Hybrid generations usually face either a heterosis advantage or a breakdown, that can be expressed by the level of parasite infection in hybrid hosts. Hybrids are less infected by parasites than parental species (especially F1 generations) or more infected than parental species (especially post-F1 generations). We performed the experiment with blood-feeding gill parasite Paradiplozoon homoion (Monogenea) infecting leuciscid species, Abramis brama and Rutilus rutilus, their F1 generation and two backcross generations. Backcross generations tended to be more parasitized than parental lines and the F1 generation. The number of differentially expressed genes (DEGs) was lower in F1 hybrids and higher in backcross hybrids when compared to each of the parental lines. The main groups of DEGs were shared among lines; however, A. brama and R. rutilus differed in some of the top gene ontology (GO) terms. DEG analyses revealed the role of heme binding and erythrocyte differentiation after infection by blood-feeding P. homoion. Two backcross generations shared some of the top GO terms, representing mostly downregulated genes associated with P. homoion infection. KEGG analysis revealed the importance of disease-associated pathways; the majority of them were shared by two backcross generations. Our study revealed the most pronounced DEGs associated with blood-feeding monogeneans in backcross hybrids, potentially (but not exclusively) explainable by hybrid breakdown. The lower DEGs reported in F1 hybrids being less parasitized than backcross hybrids is in line with the hybrid advantage.

Central European Institute of Technology Masaryk University 625 00 Brno Czech Republic

Department of Botany and Zoology Faculty of Science Masaryk University Kotlářská 2 611 37 Brno Czech Republic

Institute of Vertebrate Biology of the Czech Academy of Sciences Květná 8 603 65 Brno Czech Republic

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Thoney D.A., Hargis W.J. Monogenea (Platyhelminthes) as hazards for fish in confinement. Annu. Rev. Fish Dis. 1991;1:133–153. doi: 10.1016/0959-8030(91)90027-H. DOI

Bakke T.A., Cable J., Harris P.D. The biology of gyrodactylid monogeneans: The “Russian-doll killers“. Adv. Parasitol. 2007;64:161–376. PubMed

Tu X., Ling F., Huang A., Wang G. An infection of Gyrodactylus kobayashii Hukuda, 1940 (Monogenea) associated with the mortality of goldfish (Carassius auratus) from central China. Parasitol. Res. 2015;114:737–745. doi: 10.1007/s00436-014-4241-x. PubMed DOI

Deveney M.R., Chisholm L.A., Whittington I.D. First published record of the pathogenic monogenean parasite Neobenedenia melleni (Capsalidae) from Australia. Dis. Aquat. Org. 2001;46:79–82. doi: 10.3354/dao046079. PubMed DOI

Ogawa K. Diseases of cultured marine fishes caused by Platyhelminthes (Monogenea, Digenea, Cestoda) Parasitology. 2014;142:178–195. doi: 10.1017/S0031182014000808. PubMed DOI

Shinn A., Pratoomyot J., Bron J., Paladini G., Brooker E., Brooker A. Economic impacts of aquatic parasites on global finfish production. Glob. Aquac. Advocate. 2015;2015:82–84.

Pugachev O.N., Gerasev P.I., Gussev A.V., Ergens R., Khotenowsky I. Guide to Monogenoidea of Freshwater Fish of Palaearctic and Amur Regions. Ledizione-LediPublishing; Milan, Italy: 2010.

Khotenovsky I. The Subclass Octomacrinea Khotenovsky. Nauka; Leningrad, Russia: 1985. (In Russian)

Gelnar M., Koubková B., Pláňková H., Jurajda P. Report on metazoan parasites of fishes of the River Morava with remarks on the effects of water pollution. Helminthologia. 1994;31:47–56.

Matějusová I., Koubková B., Gelnar M., Cunningham C.O. Paradiplozoon homoion Bychowsky & Nagibina, 1959 versus P. gracile Reichenbach-Klinke, 1961 (Monogenea): Two species or phenotypic plasticity? Syst. Parasitol. 2002;53:39–47. PubMed

Nejat F., Benovics M., Řehulková E., Vukić J., Šanda R., Kaya C., Tarkan A.S., Asghar Abdoli A., Aksu S., Šimková A. Diversity, phylogeny and intraspecific variability of Paradiplozoon species (Monogenea: Diplozoidae) parasitizing endemic cyprinoids in the Middle East. Parasitology. 2023;150:705–722. doi: 10.1017/S0031182023000446. PubMed DOI PMC

