Trends in

Parasitology
Review

Advances and Discoveries in

Myxozoan Genomics
Gema Alama-Bermejo 1 and Astrid S. Holzer 1,*

Myxozoans are highly diverse and globally distributed cnidarian endoparasites Highlights
in freshwater and marine habitats. They have adopted a heteroxenous life The first myxozoan genome assembly
cycle, including invertebrate and fish hosts, and have been associated with was published in 2015, and myxozoan
diseases in aquaculture and wild fish stocks. Despite their importance, genomic genomics has since become a rapidly
progressing field, with limitations due to
resources of myxozoans have proven difficult to obtain due to their miniaturized DNA contamination from largely poly-
and derived genome character and close associations with fish tissues. The first ploid fish hosts.
‘omic’ datasets have now become the main resource for a better understanding
Myxozoan genomes are amongst the
of host–parasite interactions, virulence, and diversity, but also the evolutionary his- smallest metazoan genomes on Earth
tory of myxozoans. In this review, we discuss recent genomic advances in the field with important gene reductions and
and outline outstanding questions to be answered with continuous and improved loss of basic biological functions such
efforts of generating myxozoan genomic data. as DNA methylation and, in one case,
the mitochondrial genome.

The Myxozoa: Diverse Cnidarian Endoparasites Impacting on Fish Health The evolutionary history of myxozoans
Myxozoa is a highly diverse group of microscopic cnidarian parasites (2425 species [1]) appears to confirm a sister relationship
of the Myxozoa and Polypodium
whose representatives are strongly reduced in size and morphology when compared to their
hydriforme but their old age and derived
free-living relatives. Myxozoans are globally distributed in both marine and freshwater habitats, genome characteristics obscure evolu-
and they alternate between invertebrate hosts (annelid and bryozoan) and vertebrate hosts tionary analyses.
(predominantly fish) (Figure 1, Key Figure). Pluricellular spores are used as transmission stages
The first metagenomic study using
and contain characteristic organelles, called polar capsules. These are simplified cnidarian aquatic eDNA highlights the importance
nematocysts [2] which are responsible for host attachment. In both hosts, parasite migration and of using genomic approaches for
proliferation precedes spore formation and release. Development takes place in characteristic uncovering the strongly underestimated
cell-in-cell stages, which are found only in myxozoans, Polypodium hydriforme [3] (a myxozoan myxozoan biodiversity.

sister taxon) and in the Paramyxida [4] (an unrelated eukaryotic taxon). Most developmental stages, Comparative transcriptomics highlighted
including spores, are composed of only a few cells [5], but some spore-forming stages grow protease-encoding genes as candidate
macroscopic plasmodia releasing millions of transmission stages. gene groups for therapeutics and vac-
cine design.

Myxozoans are particularly well known for the diseases they cause in wild and aquaculture fish
stocks [6]. Recent studies on Tetracapsuloides bryosalmonae, the causative agent of proliferative
kidney disease in salmonids, have demonstrated the effects climate change can have on
myxozoan pathogens, with warmer temperatures predicted to align with escalation of disease
outbreaks in the near future [7,8].

Due to the massive growth of the aquaculture sector in the past two decades, research into fish
pathogens has intensified. Diseases represent the most significant obstacles for aquaculture
improvement and are responsible for 20% of mortalities in the sector [9]. Research into 1
Institute of Parasitology, Biology Centre
myxozoans is of particular importance due to the present lack of licensed treatments or vaccines of the Czech Academy of Sciences,
Ceske Budejovice, Czech Republic
against these parasites. A better understanding of the specific molecules used for host–parasite
interaction and immune evasion is essential for the design of antimyxozoan strategies.

Independent from commercial interests, this group of parasites is extremely attractive from an *Correspondence:
evolutionary perspective, considering that myxozoans are some of the most ancient multicellular astrid.holzer@paru.cas.cz (A.S. Holzer).

