, Ding QS, Zhang K, Chang KZ, Li SH, et al. Identification, characterization and function of orphan genes among the current Cucurbitaceae genomes. Front Plant Sci. 2022;13:872137. PubMed PMC

Holzer AS, Pimentel-Santos J. Sphaerospora Molnari. Fish parasites: a handbook of protocols for their isolation, culture and transmission. In: Sitja-Bobadilla A, Bron J, Wiegertjes G, Piazzon M, editors. European association of fish pathologists. 5 M Books Ltd.; 2021.

Born-Torrijos A, Kosakyan A, Patra S, Pimentel-Santos J, Panicucci B, Chan JTH, et al. Method for isolation of myxozoan proliferative stages from fish at high yield and purity: an essential prerequisite for in vitro, in vivo and genomics-based research developments. Cells. 2022;1:377. PubMed PMC

Alama-Bermejo G, Meyer E, Atkinson SD, Holzer AS, Wiśniewska MM, Kolísko M, et al. Transcriptome-wide comparisons and virulence gene polymorphisms of host-associated genotypes of the cnidarian parasite Ceratonova Shasta in salmonids. Genome Biol Evol. 2020;12:1258–76. PubMed PMC

Kolder ICRM, van der Plas-Duivesteijn SJ, Tan G, Wiegertjes GF, Forlenza M, Guler AT, et al. A full-body transcriptome and proteome resource for the European common carp. BMC Genomics. 2016;17:701. PubMed PMC

Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, et al. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol. 2011;29:644–52. PubMed PMC

Pruitt KD, Tatusova T, Maglott DR. NCBI Reference sequence (RefSeq): a curated non-redundant sequence database of genomes, transcripts and proteins. Nucleic Acids Res. 2005;33:D501–4. PubMed PMC

Mistry J, Chuguransky S, Williams L, Qureshi M, Salazar GA, Sonnhammer ELL, et al. Pfam: the protein families database in 2021. Nucleic Acids Res. 2021;49:D412–9. PubMed PMC

UniProt Consortium. The Universal Protein Resource (UniProt) in 2010. Nucleic Acids Res. 2010;38:D142–148. PubMed PMC

Cantalapiedra CP, Hernández-Plaza A, Letunic I, Bork P, Huerta-Cepas J. eggNOG-mapper v2: functional annotation, orthology assignments, and domain prediction at the metagenomic scale. Mol Biol Evol. 2021;38:5825–9. PubMed PMC

Kanehisa M, Sato Y, Kawashima M, Furumichi M, Tanabe M. KEGG as a reference resource for gene and protein annotation. Nucleic Acids Res. 2016;44:D457–462. PubMed PMC

Eddy SR. Accelerated Profile HMM searches. PLoS Comput Biol. 2011;7:e1002195. PubMed PMC

Armenteros JJA, Tsirigos KD, Sonderby CK, Petersen TN, Winther O, Brunak S, et al. SignalP 5.0 improves signal peptide predictions using deep neural networks. Nat Biotechnol. 2019;37:420–3. PubMed

Kanehisa M, Sato Y, Morishima K. BlastKOALA and GhostKOALA: KEGG Tools for functional characterization of genome and metagenome sequences. J Mol Biol. 2016;428:726–31. PubMed

Manni M, Berkeley MR, Seppey M, Simao FA, 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 Evol. 2021;38:4647–54. PubMed PMC

Emms DM, Kelly S. OrthoFinder: phylogenetic orthology inference for comparative genomics. Genome Biol. 2019;20:1–14. PubMed PMC

Wickham H, Averick M, Bryan J, Chang W, McGowan LD, François R, et al. Welcome to the Tidyverse. J Open Source Softw. 2019;4:1686.

Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012;9:357–9. PubMed PMC

Liao Y, Smyth GK, Shi W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics. 2014;30:923–30. PubMed

Ge SX, Son EW, Yao RN. iDEP: an integrated web application for differential expression and pathway analysis of RNA-Seq data. BMC Bioinformatics. 2018;19:1–24. PubMed PMC

Love MI, Huber W, Anders S. Moderated estimation of Fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15:1–21. PubMed PMC

Garnier S, Ross N, Rudis R, Camargo AP, Sciaini M, Scherer C. Rvision-colorblind-friendly color maps for R. R package version 06. 2021;2.

Kolde R, Kolde MR. Package ‘pheatmap’. R Package. 2015;1:790.

Stanke M, Diekhans M, Baertsch R, Haussler D. Using native and syntenically mapped cDNA alignments to improve de novo gene finding. Bioinformatics. 2008;24:637–44. PubMed

Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol. 2012;19:455–77. PubMed PMC

Almeida-Silva F, Van de Peer Y. Assessing the quality of comparative genomics data and results with the cogeqc R/Bioconductor package. MEE. 2023;14:2942–2952. Available: https://github.com/almeidasilvaf/cogeqc

Emanuelsson O, Nielsen H, Brunak S, von Heijne G. Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J Mol Biol. 2000;300:1005–16. PubMed

Horton P, Park KJ, Obayashi T, Fujita N, Harada H, Adams-Collier CJ, et al. WoLF PSORT: protein localization predictor. Nucleic Acids Res. 2007;35:W585–7. PubMed PMC

Krogh A, Larsson B, von Heijne G, Sonnhammer ELL. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol. 2001;305:567–80. PubMed

Wiśniewska MM, Kyslik J, Alama-Bermejo G, Lövy A, Kolisko M, Holzer AS et al. Comparative transcriptomics reveal stage-dependent parasitic adaptations in the myxozoan Sphaerospora Molnari, GenBank. 2024. https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1067885 PubMed PMC

Comparative transcriptomics reveal stage-dependent parasitic adaptations in the myxozoan Sphaerospora molnari