Konstanzová V., Koubková B., Kašný M., Ilgová J., Dzika E., Gelnar M. Ultrastructure of the digestive tract of Paradiplozoon homoion (Monogenea) Parasitol. Res. 2015;114:1485–1494. doi: 10.1007/s00436-015-4331-4. PubMed DOI

Krasnovyd V., Vetešník L., Gettová L., Civáňová K., Šimková A. Patterns of parasite distribution in the hybrids of non-congeneric cyprinid fish species: Is asymmetry in parasite infection the result of limited coadaptation? Int. J. Parasitol. 2017;47:471–483. doi: 10.1016/j.ijpara.2017.01.003. PubMed DOI

Kvach Y., Ondračková M., Bryjová A., Jurajda P. Parasites as biological tags of divergence in Central European gudgeon populations (Actinopterygii: Cyprinidae: Gobioninae) Biologia. 2017;72:671–679. doi: 10.1515/biolog-2017-0073. DOI

Aydogdu N., Avenant-Oldewage A., Dos Santos Q.M., Aydogdu A. Prevalence and intensity of Paradiplozoon homoion (Monogenea: Diplozoidae) from Manyas spirlin, Alburnoides manyasensis, an endemic fish of Turkey: New host and geographical record. Iran. J. Fish. Sci. 2022;19:3301–3309.

Dedic N., Vetešník L., Šimková A. Monogeneans in intergeneric hybrids of leuciscid fish: Is parasite infection driven by hybrid heterosis, genetic incompatibilities, or host-parasite coevolutionary interactions? Front. Zool. 2023;20:5. doi: 10.1186/s12983-022-00481-w. PubMed DOI PMC

Kawatsu H. Studies on the anemia of fish-IX. Nippon Suisan Gakkaishi. 1978;44:1315–1319. doi: 10.2331/suisan.44.1315. DOI

Rohlenová K., Morand S., Hyršl P., Tolarová S., Flajšhans M., Šimková A. Are fish immune systems really affected by parasites? An immunoecological study of common carp (Cyprinus carpio) Parasites Vectors. 2011;4:120. doi: 10.1186/1756-3305-4-120. PubMed DOI PMC

Hayden B., Pulcini D., Kelly-Quinn M., O’Grady M., Caffrey J., McGrath A., Mariani S. Hybridisation between two cyprinid fishes in a novel habitat: Genetics, morphology and life-history traits. BMC Evol. Biol. 2010;10:169. doi: 10.1186/1471-2148-10-169. PubMed DOI PMC

Toscano B.J., Pulcini D., Hayden B., Russo T., Kelly-Quinn M., Mariani S. An ecomorphological framework for the coexistence of two cyprinid fish and their hybrids in a novel environment. Biol. J. Linn. Soc. 2010;99:768–783. doi: 10.1111/j.1095-8312.2010.01383.x. DOI

Kuparinen A., Vinni M., Teacher A.G.F., Kähkönen K., Merilä J. Mechanism of hybridization between bream Abramis brama and roach Rutilus rutilus in their native range. J. Fish Biol. 2014;84:237–242. doi: 10.1111/jfb.12272. PubMed DOI

Konopinski M.K., Amirowicz A. Genetic composition of a population of natural common bream Abramis brama x roach Rutilus rutilus hybrids and their morphological characteristics in comparison with parent species. J. Fish Biol. 2018;92:365–385. doi: 10.1111/jfb.13506. PubMed DOI

Bartley D.M., Rana K., Immink A.J. The use of inter-species hybrids in aquaculture and fisheries. Rev. Fish Biol. Fish. 2001;10:325–337. doi: 10.1023/A:1016691725361. DOI

Šimková A., Dávidová M., Papoušek I., Vetešník L. Does interspecies hybridization affect the host specificity of parasites in cyprinid fish? Parasites Vectors. 2013;6:95. doi: 10.1186/1756-3305-6-95. PubMed DOI PMC

Šimková A., Civáňová K., Vetešník L. Heterosis versus breakdown in fish hybrids revealed by one-parental species-associated viral infection. Aquaculture. 2022;546:737406. doi: 10.1016/j.aquaculture.2021.737406. DOI

Krasnovyd V., Vetešník L., Šimková A. Distribution of host-specific parasites in hybrids of phylogenetically related fish: The effects of genotype frequency and maternal ancestry? Parasites Vectors. 2020;13:402. doi: 10.1186/s13071-020-04271-3. PubMed DOI PMC

Hayden B., Massa-Gallucci A., Caffrey J., Harrod C., Mariani S., O’Grady M., Kelly-Quinn M. Trophic dynamics within a hybrid zone—Interactions between an abundant cyprinid hybrid and sympatric parental species. Freshw. Biol. 2011;56:1723–1735. doi: 10.1111/j.1365-2427.2011.02604.x. DOI