552 Trends in Parasitology, June 2021, Vol. 37, No. 6 https://doi.org/10.1016/j.pt.2021.01.010

© 2021 Elsevier Ltd. All rights reserved.
Trends in Parasitology

Key Figure Glossary

The Myxozoan Life Cycle and Developmental Stages in Vertebrate (Fish) Benchmarking universal
single-copy orthologs (BUSCO):
and Invertebrate (Bryozoan or Annelid) Hosts, Indicating Genomic and represents a tool to assess
Transcriptomic Datasets Published to Date completeness of genome assembly,
gene set, and transcriptome. It is based
on the concept of single-copy orthologs
that should be highly conserved among
the closely related species. It uses
collections of orthologous groups with
near-universally-distributed single-copy
genes in each species, selected from
root-level orthology delineations across
arthropods (2675 genes), vertebrates
(3023 genes), metazoans (843 genes),
fungi (1438 genes), and eukaryotes
(429 genes). The expectations for these
genes to be found in a genome, and to
be found only in a single copy, are
evolutionarily sound.
Core eukaryotic genes (CEGs):
defines a set of 458 core proteins that
are present in a wide range of eukaryotic
taxa. Since these proteins are highly
conserved, sequence alignment
methods can reliably identify their
exon–intron structures in genomic
sequences. This is performed by using a
computational method called CEGMA
(Core Eukaryotic Genes Mapping
Approach [27]). The resulting dataset
can be used to train a gene finder or to
assess the completeness of the genome
or annotations. This method has now
been outdated by BUSCO
Trends in Parasitology (see preceding text).
Environmental DNA (eDNA): DNA
Figure 1. Dataset identifiers can be found in Table 1 (genomes, Roman numerals, green) and Table 2 (transcriptomes, Arabic content of environmental samples,
numerals, red). Letters refer to different datasets of the same species. Lines to different parasite stages indicate comparative such as soil, water, sediments, etc.,
studies. Transcriptomic and genomic datasets of the same study are listed together (e.g., 4 II, 9 III, etc.). 2B is unspecified which may contain whole organisms
with regard to parasite stages but the sample was taken 140 days postexposure of the fish host. (bacteria, protists, and metazoans,
including parasite transmission
stages), materials derived from
organisms (e.g., sloughed cells or
parasites on our planet. They successfully diversified massively, exploiting a large number of feces), and extracellular DNA. eDNA
hosts with a high degree of flexibility, switching even to distant groups such as molluscs, mono- can be extracted and sequenced
geneans, and trematodes (summarized in [10]). directly after library preparation or after
PCR amplification of specific gene
regions, using more or less specific
The first high-throughput sequencing (HTS) (see Glossary) dataset of myxozoans [11], and primers matching all organisms,
especially the first annotated genomes [12,13], initiated a new era of research into myxozoans. restricted groups, or those targeting
With missing in vitro culture models, myxozoan ‘omics’ has become a rapidly progressing field individual genera and species.
Functional genomics: refers to the
that has provided surprising new insights into their derived genomes and has become the primary
attempt to describe a gene function,
source for an improved understanding of myxozoan evolution, biodiversity, and host exploitative integrating genomic, transcriptomic,
mechanisms. In this review, we discuss recent genomic advances and their findings, address proteomic, and metabolomic
the current challenges and limitations of sequencing myxozoan genomes and transcriptomes, information. The aim is to understand the
relationship between genotype and
and highlight methodologies and approaches that could generate improved datasets. We further phenotype – for example, in myxozoans,
outline outstanding questions in myxozoan research that should be addressed to solve key connecting a specific gene or group of
questions regarding the biology and evolution of these basal metazoan parasites and to advance genes with increased virulence or fatal
the development of targeted antiparasitic strategies for aquaculture. outcome in a fish infection.

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High-throughput sequencing (HTS):

The Myxozoan Genome and Transcriptome Datasets and Their Characteristics
also known as next-generation
To date, genomes of varying gene coverage have been sequenced for nine myxozoans, with sequencing (NGS), HTS is a
assemblies available for six species (Table 1). RNA-seq data have been produced for 14 species, comprehensive term used to describe
but assemblies have been made available for only 8 species (Table 2) (life cycle stages indicated in technologies that sequence organismal
DNA and RNA in a rapid and cost-
Figure 1). Genome sequencing demonstrated that the transition from a free-living ancestor to a
effective manner.
typical microscopic myxozoan parasite is concomitant with an extreme reduction in genome Long-branch attraction (LBA): refers
size and gene content [12–14] (Box 1). Presently known myxozoan genomes range from 22.5 to a systematic error whereby distantly
to 188.5 Mb, which represents approximately 10% of the size of their free-living cnidarian relatives related lineages are incorrectly inferred
to be closely related. LBA arises when
[15–18]. Thus, myxozoans have some of the smallest genomes in the animal kingdom. Hence, it two or more lineages have undergone a
appears that selective pressures associated with parasitism have placed evolutionary constraints large amount of molecular change
on the genomes of myxozoans similar to those on other parasite groups, leading to a conver- (e.g., they accumulated a large number
of nucleotide or amino acid changes),
gence towards genome reduction or compaction [19,20]. Interestingly, P. hydriforme also para-
which causes them to appear similar
sitizes fish during part of its life cycle but has an estimated genome size that is 20 times larger (561 (thus closely related), though they are
Mb) than that of Kudoa iwatai, containing three times as many protein-coding genes [12]. not related by descent.
Myxozoan genomes further show a characteristically low GC content, when compared with Metabarcoding: barcoding is a term
that refers to taxonomic identification of
P. hydriforme or free-living cnidarians, which, in some organisms, has been found to be associ-
species based on single-specimen
ated with reduced genome size [21]. sequencing of diagnostic barcoding
markers (e.g., COI). Metabarcoding
In myxozoans, gene reductions seem to affect predominantly development, cell differentiation, refers to the taxonomic identification of
multiple species extracted from a mixed
and cell-to-cell communication [12]. Consistent with the lack of a complex multicellular body sample (community DNA or eDNA)
in almost all myxozoans, with the exception of malacosporeans, which can form worm-like which have been PCR-amplified and
bodies with tetraradially arranged muscle strands [22], myxozoan genomes appear to lack sequenced on a high-throughput
key genes and signaling pathways such as those related to body plans (Hox-like, Runx, platform.
Operational taxonomic unit (OTU): a
Wnt pathway, Hedgehog pathway; [12,23]). Only in T. bryosalmonae, some homeobox term used to classify groups of closely
proteins, distinct from the classical Hox cluster, and a frizzled receptor (part of the Wnt related individuals. OTUs are cluster of
pathway), were identified and found to display bryozoan-specific expression profiles [23]. similar sequence variants of a specific
marker gene sequence (often 16S or
However, the roles of these genes in myxozoan development await clarification as they can
18S rDNA). Each of these clusters is
function independently from Hox and Wnt pathways [23]. Myxozoan genomes also show a intended to represent a taxonomic unit
streamlined metabolic repertoire lacking classic and complementary anaerobic pathways (species, subspecies, or genus),
and gluconeogenesis [13]. Extreme reductions further include the loss of DNA cytosine depending on the sequence similarity
threshold given for analysis. In
methylation [24], which has been related to genome compression [25] and is often observed
myxozoans, typically, OTU clusters are
in persistently large populations, likely present in myxozoans. A recent exciting discovery defined by a 99% identity threshold of
showed that Henneguya salminicola lost its mitochondrial genome and nearly all nuclear the 18S rDNA gene sequences to
genes involved in its transcription and replication [14]. The loss of this core animal feature distinguish species at the genus level,
though this threshold may vary
shows how remarkably flexible myxozoan genomes are in their adaption to changing environ-
according to the subclade of myxozoans
mental conditions such as an anaerobic habitat. investigated. 'OTU' in myxozoan studies
is hence mostly a pragmatic proxy for
As a consequence of such rapid changes, genomes of myxozoans show a considerably accel- 'species'.

