bioRxiv preprint doi: https://doi.org/10.1101/2020.09.28.312801; this version posted September 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Comparative transcriptomics and host-specific parasite gene expression profiles 2 inform on drivers of proliferative kidney disease 3 4 Marc Faber1, Sohye Yoon1,2, Sophie Shaw3, Eduardo de Paiva Alves3,4, Bei Wang1,5, Zhitao 5 Qi1,6, Beth Okamura7, Hanna Hartikainen8, Christopher J. Secombes1, Jason W. Holland1 * 6 7 1 Scottish Fish Immunology Research Centre, University of Aberdeen, Aberdeen AB24 2TZ, UK. 8 2 Present address: Genome Innovation Hub, Institute for Molecular Bioscience, The University of 9 Queensland, Brisbane, QLD 4072, Australia. 10 3 Centre for Genome Enabled Biology and Medicine, University of Aberdeen, Aberdeen AB24 2TZ, 11 UK. 12 4 Aigenpulse.com, 115J Olympic Avenue, Milton Park, Abingdon, Oxfordshire, OX14 4SA, UK. 13 5 Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic 14 Animal, Key Laboratory of Control for Disease of Aquatic Animals of Guangdong Higher Education 15 Institutes, College of Fishery, Guangdong Ocean University, Zhanjiang, P.R. China. 16 6 Key Laboratory of Biochemistry and Biotechnology of Marine Wetland of Jiangsu Province, Yancheng 17 Institute of Technology, Jiangsu, Yancheng, 224051, China. 18 7 Department of Life Sciences, The Natural History Museum, London SW7 5BD, UK. 19 8 School of Life Sciences, University of Nottingham, Nottingham, NG7 2RD, UK. 20 21 22 23 *Author for correspondence. 24 Tel: +44 1224 438047 25 Email: [email protected] 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.28.312801; this version posted September 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 43 Abstract 44 45 The myxozoan parasite, Tetracapsuloides bryosalmonae has a two-host life cycle alternating 46 between freshwater bryozoans and salmonid fish. Infected fish can develop Proliferative 47 Kidney Disease (PKD), characterised by a gross lymphoid-driven kidney pathology in wild 48 and farmed salmonids. To facilitate an in-depth understanding of T. bryosalmonae-host 49 interactions, we have adopted a two-host parasite transcriptome sequencing approach to 50 minimize host contamination in the absence of a complete T. bryosalmonae genome. 51 Parasite contigs common to both infected hosts (the intersect transcriptome; 7,362 contigs) 52 were typically AT-rich (60-75% AT). 5,432 contigs within the intersect were annotated with 53 1,930 unannotated contigs encoding for unknown transcripts. We have focused on 54 transcripts encoding proteins involved in; nutrient acquisition, host-parasite interactions, 55 development, and cell-to-cell communication or proteins of unknown function, establishing 56 their potential importance in each host by RT-qPCR. Host-specific expression profiles were 57 evident, particularly in transcripts encoding proteases and proteins involved in lipid 58 metabolism, cell adhesion, and development. We confirm for the first time the presence of 59 homeobox proteins and a frizzled homologue in myxozoan parasites. 60 The novel insights into myxozoan biology that this study reveals will help to focus research in 61 developing future disease control strategies. 62 63 64 65 66 Keywords: Tetracapsuloides bryosalmonae, myxozoan, freshwater bryozoan, rainbow trout, 67 metabolism, development, virulence, gene expression 68 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.28.312801; this version posted September 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 69 Introduction 70 71 Proliferative Kidney Disease (PKD) is an economically and ecologically important disease 72 that impacts salmonid aquaculture and wild fish populations in Europe and North America1. 73 The geographic range of PKD is broad and recent disease outbreaks in a wide range of 74 salmonid hosts underlines the status of PKD as an emerging disease. The occurrence and 75 severity of PKD is temperature driven, with projections of warmer climates predicted to align 76 with escalation of disease outbreaks in the future2,3. 