Nzau Matondo B., Ovidio M., Poncin P., Kakesa T.A., Wamuini L.S., Philippart J.C. Hybridization success of three common European cyprinid species, Rutilus rutilus, Blicca bjoerkna and Abramis brama and larval resistance to stress tests. Fish. Sci. 2007;73:1137–1146. doi: 10.1111/j.1444-2906.2007.01445.x. DOI

Hayden B., McLoone P., Coyne J., Cafrey J.M. Extensive hybridization between roach, Rutilus rutilus L., and common bream, Abramis brama L. in Irish lakes and rivers. Biol. Environ. 2014;114:35–39. doi: 10.3318/bioe.2014.04. DOI

Šimková A., Gettová L., Civáňová K., Seifertová M., Janáč M., Vetešník L. Diversity of MHC IIB genes and parasitism in hybrids of evolutionarily divergent cyprinoid species indicate heterosis advantage. Sci. Rep. 2021;11:16860. doi: 10.1038/s41598-021-96205-x. PubMed DOI PMC

Dobzhansky T. Genetics and the Origin of Species. Columbia University Press; New York, NY, USA: 1937.

Muller H.J. Isolating mechanisms, evolution, and temperature. Biol. Symp. 1942;6:71–125.

Rand D.M., Haney R.A., Fry A.J. Cytonuclear coevolution: The genomics of cooperation. Trends Evol. Ecol. 2004;19:645–653. doi: 10.1016/j.tree.2004.10.003. PubMed DOI

Renaut S., Nolte A.W., Bernatchez I. Gene expression divergence and hybrid misexpression between lake whitefish species pairs (Coregonus spp. Salmonidae) Mol. Biol. Evol. 2009;26:925–936. doi: 10.1093/molbev/msp017. PubMed DOI

Renaut S., Bernatchez L. Transcriptome-wide signature of hybrid breakdown associated with intrinsic reproductive isolation in lake whitefish species pairs (Coregonus spp. Salmonidae) Heredity. 2011;106:1003–1011. doi: 10.1038/hdy.2010.149. PubMed DOI PMC

Stelkens R.B., Schmid C., Seehausen O. Hybrid breakdown in cichlid fish. PLoS ONE. 2015;10:e0127207. doi: 10.1371/journal.pone.0127207. PubMed DOI PMC

Tichopád T., Vetešník L., Šimková A., Rodina M., Franěk R., Pšenička M. Spermatozoa morphology and reproductive potential in F1 hybrids of common carp (Cyprinus carpio) and gibel carp (Carassius gibelio) Aquaculture. 2020;521:735092. doi: 10.1016/j.aquaculture.2020.735092. DOI

Stolbunova V.V., Pavlova V.V., Kodukhova Y.V. Asymmetric hybridization of roach Rutilus rutilus and common bream Abramis brama in controlled reverse crosses: Genetic and morphological patterns. Biosyst. Divers. 2020;28:376–383. doi: 10.15421/012048. DOI

Zhi T., Huang C., Sun R., Zheng Y., Chen J., Xu X., Brown C.L., Yang T. Mucosal immune response of Nile tilapia Oreochromis niloticus during Gyrodactylus cichlidarum infection. Fish Shellfish Immunol. 2020;106:21–27. doi: 10.1016/j.fsi.2020.07.025. PubMed DOI

Lu C., Ling F., Ji J., Kang Y.-J., Wang G.-X. Expression of immune related genes in goldfish gills induced by Dactylogyrus intermedius infections. Fish Shellfish Immunol. 2013;34:372–377. doi: 10.1016/j.fsi.2012.11.004. PubMed DOI

Tu X., Qi X., Huang A., Ling F., Wang G. Cytokine gene expression profiles in goldfish (Carassius auratus) during Gyrodactylus kobayashii infection. Fish Shellfish Immunol. 2019;86:116–124. doi: 10.1016/j.fsi.2018.11.035. PubMed DOI

Zhou S., Liu Y.T., Dong J., Yang Q.H., Xu N., Yang Y.B., Gu Z.M., Ai X.H. Transcriptome analysis of goldfish (Carassius auratus) in response to Gyrodactylus kobayashii infection. Parasitol. Res. 2021;120:161–171. doi: 10.1007/s00436-020-06827-9. PubMed DOI