erated rate of molecular evolution, possibly the fastest known among eukaryotes [10]. This
hampers the identification of gene homologs and leads to low genome completeness scores
when assessing genome assembly and annotation completeness based on benchmarking
universal single-copy orthologs, using BUSCO [26] (and prior by search for core eukary-
otic genes (CEGs), using CEGMA [27]). Even in genomes where targeted searches identified
75/78 nuclear ribosomal protein genes, suggesting a completeness of >90%, only 37.5–73%
of CEGs were discovered [14]. This hinders comparative and functional analyses between dif-
ferent phylogenetic lineages and with other organisms, largely rendering myxozoan genomes
‘black boxes’.

Likely due to similar reasons, functional RNA-seq studies of myxozoan-infected host tissues
(Table 2) have so far provided only a small number of myxozoan transcriptomic studies

554 Trends in Parasitology, June 2021, Vol. 37, No. 6

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Table 1. Genomic Datasets of Myxozoansa

Lineage Oligochaete-infecting myxozoans
Speciesb Myxobolus cerebralis (I) Thelohanellus kitauei (II) Myxobolus squamalis (III) Henneguya salminicola (IV)
Host Tubifex tubifex Cyprinus carpio Oncorhynchus kisutch Oncorhynchus tschawytscha
Sample Released spores Intestine Scales Skeletal muscle
Parasite stage Actinospores Myxospores Myxospores Myxospores
Geographic location Laboratory China USA USA
Acc. Number Reads (SRA Study) SRP017405 SRP020474 SRP158957 SRP158827
Platform 454 GS FLX Titanium 454 GS FLX + Illumina Genome Illumina HiSeq 3000 Illumina HiSeq 3000
Analyzer II
Library strategy WGS WGS WGS WGS
Library selection – Random Random Random
Read length – 8K paired + 100 bp (PE) 150 bp (PE) 150 bp (PE)

Acc. Number Genome Assembly – GCA_000827895.1 GCA_010108815.1 GCA_009887335.1

Estimated genome size (Mb) – 188.5 53.1 60.0

Genome coverage – 8× (454) + 29×+13× 86.1× 311×
(Illumina) = 50×
Total length assembly (Mb) – 150.7 43.7 61.4
No. of scaffolds or contigs – 5610 scaffolds 37 919 contigs 18 330 scaffolds

N50 – 170Kb 1286 7570

Number of predicted proteins – 16 638 5725 8188
% GC content – 37.5 27 29
Used for mt genome mining – – Yes Yes
Completeness (CEGMA) – 46.8% c
37.5% 53.6%
Acc. Number Mitoch. genome – – – –
(RefSeq)

Refs [42] [13] [14] [14]

a
Abbreviations: SRA, Sequence Read Archive; WGS, whole-genome sequencing; WXS, whole-exome sequencing; bp, base pair; PE, paired-end reads; RG, represen-
tative genome; Mb, megabase; CEGMA, core eukaryotic genes mapping approach (no BUSCO scores published to date); RefSeq, NCBI Reference Sequence Database;
INSDC, International Nucleotide Sequence Database Collaboration. Data reported in this table were obtained from literature and NCBI databases.
b
Roman numbers next to parasite names provide a reference to Figure 1, where they are mapped to parasite life cycle stages.
c
From [14].

characterized by limited depth, while most analyses have focused on characterizing host
responses in fish [28–33] and recently, in two invertebrate hosts [34,35]. Nevertheless, the limited
data available have been the source of exciting novelties and have enabled identification of key
genes and proteins for host exploitation (see the section ‘Functional Genomics of Myxozoans:
Finding Candidates for Disease Control’). Most importantly, recent datasets include highly valu-
able comparative approaches, such as a comparison of host-specific genotypes of Ceratonova
shasta characterized by different virulence levels [36], differential gene expression in vertebrate
vs invertebrate hosts of T. bryosalmonae [23], and a dataset characterizing exclusively prolifera-
tive parasite stages [37], rather than late-stage spore-forming stages or a mixture of different
developmental stages (Figure 1).

