Ceratonova shasta is an important myxozoan pathogen affecting the health of salmonid fishes in the Pacific Northwest of North America. C. shasta exists as a complex of host-specific genotypes, some with low to moderate virulence, and one that causes a profound, lethal infection in susceptible hosts. High throughput sequencing methods are powerful tools for discovering the genetic basis of these host/virulence differences, but deep sequencing of myxozoans has been challenging due to extremely fast molecular evolution of this group, yielding strongly divergent sequences that are difficult to identify, and unavoidable host contamination. We designed and optimized different bioinformatic pipelines to address these challenges. We obtained a unique set of comprehensive, host-free myxozoan RNA-seq data from C. shasta genotypes of varying virulence from different salmonid hosts. Analyses of transcriptome-wide genetic distances and maximum likelihood multigene phylogenies elucidated the evolutionary relationship between lineages and demonstrated the limited resolution of the established Internal Transcribed Spacer marker for C. shasta genotype identification, as this marker fails to differentiate between biologically distinct genotype II lineages from coho salmon and rainbow trout. We further analyzed the datasets based on polymorphisms in two gene groups related to virulence: cell migration and proteolytic enzymes including their inhibitors. The developed SNP-calling pipeline identified polymorphisms between genotypes and demonstrated that variations in both motility and protease genes were associated with different levels of virulence of C. shasta in its salmonid hosts. The prospective use of proteolytic enzymes as promising candidates for targeted interventions against myxozoans in aquaculture is discussed. We developed host-free transcriptomes of a myxozoan model organism from strains that exhibited different degrees of virulence, as a unique source of data that will foster functional gene analyses and serve as a base for the development of potential therapeutics for efficient control of these parasites.

Host filtering at read level – Ceratonova shasta and neither reads lists

Lists of Ceratonova shasta mapping reads and reads that did not map neither to host (Oncorhynchus mykiss) nor to parasite (C. shasta). Reads belong to three different genotypes (I, IIC, IIR) and to five different libraries (I, IIC, IIR-RBTJ7, IIR-RBTC16, IIR-RBT6).

Lists of reads, 20 .list files.

I_csR1.list

I_csR2.list

I_neitherR1.list

I_neitherR2.list

IIC_csR1.list

IIC_csR2.list

IIC_neitherR1.list

IIC_neitherR2.list

IIR_RBTJ7_csR1.list

IIR_RBTJ7_csR2.list

IIR_RBTJ7_neitherR1.list

IIR_RBTJ7_neitherR2.list

IIR_RBTC16_csR1.list

IIR_RBTC16_csR2.list

IIR_RBTC16_neitherR1.list

IIR_RBTC16_neitherR2.list

IIR_RBT6_csR1.list

IIR_RBT6_csR2.list

IIR_RBT6_neitherR1.list

IIR_RBT6_neitherR2.list

Genotype IIR (RBT6) assembled transcriptomes

Two assembled transcriptomes using IIR RBT6 C. shasta only reads or C. shasta only + neither reads.

2 .fasta files

IIR_RBT6_cs_only.fasta

IIR_RBT6_cs_neither.fasta

Host and other contaminants (other microorganisms) IIR RBT6 filtered assemblies

Filtered assembled transcriptomes for host (Oncorhynchus mykiss) and other microorganisms (virus, fungi, bacteria) using Taxon ID annotations.

2 .fasta files

IIR_RBT6_cs_only_extra_clean.fasta

IIR_RBT6_cs_neither_extra_clean.fasta

Longest representatives reference IIR RBT6 cs+neither assembly used for SNPs analyses

1 . fasta file

IIR_RBT6_cs_neither_extra_clean_woreps.fasta

SNPs tables

Genotypes called from nucleotide frequencies with a minimum coverage of 5 and 0.25 heterozygosity threshold.

5 .tab files

I_genotypes.tab

IIC_genotypes.tab

IIR_RBTJ7_genotypes.tab

IIR_RBTC16_genotypes.tab

IIR_RBT6_genotypes.tab

Genotypes called from nucleotide frequencies with a minimum coverage of 20 reads and 0.1 heterozygosity threshold.

5 .tab files

I_genotypes2000101.tab

IIC_genotypes2000101.tab

IIR_RBTJ7_genotypes2000101.tab

IIR_RBTC16_genotypes2000101.tab

IIR_RBT6_genotypes2000101.tab

Phylogenomic and SNPs-based phylogenetic alignments

9 .nex and 51 .fasta files

cmkb_permissive.nex

cmkb_permissive_filtered.nex

combined.nex

combined_cmkb_curated.nex

combined_merops_curated.nex

combined_only_available_all.nex

merops_permissive.nex

merops_permissive_filtered.nex

phylogenomic_tree.nex

51 individual genes alignments for phylogenomics in .fasta