Zhou S., Li W.X., Zou H., Zhang J., Wu S.G., Li M., Wang G.T. Expression analysis of immune genes in goldfish (Carassius auratus) infected with the monogenean parasite Gyrodactylus kobayashii. Fish Shellfish Immunol. 2018;77:40–45. doi: 10.1016/j.fsi.2018.03.033. PubMed DOI

Zhi T., Xu X., Chen J., Zheng Y., Zhang S., Peng J., Brown C.L., Yang T. Expression of immune related genes of Nile tilapia Oreochromis niloticus after Gyrodactylus cichlidarum and Cichlidogyrus sclerosus infections demonstrating immunesuppression in coinfection. Fish Shellfish Immunol. 2018;80:397–404. doi: 10.1016/j.fsi.2018.05.060. PubMed DOI

Gela D., Kocour M., Rodina M., Flajšhans M., Beránková P., Linhart O. Controlled Reproduction Technology of Common Carp (Cyprinus carpio L.) Volume 99 University of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Waters; Vodňany, Czech Republic: 2009. Methodology Editon.

Linhart O., Rodina M., Bastl J., Cosson J. Urinary bladder, ionic composition of seminal fluid and urine with characterization of sperm motility in tench (Tinca tinca L.) J. Appl. Ichthyol. 2003;19:177–181. doi: 10.1046/j.1439-0426.2003.00470.x. DOI

Pečínková M., Matějusová I., Koubková B., Gelnar M. Investigation of Paradiplozoon homoion (Monogenea, Diplozoidae) life cycle under experimental conditions. Parasitol. Int. 2007;56:179–183. doi: 10.1016/j.parint.2007.01.010. PubMed DOI

Andrews S. FastQC: A Quality Control Tool for High Throughput Sequence Data. 2010. [(accessed on 1 May 2023)]. Available online: http://www.bioinformatics.babraham.ac.uk/projects/fastqc/

Chu J., Sadeghi S., Raymond A., Jackman S.D., Nip K.M., Mar R., Mohamadi H., Butterfield Y.S., Robertson A.G., Birol I. BioBloom tools: Fast, accurate and memory-efficient host species sequence screening using bloom filters. Bioinformatics. 2014;30:3402–3404. doi: 10.1093/bioinformatics/btu558. PubMed DOI PMC

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

Dobin A., Davis C.A., Schlesinger F., Drenkow J., Zaleski C., Jha S., Batut P., Chaisson M., Gingeras T.R. STAR: Ultrafast universal RNA-seq aligner. Bioinformatics. 2013;29:15–21. doi: 10.1093/bioinformatics/bts635. PubMed DOI PMC

Broad Institute Picard Toolkit. 2019. [(accessed on 15 June 2023)]. Available online: https://broadinstitute.github.io/picard/

Li H., Handsaker B., Wysoker A., Fennell T., Ruan J., Homer N., Marth G., Abecasis G., Durbin R. and 1000 Genome Project Data Processing Subgroup. The sequence alignment/map format and SAMtools. Bioinformatics. 2009;25:2078–2079. doi: 10.1093/bioinformatics/btp352. PubMed DOI PMC

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

Ewels P., Magnusson M., Lundin S., Käller M. MultiQC: Summarize analysis results for multiple tools and samples in a single report. Bioinformatics. 2016;32:3047–3048. doi: 10.1093/bioinformatics/btw354. PubMed DOI PMC

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

Camacho C., Coulouris G., Avagyan V., Ma N., Papadopoulos J., Bealer K., Madden T.L. BLAST+: Architecture and applications. BMC Bioinform. 2009;10:421. doi: 10.1186/1471-2105-10-421. PubMed DOI PMC

Song L., Florea L. Rcorrector: Efficient and accurate error correction for Illumina RNA-seq reads. GigaScience. 2015;4:48. doi: 10.1186/s13742-015-0089-y. PubMed DOI PMC

Haas B.J., Papanicolaou A., Yassour M., Grabherr M., Blood P.D., Bowden J., Couger M.B., Eccles D., Li B., Lieber M., 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. doi: 10.1038/nprot.2013.084. PubMed DOI PMC

Bushmanova E., Antipov D., Lapidus A., Prjibelski A.D. rnaSPAdes: A de novo transcriptome assembler and its application to RNA-Seq data. GigaScience. 2019;8:giz100. doi: 10.1093/gigascience/giz100. PubMed DOI PMC

Li D., Liu C.M., Luo R., Sadakane K., Lam T.W. MEGAHIT: An ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph. Bioinformatics. 2015;31:1674–1676. doi: 10.1093/bioinformatics/btv033. PubMed DOI

Gilbert D. EvidentialGene: tr2aacds, mRNA Transcript Assembly Software. 2013. [(accessed on 1 May 2023)]. Available online: https://arthropods.eugenes.org/EvidentialGene.