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 Trends in Parasitology

Polychaete-infecting myxozoans
Kudoa iwatai (V) Sphaeromyxa Enteromyxum Kudoa Kudoa K. iwatai (X)
zaharoni (VI) leei (VII) septempunctata (VIII) hexapunctata (IX)
Sparus aurata Pterois miles Sparus aurata Paralichthys olivaceus Thunnus orientalis Acanthopagrus
latus
Eye Gall bladder Intestine Muscle Muscle Muscle
Myxospores Plasmodia Trophozoites Myxospores Myxospores Myxospores
Israel Israel Israel Japan Japan Japan
SRP047215/SRP047291 SRP058559 SRP058560 – – –
Illumina HiSeq 2000/ Illumina HiSeq Illumina HiSeq 2000 Illumina Genome Analyzer IIx/ Illumina Genome Illumina MiSeq
Illumina Genome 2000 Illumina MiSeq/ Analyzer IIx
Analyzer IIx PacBio RSII
WGS WXS WGS – – –
Random Random Random – – –
100 bp (PE) /76 bp (PE) 100 bp (PE) 100 bp (PE) 125 bp (PE), 81 bp (PE)/ 81 bp (PE) 300 bp (PE)
300-200 bp (PE)/ ~10kb
3 assemblies: GCA_001455285.1 2 assemblies: – – –
GCA_001407235.2 GCA_001455295.2 (RG)/
(RG)/ GCA_001455295.1
GCA_001407235.1/
GCA_001407335.1
22.5 – – – – –
1000× 500× 40× – – –
31.2/21.6 173.6 68 – – –
1639 (v2)/ 1637 (v1)/ 70 914 scaffolds 69 053/69 045 scaffolds – – –
5700 scaffolds
40195 / 6350 4474 998/996 – – –
5533 – – – – –
28 – – – – –
Yes – Yes Yes Yes Yes
73% – – – – –
LT671462 (Chr. 1), – LN868201–LN868207, NC_027523 NC_027524 NA
LT671463 (Chr. 2) LT714695 (8 chromosomes)
(INSDC) (INSDC)
[12,54] [12] [12,54] [55] [55] [55]

Major Obstacles for the Generation of HTS Datasets of Myxozoans

Apart from the issues associated with their strongly derived genomes, progress in myxozoan
gene discovery has been majorly impaired due to host contamination in almost all datasets
[38]. The reduced myxozoan genome size aggravates this problem as fish host genomes
can be 10–50× larger (usually >1 Gb), due to their mostly polyploid character [39,40]. Not
surprisingly, initial HTS datasets of myxozoans had low coverage and prominent but
unaddressed host contamination. Contamination has led to misidentification of myxozoan
genes even before the genomic era. In 1998, Hox genes allegedly belonging to myxozoans
lead to speculations about a tripoblast origin of myxozoans but were later found to be of
host origin [11,41].

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 Table 2. Transcriptomic Datasets of Myxozoans
Lineage Malacosporea Sphaerospora Oligochaete-infecting myxozoans
s.s.
Speciesa Buddenbrockia Tetracapsuloides bryosalmonae (2) Sphaerospora Thelohanellus Myxobolus Myxobolus Myxobolus
plumatellae (1) molnari (3) kitauei (4) pendula (5) cerebralis (6) honghuensis (7)
Host Plumatella sp. Fredericella Oncorhynchus Salmo trutta F. sultana F. sultana/O. Cyprinus carpio C. carpio Semotilus Tubifex Carassius
sultana (2A) mykiss (2B) (2C) (2D) mykiss (2E) atromaculatus tubifex auratus gibelio
Sample Zooid Bryozoan Posterior Posterior Zooids Zooids/kidney Blood Intestine Gills Spores Pharynx
stage kidney kidney
Parasite stage Worm Spore sacs 130 days p.i. Presporogonic, Spore sacs Spore sacs/ Mixed Myxospores Myxospores Actinospores Unknown
12 weeks p.e. extrasporogonic extrasporogonic
stages stages
Trends in Parasitology

Geographic – UK Laboratory Laboratory Laboratory UK Hungary China Canada USA China

location
Acc. Number – – Not SRP197993 SRP261017 PRJEB19471 SRP186121 SRP020474 SRP063943 SRP045736 SRP193901
reads (SRA study) deposited (ENA)
Platform – – Illumina Illumina HiSeq Illumina Illumina HiSeq Illumina HiSeq Illumina GA Illumina HiSeq Illumina Illumina HiSeq
HiSeq 2500 2500 NextSeq550 2000 2000 2500 HiSeq 2000 2500
Library strategy – – RNA-seq RNA-seq RNA-seq RNA-seq RNA-seq RNA-seq RNA-seq miRNA-seq RNA-seq
Library selection cDNA cDNA Unknown cDNA cDNA cDNA Unspecified Size PolyA Unspecified cDNA
fractionation
Read length – – Unknown 100 bp (SE) 150 bp (PE) 100 bp (PE) 90 bp (PE) 101 bp (PE) 125 bp (PE) 100 bp (PE) 150 bp (PE)
Host % (mapping 32.6% ESTsb 18% ESTsb – 89.5–91.5% 39.4–51.2% 89.7% reads 28.5% reads/ – 17.4% 36.8% Not provided
reads or blast reads reads (kidney) 81.1% contigs contigs contigsb
contigs)
Accession ES599040- FN555710- No assembly No assembly No assembly 10.6084/ https://doi.org/ Not available Only Myxozoa GBKL01 No assembly
number or site of ES599804 FN5559871 m9.figshare. 10.5061/dryad. (37482 (TSA)
deposit of (Nucleotide) (Nucleotide) 11889672 j3tx95x9c transcripts
parasite ESTs or Addt. file 7)
assembly
No. ESTs or 768 ESTs 278 ESTs – – – 21 720 (from 29 765 (no – 272 047 52 972 –
contigs (Parasite bryozoan)/ carp) Myxozoa
assembly) 5384 (from fish) (232 396 only
/7362 contigs Myxozoa +
(intersect) 39651
stronger
match to
Myxozoa)
Assembly size – – – – – Not provided Not provided Not provided Not provided 42.9 –
(Mb)
N50 – – – – – 1772/1415/ Not provided Not provided Not provided 994 –
2479
Number of – – – – – Not provided 29 588 8659 267 995 Not provided –

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predicted proteins (ORF)
(continued on next page)