77 PKD is caused by the myxozoan parasite, Tetracapsuloides bryosalmonae. T. bryosalmonae 78 spores are released from the definitive bryozoan host, Fredericella sultana4,5 and, following 79 attachment, invade fish hosts via skin epidermal mucous cells (in gills and elsewhere) and 80 migrate through the vascular system to the kidney and other organs, including spleen and 81 liver6. Extrasporogonic proliferation of T. bryosalmonae in kidney tissues elicits a chronic 82 tissue pathology, characterised by lymphoid hyperplasia, granulomatous lesions, renal 83 atrophy, anaemia7,8 and hyper secretion of immunoglobulins9,10. The severity and 84 development of these hallmark symptoms of PKD are modified by a variety of biological, 85 environmental and chemical stressors, impacting on parasite load, host immunity, and 86 disease recovery10,11. A genetic basis for such pathological and developmental variation is 87 evidenced by introduced, non-co-evolved salmonid hosts in Europe, such as rainbow trout, 88 acting as dead-end hosts. In contrast to native salmonids, the European strains of T. 89 bryosalmonae are unable to produce viable sporogonic renal stages, which are infective to 90 the bryozoan host5. Despite these recent advances in understanding the host responses to 91 PKD, the molecular bases of the host-parasite interactions that drive PKD development are 92 currently poorly known and there are no therapeutic measures for disease control. 93 An increasing number of genomic, transcriptomic and targeted gene studies now show that 94 myxozoans, belong to the Phylum Cnidaria12–14. As adaptations to parasitism, myxozoans 95 exhibit extreme morphological simplification and drastically reduced genome sizes relative to 96 free-living cnidarians15,16. Nevertheless, polar capsules homologous to the stinging 97 nematocysts of cnidarians have been retained and are used for host attachment. Whilst 98 myxozoans exhibit an apparent streamlining of metabolic and developmental processes 99 compared to free-living cnidarians, not surprisingly, they have retained large numbers of 100 proteases18. Likewise, low density lipoprotein receptor class A domain-containing proteins 101 (LDLR-As) are also numerous. This along with the apparent dominance of myxozoan lipases 102 in infected fish, revealed in this and previous studies, suggests that host lipids are an 103 important source of nutrients for myxozoans18,19. LDLR-As may, thus, be particularly 104 important in virulence processes in myxozoan parasites, but to date have not been 105 characterised in T. bryosalmonae. 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.28.312801; this version posted September 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 106 T. bryosalmonae belongs to the relative species-poor, early diverging myxozoan clade 107 Malacosporea, which has retained primitive features, such as epithelial layers and, in some 108 cases, musculature20. Malacosporeans alternate between fish and freshwater bryozoan 109 hosts17. There are clear host-specific developmental differences, including meiosis in 110 bryozoan hosts and morphologies of spores released from fish and bryozoan hosts21. Host 111 specific differences in gene expression may provide avenues for the development of targeted 112 future therapeutics and are an important prerequisite in understanding the parasite’s biology. 113 However, biological characterization of myxozoans via transcriptome and genome data has 114 for various reasons been hampered. Provision of sufficient and appropriate material may be 115 problematic. For example, sporogonic stages (henceforth referred to as spore sacs) of T. 116 bryosalmonae can be released from the body cavity of bryozoan hosts by dissection and can 117 occasionally be collected in substantial quantity (e.g. hundreds of spore sacs) from infected 118 bryozoans maintained in laboratory mesocosms22 or from field-collected material3. However, 119 attempts to purify parasite stages from infected fish kidney tissues have been unsuccessful. 120 Although dual RNA-Seq approaches and selective enrichment of parasite stages from host 121 tissues can be attempted, most myxozoan genomes and transcriptomes still carry host 122 contamination23. In situ expression experiments are hindered by low parasite to host tissue 123 ratios in infected tissues and the subsequent very low coverage of parasite transcripts. 124 In the absence of a complete host-free T. bryosalmonae genome, we used transcriptomes 125 from the fish and bryozoan hosts to develop an intersect transcriptome for comparative 126 transcriptomics. After maximising parasite