Smith-Unna R., Boursnell C., Patro R., Hibberd J.M., Kelly S. TransRate: Reference-free quality assessment of de novo transcriptome assemblies. Genome Res. 2016;26:1134–1144. doi: 10.1101/gr.196469.115. PubMed DOI PMC

Bushmanova E., Antipov D., Lapidus A., Suvorov V., Prjibelski A.D. rnaQUAST: A quality assessment tool for de novo transcriptome assemblies. Bioinformatics. 2016;32:2210–2212. doi: 10.1093/bioinformatics/btw218. PubMed DOI

Simão F.A., Waterhouse R.M., Ioannidis P., Kriventseva E.V., Zdobnov E.M. BUSCO: Assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics. 2015;31:3210–3212. doi: 10.1093/bioinformatics/btv351. PubMed DOI

Haas B.J. TransDecoder. 2015. [(accessed on 1 May 2023)]. Available online: https://github.com/TransDecoder/TransDecoder.

Bryant D.M., Johnson K., DiTommaso T., Tickle T., Couger M.B., Payzin-Dogru D., Lee T.J., Leigh N.D., Kuo T.H., Davis F.G., et al. A tissue-mapped axolotl de novo transcriptome enables identification of limb regeneration factors. Cell Rep. 2017;18:762–776. doi: 10.1016/j.celrep.2016.12.063. PubMed DOI PMC

UniProt Consortium UniProt: A hub for protein information. Nucleic Acids Res. 2015;43:D204–D212. doi: 10.1093/nar/gku989. PubMed DOI PMC

Rawlings N.D., Waller M., Barrett A.J., Bateman A. MEROPS: The database of proteolytic enzymes, their substrates and inhibitors. Nucleic Acids Res. 2014;42:D503–D509. doi: 10.1093/nar/gkt953. PubMed DOI PMC

O’Leary N.A., Wright M.W., Brister J.R., Ciufo S., Haddad D., McVeigh R., Rajput B., Robbertse B., Smith-White B., Ako-Adjei D., et al. Reference sequence (RefSeq) database at NCBI: Current status, taxonomic expansion, and functional annotation. Nucleic Acids Res. 2016;44:D733–D745. doi: 10.1093/nar/gkv1189. PubMed DOI PMC

Nucleotide [Internet] National Library of Medicine (US), National Center for Biotechnology Information; Bethesda, MD, USA: 1988. [(accessed on 15 June 2023)]. Available online: https://www.ncbi.nlm.nih.gov/nuccore.

Finn R.D., Bateman A., Clements J., Coggill P., Eberhardt R.Y., Eddy S.R., Heger A., Hetherington K., Holm L., Mistry J., et al. Pfam: The protein families database. Nucleic Acids Res. 2014;42:D222–D230. doi: 10.1093/nar/gkt1223. PubMed DOI PMC

Eddy S.R. Accelerated profile HMM searches. PLoS Comp. Biol. 2011;7:e1002195. doi: 10.1371/journal.pcbi.1002195. PubMed DOI PMC

Nielsen H. Protein Function Prediction: Methods and Protocols. Humana; New York, NY, USA: 2017. Predicting Secretory Proteins with SignalP; pp. 59–73. PubMed

Patro R., Duggal G., Love M.I., Irizarry R.A., Kingsford C. Salmon provides fast and bias-aware quantification of transcript expression. Nat. Methods. 2017;14:417–419. doi: 10.1038/nmeth.4197. PubMed DOI PMC

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

Kanehisa M., Furumichi M., Tanabe M., Sato Y., Morishima K. KEGG: New perspectives on genomes, pathways, diseases and drugs. Nucleic Acids Res. 2017;45:D353–D361. doi: 10.1093/nar/gkw1092. PubMed DOI PMC

Carbon S., Ireland A., Mungall C.J., Shu S., Marshall B., Lewis S. AmiGO Hub and Web Presence Working Group. AmiGO: Online access to ontology and annotation data. Bioinformatics. 2009;25:288–289. doi: 10.1093/bioinformatics/btn615. PubMed DOI PMC

Moriya Y., Itoh M., Okuda S., Yoshizawa A.C., Kanehisa M. KAAS: An automatic genome annotation and pathway reconstruction server. Nucleic Acids Res. 2007;35:W182–W185. doi: 10.1093/nar/gkm321. PubMed DOI PMC