557
 Table 2. (continued)

558
Lineage Malacosporea Sphaerospora Oligochaete-infecting myxozoans
s.s.
Speciesa Buddenbrockia Tetracapsuloides bryosalmonae (2) Sphaerospora Thelohanellus Myxobolus Myxobolus Myxobolus
plumatellae (1) molnari (3) kitauei (4) pendula (5) cerebralis (6) honghuensis (7)
% GC content – – – – – 38.7/31.3/ 35.7% 39% (coding 33.3% Not provided –
33.1% region) (of the total
assembly –
930 419)
Completeness – – – – – –/66.7% –/53% Not provided Not provided 39%/ –
(CEGMA/BUSCO)c –/41.6% 49.5%d
–/50.1%
Refs [11] – [31] [32] [35] [23] [37] [13] [38] [12] [29]

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Lineage Oligochaete-infecting myxozoans Polychaete-infecting myxozoans
Species Myxobolus Myxobolus Henneguya Enteromyxum Enteromyxum Kudoa iwatai Kudoa Ceratonova shasta (15)
cultus (8) squamalis salminicola (10) scophthalmi (11) leei (12) (13) septempunctata
IIR IIC I
(9) (14)
Host Branchiura O. mykiss Oncorhynchus Scophthalmus S. maximus Sparus S. aurata Paralichthys O. mykiss Oncorhynchus O.
sowerbyi tschawytscha maximus (11A) (11B) aurata olivaceus kisutch tschawytscha
Sample Worm Scales Skeletal Head kidney, Head kidney, Intestine – Muscle Ascites Intestine Intestine
muscle spleen, pyloric spleen, pyloric
caeca caeca
Parasite stage Unknown Myxospores Myxospores 42 days p.i. 24 days p.i. Stages Myxospores Myxospores Developmental Developmental Developmental
stages and stages and stages and
myxospores myxospores myxospores
Geographic China USA USA Laboratory Laboratory Laboratory Israel Japan USA USA USA
location
Acc. Number SRP212980 SRP158957 SRP158827 SRP050607 SRP065375 SRP270898 SRP042325 Not provided SRP040506 SRP040506 SRP040506
Reads (SRA
study)
Platform Illumina Illumina Illumina HiSeq Illumina HiSeq Illumina Illumina Illumina Illumina Illumina HiSeq Illumina HiSeq Illumina HiSeq
HiSeq 2000 HiSeq 3000 2000 HiSeq 2000 MiSeq HiSeq 2000 Genome 2000 & 3000 2000 2000
3000 Analyzer IIx
Library strategy RNA-seq RNA-seq RNA-seq RNA-seq RNA-seq Amplicon RNA-seq Not provided RNA-seq RNA-seq RNA-seq
Library selection cDNA Random Random cDNA PolyA PCR PolyA Not provided PolyA PolyA PolyA
Read length 100 bp 150 bp 150 bp (PE) 100 bp (PE) 100 bp (PE) 100 bp (PE) 100 bp (PE) 81 bp (PE) 101 bp (PE) 101 bp (PE) 101 bp (PE)
(PE) (PE)
Host % (mapping Not Not Not provided 65–90% 90% reads – Not provided Not provided 9.5–32.2% 56.8% reads 70.2% reads
reads or blast provided provided reads (pyloric reads/0.3%–
contigs) caeca) 15.5% contigs
Accession No GHBR01 GHBP01 No assembly Not available No assembly GBGI01 Not available https://doi.org/ No assembly No assembly
Number or site of assembly (TSA) (TSA) (non-fish (TSA) 10.5061/dryad.
deposit of ESTs assembly) tx95x69tt
or assembly
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 No. ESTs or – 11 236 31 825 – Not provided – 6528 contigs Not provided 39 407/56 876 – –
contigs (Parasite contigs contigs contigs (IIR
assembly) RBT6 cs only/
cs+neither)
Assembly size – 7.2 18.3 – Not provided – 30 Not provided 22.4/32.9 – –
(Mb)
N50 – 714 600 – Not provided – 1662 Not provided 825/870 – –
Trends in Parasitology

Number of – 4105 3518 – Not provided – Not provided Not provided Not provided – –
predicted proteins
% GC content – Not Not provided – Not provided – Not provided Not provided Not provided – –
provided
Completeness – 23.8% 26.6% – Not provided – 77%/48.2%® Not provided 46% & 71%/– – –
(CEGMA/BUSCO)c
Refs [34] [14] [14] [33] [28] [30] [12] [55] [36] [36] [36]

Abbreviations: p.i., postinfection; SRA, Sequence Read Archive; ENA, European Nucleotide Archive; bp, base pair; SE, single-end reads; PE, paired-end reads; ESTs, expressed sequence tags; TSA, tran-
scriptome shotgun assembly database; Mb, Megabase; ORF, open reading frame; CEGMA, core eukaryotic genes mapping approach; BUSCO, benchmarking universal single-copy orthologs. Data reported
in this table were obtained from the literature and NCBI databases.
a
Arabic numbers next to parasite names provide a reference to Figure 1, where they are mapped to parasite life cycle stages.
b
Host contamination percentage from [36].
c
CEGMA: 248 genes, BUSCO: 978 genes; ® values from [35].

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Box 1. Numerical Characteristics of Myxozoan Genomes at a Glance

• Myxozoan genomes have a size of only 22.5–188.5 Mb [12–14] while their free-living relatives are generally >200 Mb
[15] to approximately 1 Gb in Hydra [16] and few exceptionally large genomes [17,18].
• Protein-coding genes are reduced to 5533–16 638, while P. hydriforme has 17 440 and free-living species have
20 000–66 156 [14,17].
• A general reduction of the number of orthologous gene groups is common in Myxozoa, with 2735 in K. iwatai vs
5451–11 162 [14,17] in other cnidarians. Numerous gene ontology categories are depleted [13].
• Myxozoan genomes are very compacted and are characterized by smaller intron/exon sizes (82–240 bp/102–235 bp)
than those of free-living cnidarians (799–2673 bp/208–218 bp) [12,14].
• Genomes of myxozoans show an extremely fast evolutionary rate and are hence highly derived, resulting in low genome
completeness scores with 37.5–73% CEGs in myxozoans vs 70–97% CEGs in free-living cnidarians [14].
• The GC content of myxozoan genomes is 23.6–37.5% vs 29–43% in free-living cnidarians.

Due to the present lack of myxozoan in vitro cultures, and to enrich parasite DNA, macroscopic
plasmodia filled with spores that can be relatively easily excised have become the preferred study
material [11–14,38,42]. However, not all myxozoan species form macroscopic plasmodia, and
with growing interest in other developmental stages and gene expression related to invasion,
migration, proliferation, and their interaction with hosts, a need for their physical concentration and
isolation emerged. This has been successfully achieved by centrifugation/density centrifugation
[36,37] and by ion-exchange separation [43], while polar capsules of C. shasta spores were isolated
using a tailored dielectrophoresis-based microfluidic chip [44]. Enrichment of the parasite-to-host-
cell ratio is essential, hence physical separation methods urgently require further investigation.

Even in the best parasite prep, contamination remains, likely also due to the parasite feeding on its
host by relatively unspecific endocytosis [45]. Hence, bioinformatic filtration of myxozoan HTS
data is an indisputable necessity. Sequences are commonly filtered after assembly, using NCBI
BLAST homology searches using a host or close relative-of-host reference genome (see
Table S1 in the supplemental information online). The transcriptome of Myxobolus pendula was
one of the first to apply a complex in silico hybridization pipeline that revealed 17–36.8% host
contamination [38]. A number of genomes and transcriptomes were subsequently filtered using
host genomes [12,14], host and parasite genomes [36], and even additional databases such
as the NCBI nr database [14], with increasingly thorough and complex approaches in recent
studies (Table S1). Three studies further implemented prior-to-assembly filtration strategies
[14,36,37], which have proven to reduce the formation of host–pathogen chimeras during
assembly [46]. While host filtration is essential, it has to be kept in mind that it is likely detrimental
to the detection of horizontal gene transfer events between fish and myxozoans. It was further
shown that using cnidarian genomes for filtration is a strategy of limited success, characterized
by poor recovery rate, likely due to the fast rate of myxozoan genome evolution.

In-house models using specific pathogen-free hosts and isolated laboratory lines of myxozoan
parasites would be highly desirable for improving the ‘cleanliness’ of datasets. Future approaches
should further consider single-cell sequencing which would not only help to reduce host
contamination but also allow us to gain insights into cell type specificity during development
[47]. Long-read sequencing technology would improve myxozoan genome assemblies, mapping
certainty, transcript isoform identification, and the detection of structural variants [48]. Finally, it
is essential that all analytical and filtration steps are well documented and that datasets are
published as raw data and assemblies, potentially also including intermediate files.

Origins and Evolutionary History of Myxozoans

Molecular evidence confirming a cnidarian origin of the Myxozoa was first provided with the
phylogenetic analysis of Buddenbrockia expressed sequence tag libraries [11] and the discovery

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of minicollagen genes [49], a taxonomically restricted gene family specific for the Cnidaria.
While their morphological simplicity makes it impossible to relate myxozoans to free-living
cnidarians, phylogenomic evidence frequently places the Myxozoa as a medusozoan sister
group [12,42,50], with high bootstrap support. Furthermore, large-scale phylogenomic analyses
support a sister-clade relationship of the Myxozoa with another fish-parasitic cnidarian,
P. hydriforme [12], together classified as Endocnidozoa. However, it has to be kept in mind
that both lineages are characterized by an accelerated molecular clock, leading to long branches
in phylogenomic studies [12], likely causing long-branch attraction (LBA) artifacts (reviewed by
Bergsten [51]). These may hamper a definitive resolution of their relationship [10].

Independently from phylogeny, a common origin of the Endocnidozoa appears to be confirmed

by a number of shared genome features, one of them being the loss of the genes related to
DNA cytosine methylation [24]. While myxozoans lack almost all relevant genes, P. hydriforme
appears to have a partial subset. However, lack of gene discovery in P. hydriforme may be
partially attributed to missing data (only transcriptomic data available). It can also not be
completely excluded that such reductions are the result of convergent developments, based
on parasitic lifestyle. Other drastic modifications, such as the loss of the mitochondrial genome,
appear to be a spontaneous adaptation known from H. salmonicola, while a very close relative
of this species is fully equipped for, and capable of, aerobic respiration [14].

The mitochondrial (mt) genome structure provides another independent character trait to
investigate the phylogenetic position of P. hydriforme and myxozoans. As most animals [52],
Polypodiumi, and all myxozoans have circular mt genomes, that contrasts to the linear mt
genomes of their sister group, the Medusozoans [53]. Myxozoan mt genomes are partitioned
into multiple circles, two in Kudoa [54,55], six in Myxobolus squamalis [14], and eight in
Enteromyxum leei [54]. Another synapomorphy is the massive expansion of noncoding regions
leading to the largest circular mt genomes sequenced to date (86 kb bp in Polypodium and
165.8 kb in E. leei) [54,56].