Suzuki S., Kakuta M., Ishida T., Akiyama Y. Faster sequence homology searches by clustering subsequences. Bioinformatics. 2015;31:1183–1190. doi: 10.1093/bioinformatics/btu780. PubMed DOI PMC

Wu T., Hu E., Xu S., Chen M., Guo P., Dai Z., Feng T., Zhou L., Tang W., Zhan L., et al. Cluster Profiler 4.0: A universal enrichment tool for interpreting omics data. Innovation. 2021;2:100141. PubMed PMC

Dowle M., Srinivasan A. data.table: Extension of ‘data.frame’. 2023. [(accessed on 15 June 2023)]. Available online: https://Rdatatable.gitlab.io/data.table.

Kolde R., Kolde M.R. Package ‘pheatmap’. 2023. [(accessed on 15 June 2023)]. Available online: https://cran.r-project.org/web/packages/pheatmap/index.html.

Wickham H. ggplot2. Wiley Interdiscip. Rev. Comput. Stat. 2011;3:180–185. doi: 10.1002/wics.147. DOI

Pfaffl M.W. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001;29:2002–2007. doi: 10.1093/nar/29.9.e45. PubMed DOI PMC

Vandesompele J., De Preter K., Pattyn F., Poppe B., Van Roy N., De Paepe A., Speleman F. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002;3:research0034.1. doi: 10.1186/gb-2002-3-7-research0034. PubMed DOI PMC

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

Muduli C., Rathore G., Singh A., Srivastava R. Identification of reference genes for quantitative expression analysis in Indian catfish, Clarias magur, under physiological and pathological conditions. Aquac. Res. 2022;53:2785–2795. doi: 10.1111/are.15793. DOI

Potrok A. Master’s Thesis. Faculty of Science, Masaryk University; Brno, Czech Republic: 2020. [(accessed on 1 March 2023)]. The Effect of Single-Species Infection by Dactylogyrus Parasites on Expression of Selected Immune Genes in Goldfish (Carassius auratus gibelio) Available online: https://is.muni.cz/th/gjmad/?studium=246504;lang=cs.

Zheng W.J., Sun L. Evaluation of housekeeping genes as references for quantitative real time RT-PCR analysis of gene expression in Japanese flounder (Paralichthys olivaceus) Fish Shellfish Imunol. 2011;30:638–645. doi: 10.1016/j.fsi.2010.12.014. PubMed DOI

Mo F., Zhao J., Liu N., Cao L.H., Jiang S.X. Validation of reference genes for RT-qPCR analysis of CYP4T expression in crucian carp. Genet. Mol. Biol. 2014;37:500–507. doi: 10.1590/S1415-47572014000400005. PubMed DOI PMC

Jirawatnotai S., Hu Y., Livingston D.M., Sicinski P. Proteomic identification of a direct role for cyclin d1 in DNA damage repair. Cancer Res. 2012;72:4289–4293. doi: 10.1158/0008-5472.CAN-11-3549. PubMed DOI PMC

Jiang W.D., Hu K., Liu Y., Jiang J., Wu P., Zhao J., Zhang Y.A., Zhou X.Q., Feng L. Dietary myo-inositol modulates immunity through antioxidant activity and the Nrf2 and E2F4/cyclin signalling factors in the head kidney and spleen following infection of juvenile fish with Aeromonas hydrophila. Fish Shellfish Immunol. 2016;49:374–386. doi: 10.1016/j.fsi.2015.12.017. PubMed DOI

Dodd M.E., Hatzold J., Mathias J.R., Walters K.B., Bennin D.A., Rhodes J., Kanki J.P., Look A.T., Hammerschmidt M., Huttenlocher A. The ENTH domain protein Clint1 is required for epidermal homeostasis in zebrafish. Development. 2009;136:2591–2600. doi: 10.1242/dev.038448. PubMed DOI PMC

He G.H., Helbing C.C., Wagner M.J., Sensen C.W., Riabowol K. Phylogenetic analysis of the ING family of PHD finger proteins. Mol. Biol. Evol. 2005;22:104–116. doi: 10.1093/molbev/msh256. PubMed DOI

Dantas A., Al Shueili B., Yang Y., Nabbi A., Fink D., Riabowol K. Biological functions of the ING proteins. Cancers. 2019;11:1817. doi: 10.3390/cancers11111817. PubMed DOI PMC

Roesner A., Hankeln T., Burmester T. Hypoxia induces a complex response of globin expression in zebrafish (Danio rerio) J. Exp. Biol. 2006;209:2129–2137. doi: 10.1242/jeb.02243. PubMed DOI

Peixoto D., Machado M., Azeredo R., Costas B. Chronic Inflammation Modulates Opioid Receptor Gene Expression and Triggers Respiratory Burst in a Teleost Model. Biology. 2022;11:764. doi: 10.3390/biology11050764. PubMed DOI PMC

Heo M.J., Kim A., Park C.I. Data on molecular characterization and gene expression analysis of secretory carrier-associated membrane protein 5 (SCAMP5) from the red sea bream (Pagrus major) Data Brief. 2019;25:103901. doi: 10.1016/j.dib.2019.103901. PubMed DOI PMC

Azimzadeh K., Mohammadisefat P. Alterations of Cystatin c, Gelsolin, Hepcidin and Sphingosine 1 phosphate in rainbow trout (Oncorhynchus mykiss) with naturally infected Ichthyophthirius multifiliis in Urmia: Determination of Possible Potential Diagnostic Biomarker. Turk. J. Fish. Aquat. Sci. 2022;22:TRJFAS19913. doi: 10.4194/TRJFAS19913. DOI

Yin Z., Nie H., Jiang K., Yan X. Molecular mechanisms underlying Vibrio tolerance in Ruditapes philippinarum revealed by comparative transcriptome profiling. Front. Immunol. 2022;13:879337. doi: 10.3389/fimmu.2022.879337. PubMed DOI PMC

Fritz R.S., Nichols-Orians C.M., Brunsfeld S.J. Interspecific hybridization of plants and resistance to herbivores: Hypotheses, genetics, and variable responses in a diverse herbivore community. Oecologia. 1994;97:106–117. doi: 10.1007/BF00317914. PubMed DOI

Artamonovaa V.S., Makhrova A.A., Shulmanb B.S., Khaiminac O.V., Yurtsevad A.O., Lajusd D.L., Shirokove V.A., Shurov I.L. Response of the Atlantic Salmon (Salmo salar L.) Population of the Keret River to the Invasion of Parasite Gyrodactylus salaris Malmberg. Russ. J. Biol. Invasions. 2011;2:73–80. doi: 10.1134/S2075111711020020. DOI

Zhigileva O.N., Uslamina I.M. Helminths’ Infestation of Various Mitochondrial Lines of the Sable Martes zibellina and the Pine Marten Martes martes. Russ. J. Genet. Appl. Res. 2017;7:648–653. doi: 10.1134/S2079059717060156. DOI

Toh S.Q., Glanfield A., Gobert G.N., Jones M.K. Heme and blood-feeding parasites. Friends or foes? Parasites Vectors. 2010;3:108. PubMed PMC

Martínez-Sernández V., Mezo M., González-Warleta M., Perteguer M.J., Muino L., Guitián E., Gárate T., Ubeira F.M. The MF6p/FhHDM-1 Major Antigen Secreted by the Trematode Parasite Fasciola hepatica Is a Heme-binding Protein. J. Biol. Chem. 2014;289:1441–1456. doi: 10.1074/jbc.M113.499517. PubMed DOI PMC

Vorel J., Cwiklinski K., Roudnický P., Ilgová J., Jedličková L., Dalton J.P., Mikeš L., Gelnar M., Kašný M. Eudiplozoon nipponicum (Monogenea, Diplozoidae) and its adaptation to haematophagy as revealed by transcriptome and secretome profiling. BMC Genom. 2021;22:274 PubMed PMC

Morgan W.T., Smith A. Binding and transport of iron-porphyrins by hemopexin. Adv. Inorg. Chem. 2001;51:205–241.

Vincent S.H., Grady R.W., Shaklai N., Snider J.M., Mullereberhard U. The influence of heme-binding proteins in heme-catalyzed oxidations. Arch. Biochem. Biophys. 1988;265:539–550. doi: 10.1016/0003-9861(88)90159-2. PubMed DOI

Pleic I.L., Buselic I., Trumbic Z., Bocina I., Sprung M., Mladineo I. Expression analysis of the Atlantic bluefin tuna (Thunnus thynnus) pro-inflammatory cytokines, IL-1 beta, TNF alpha 1 and TNF alpha 2 in response to parasites Pseudocycnus appendiculatus (Copepoda) and Didymosulcus katsuwonicola (Digenea) Fish Shellfish Immunol. 2015;45:946–954. doi: 10.1016/j.fsi.2015.06.008. PubMed DOI

Parida S., Mohapatra A., Mohanty J., Sahoo P. Labeo rohita and Argulus siamensis infection: Host size, local inflammatory reaction and immunity modulate ectoparasite load on fish. Aquac. Res. 2018;49:757–766. doi: 10.1111/are.13506. DOI