Large-scale relationships among myxozoan taxa are relatively well established based on small-
subunit (SSU) rDNA sequences [10,57], and the SSU rDNA clustering is reflected in
phylogenomic analyses [12,58], though these include only few lineages due to limited data
availability at the time (Table 1). Myxozoans cluster into four major lineages (Figure 2), defined
by their invertebrate hosts (confirmed life cycles in parenthesis). These are the most basal
bryozoan-infecting Malacosporea (three life cycles; [59]) and three annelid-infecting lineages
forming the Myxosporea (55 life cycles). These three lineages are (i) polychaete-infecting
myxozoans (PIMs, ten life cycles, summarized in [10]); (ii) oligochaete-infecting myxozoans
(OIMs, 44 life cycles, summarized in [10,60,61]); and (iii) the Sphaerospora sensu stricto clade
(one life cycle, [62]), which infects oligochaetes and likely older annelid groups [63]. Within the
major clades, species cluster according to organs they infect in their fish hosts (Figure 2; [57]).
There is compelling evidence from phylogeny (seen also in Figure 2), host–parasite co-
phylogeny, and molecular dating, that, during myxozoan evolution, all lineages first infected inver-
tebrates and later adapted secondary vertebrate hosts on multiple occasions [10,64].
Cospeciation patterns of myxozoans and their vertebrate hosts have been suggested as the cen-
tral reason for the massive diversification rates observed in myxozoans [10].

Overall, recent HTS studies have demonstrated that myxozoan genome contents are extremely
flexible and adaptive to changes, which explains their long and successful evolutionary history
but obscures the analysis of genomic adaptations in the light of evolution. Several genomes
and transcriptomes of myxozoans have been published in the last year (Tables 1 and 2) and

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Figure 2. Phylogeny and Evolutionary History of Myxozoans. The tree indicates four major phylogenetic lineages
(Malacosporea, Sphaerospora, oligochaete- and polychaete-infecting myxozoans), which are defined by invertebrate host
groups. All lineages but the ones labelled 'tetrapod hosts', 'chimaera host', and 'shark and ray hosts' infect teleost fishes
as secondary hosts. The color of the branches indicates diversification in different host organ systems (yellow –urinary
system, green – biliary system, and blue – within tissues). The closest relatives to the Myxozoa are Polypodium hydriforme
(sister clade) and the Medusozoa.

the analyses of additional datasets are under way, offering a wealth of data for future large-scale
analyses. Comparative genomic approaches will allow us to characterize lineage-specific adap-
tive changes in myxozoan genomes. Genomes of malacosporeans would be of particular interest
in this respect, as similarities and differences in gene family expansions can help to gain further
insights into the adaptations characterizing their independent historic invertebrate and vertebrate
host acquisition events. To further confirm or refute a common origin of the Endocnidozoa and
decipher their origins within the Cnidaria, a complete genome of Polypodium is required, and
solutions for dealing with varying evolutionary rates across different cnidarian lineages must be
developed and applied [10,65,66].

Exploring Myxozoan Diversity by eDNA Metabarcoding

Myxozoans represent a spectacular radiation of cnidarian endoparasites, accounting for about
one fifth of the species of the whole phylum [67]; however, their true biodiversity is still unknown
and very likely largely underestimated [68].

The detection of myxozoans has traditionally been performed by morphological screening of fish,
resulting in punctual and patchy observations, depending on available hosts and temporary
occurrences of spores within them. In contrast, most transmission stages in the environment
are known to survive and to be infective for up to 6 months [69], except for the taxonomically
deprived group of malacosporeans. Access and conservation issues related to the study of fish

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provide further reasons why it is more desirable to estimate myxozoan diversity from the environ-
ment rather than from their hosts.

The genomic revolution has fundamentally changed the way we survey biodiversity on earth [70].
HTS platforms now enable rapid sequencing of DNA from a diversity of environmental DNA
(eDNA) samples. eDNA metabarcoding offers a powerful molecular tool capable of noninva-
sively surveying species richness from many habitats. This research has recently expanded
considerably, also with regard to parasitological research, including myxozoans, for which it is
clearly relevant [71] due to its power for identification at an increased resolution and quantification
of even the smallest transmission stages.

Myxozoan operational taxonomic units (OTUs) have been detected in a number of large-scale
eDNA studies using universal SSU rDNA primers and investigating a variety of aquatic habitats,
such as oceans, rivers, lakes, and ponds (e.g., [72,73]). Curiously, eDNA approaches have also
uncovered myxozoan OTUs in unexpected hosts and habitats, such as in the gut of the desert
night lizard Xantusia vigilis [74], hence demonstrating the power of the approach for the discovery
of new hosts or food chains. Though often used for monitoring purposes of various myxozoan
pathogens in species-specific quantitative assays [7,75,76], so far only one study specifically
targeted the discovery of myxozoan diversity from eDNA [77]. The authors found only 7% of the
recovered myxozoan OTUs to match previously known SSU rDNA sequences, though it has been
estimated that some 23% of described myxozoan species have SSU rDNA sequences available
[78]. The data were obtained from predominantly well-studied habitats with known myxozoan
hosts and parasites, yet it underestimated myxosporean diversity more than three times. This
suggests that the lineage discovery rate is substantially higher in unexplored areas, such as different
habitats and depths of the ocean, where many parasite groups are poorly sampled [79,80].