Mo Z.Q., Wu H.K., Hu Y.T., Lu Z.J., Lai X.L., Chen H.P., He Z.K., Luo X.C., Lee Y.V., Dan X.M. Transcriptomic analysis reveals innate immune mechanisms of an underlying parasite-resistant grouper hybrid (Epinephelus fuscogutatus × Epinephelus lanceolatus) Fish Shellfish Immunol. 2021;119:67–75. doi: 10.1016/j.fsi.2021.09.041. PubMed DOI

Niu J.J., Sun M.M., Li Z.Y., Wang Z.Y., Kong M., Wang Y.F., Song J.Q., Zhang Q.Q., He Y., Qi J. Whole transcriptome analysis provides new insight on immune response mechanism of golden pompano (Trachinotus ovatus) to Amyloodinium ocellatum infestation. Aquaculture. 2022;560:738396.

Lindenstrøm T., Buchmann K., Secombes C.J. Gyrodactylus derjavini infection elicits IL-1β expression in rainbow trout skin. Fish Shellfish Immunol. 2003;15:107–115. PubMed

Tu X., Liu L., Qi X., Chen W., Wang G., Ling F. Characterization of Toll-like receptor gene expression in goldfish (Carassius auratus) during Dactylogyrus intermedius infection. Dev. Comp. Immunol. 2016;63:78–83. PubMed

Reyes-Becerril M., Alamillo E., Trasviña A., Hirono I., Kondo H., Jirapongpairoj W., Ascencio-Valle F., Angulo C. In vivo and in vitro studies using larval and adult antigens from Neobenedenia melleni on immune response in yellowtail (Seriola lalandi) J. Fish Dis. 2017;40:1497–1509. PubMed

Duan C., Ma Z., Wu S., Ding X.J., Tu X., Ye J. Functional characterization of complement factor D on the defence against Gyrodactylus kobayashii (Monogenea) infection in goldfish (Carassius auratus) Aquaculture. 2021;545:737214.

Roudnický P., Vorel J., Ilgová J., Benovics M., Norek A., Jedličková L., Mikeš L., Potešil D., Zdráhal Z., Dvořák J., et al. Identification and partial characterization of a novel serpin from Eudiplozoon nipponicum (Monogenea, Polyopisthocotylea) Parasite. 2018;25:61. PubMed PMC

Jedličková L., Dvořak J., Hrachovinová I., Ulrychová L., Kašný M., Mikeš L. A novel Kunitz protein with proposed dual function from Eudiplozoon nipponicum (Monogenea) impairs haemostasis and action of complement in vitro. Int. J. Parasitol. 2019;49:337–346. PubMed

Tan E., Low K.W., Wong W.S.F., Leung K.Y. Internalization of Aeromonas hydrophila by fish epithelial cells can be inhibited with a tyrosine kinase inhibitor. Microbiology. 1998;144:299–307. PubMed

Li Z.X., Li Y.W., Xu S., Xu Y., Mo Z.Q., Dan X.M., Luo X.C. Grouper (Epinephelus coioides) TCR signaling pathway was involved in response against Cryptocaryon irritans infection. Fish Shellfish Immunol. 2017;64:176–184. PubMed

Howe K., Matthew D.C., Torroja C.F., Torrance J., Berthelot C., Stemple D.L. The zebrafish reference genome sequence and its relationship to the human genome. Nature. 2013;496:498–503. PubMed PMC

Shin J.T., Fishman M.C. From zebrafish to human: Modular medical models. Annu. Rev. Genom. Hum. Genet. 2002;3:311–340. PubMed

Giardoglou P., Beis D. On Zebrafish Disease Models and Matters of the Heart. Biomedicines. 2019;7:15. PubMed PMC

Das B.K., Chakraborty H., Rout A.K., Behera B.K. De novo whole transcriptome profiling of Edwardsiella tarda isolated from infected fish (Labeo catla) Gene. 2019;701:152–160. PubMed

Hung K.S., Chen S.Y., Hsu P.H., Lin B.A., Hu C.H., Yang C.H., Pai T.W., Tzou W.S., Chung H.Y. Comparative transcriptome analysis of organ-specific adaptive responses to hypoxia provides insights to human diseases. Genes. 2022;13:1096. PubMed PMC

Edgar R., Domrachev M., Lash A.E. Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res. 2002;30:207–210. PubMed PMC

Host-specific monogeneans parasitizing freshwater fish: The ecology and evolution of host-parasite associations