Other than providing insights into a vastly undiscovered diversity, the first eDNA study in
myxozoans has also highlighted major methodological pitfalls. In general, challenges to species
discovery and accurate abundance estimation through eDNA metabarcoding stem from multiple
factors in the field, the laboratory, and the keyboard [81]. However, in myxozoans, additional
components, based on specific molecular and biological characteristics, require further method-
ological considerations (Figure 3). Firstly, the dilution effect in plankton-rich water poses a major
problem for detection, especially when using general SSU rDNA primers. This is further aggra-
vated by the presence of PCR inhibitors [82]. Inhibitor removal combined with lineage-specific
amplicon sequencing currently represents the most successful approach [77]. Interestingly, the
highest myxozoan diversity recovery rate was obtained from natural filters such as the feces of
piscivorous animals [77].

eDNA surveys provide a nondestructive and nonbiased means for characterizing true myxozoan
diversity in aquatic habitats [71], achieved with incomparable scale, speed, and comprehensive-
ness of information [70]. We anticipate that species richness estimates deriving from unique
OTUs will outstrip the number of traditionally described myxozoan species in the future. eDNA
could further be an important tool in conservation and monitoring past and present myxozoan
biodiversity [83], especially in the light of environmental degradation [84] and invasions [85]. An
essential future requirement is the establishment of a robust methodology, including comparative
approaches for sampling sites, timings, and methods, processing, and marker-based diversity
yield (Figure 3). Based on an optimized methodology, and the generation of numerous datasets
from different ecosystems, important ecological questions can eventually be addressed, insights
into myxozoan life cycles, transmission and dispersion can be gained, and evolutionary origins
and trajectories may be recreated.

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Outstanding Questions
What is the true size of myxozoan
genomes once all contamination has
been removed, and does genome size
vary between the major phylogenetic
lineages?

Can large-scale comparisons of

myxozoan genomes from different line-
ages provide further insights into the
history of host acquisitions and differ-
ent strategies of host exploitation?

What are the major groups of reduced

genes in myxozoan genomes, and
which gene families have expanded?
What is the importance of enriched
gene families to parasite survival and
propagation in fish hosts?

Are the Endocnidozoa truly monophyletic

or does the ancient and derived character
of their two lineages cause them to cluster
together? Are modifications in their
genomes convergent developments or
did they evolve in the common ancestor?

Just how much do we underestimate

myxozoan biodiversity by studying
infections in fish and when comparing
these data with eDNA metabarcoding
Trends in Parasitology
approaches?
Figure 3. Flow Chart Providing Necessary Considerations for the Estimation of Myxozoan Biodiversity and
Abundance from Environmental DNA (eDNA). The experimental design should consider multiple parameters in the What are the roles of myxozoan
field, the laboratory, and the keyboard based on the specific molecular and biological characteristics of myxozoan proteases during parasite invasion,
parasites. Abbreviations: Lab, laboratory; OTU, operational taxonomic unit; SSU, small subunit. nutrition and host–parasite interaction?

What are the genetic mechanisms of

Functional Genomics of Myxozoans: Finding Candidates for Disease Control myxozoan immune evasion in their
Functional genomics opens new venues for myxozoan research that provide important fish hosts? Do myxozoans modify
their antigens during their intrapiscine
perspectives for disease control. With the continuously increasing quality of myxozoan development, and can we use the
genomes and transcriptomes, and targeted comparative studies, we are now able to pinpoint relevant molecules for vaccine design?
genes that could constitute good vaccine candidates or drug targets. As such, proteolytic
enzymes and their inhibitors, which amount to 2.5% of the proteome of Thelohanellus kitauei
[13], 2.6% of the transcriptome of Sphaerospora molnari proliferative blood stages [37], and
6.1% of the proteome of C. shasta polar capsules [2], are prime candidates requiring further
research. While the percentage of proteolytic enzymes in T. kitauei represents only 42.8% and
52.6% of those in free-living Nematostella vectensis and Hydra magnipapillata, respectively, they
are much less reduced than their lipid (16.3% and 37.3%) and carbohydrate (8.5% and 19.4%)
digestive enzymes [13].

Importantly, in parasites, proteases play key roles in pathogenesis due to their involvement in host
invasion, parasite migration through tissue barriers, degradation of hemoglobin and other
proteins, immune evasion, and activation of inflammation [86]. Proteases are ancient molecules
that likely arose at the earliest stages of protein evolution [87] and they appear to have persisted
in myxozoans despite their massive genome reductions, with latest genomic and transcriptomic
studies uncovering vast proteolytic arsenals (100–422 homologs), present in all myxozoan
lineages [13,23,37], indicating their essential roles in parasite survival and propagation.

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Despite their universal occurrence in myoxzoans, the particular functions of specific proteases
in different taxa are scarcely known. The first proteases investigated were cysteine and serine
proteases – used for enzymatic destruction of host muscle (Kudoa spp.; [88,89]) and cartilage
(Myxobolus cerebralis; [90,91]) – produced by spore-forming parasite stages in fish. With the
onset of myxozoan genomics, full protease repertoires were analyzed for the first time
[13,23,37]. Comparative transcriptomics demonstrated that proteases are strongly associated
with virulence in host-associated genotypes of C. shasta [36], and differential expression in
vertebrate vs invertebrate hosts of T. bryosalmonae showed that most secreted proteases
show higher expression levels in their fish host [23]. This suggests key roles in immune defense,
evasion, or modulation, as vertebrates, in contrast to invertebrates, possess a sophisticated
system of antigen-specific B and T cells driving immunological memory towards myxozoan-
borne diseases [92].

Protease inhibitors could be another key group for the design of specific antimyxozoan treat-
ments as they can have immunomodulatory roles during parasite invasion, dissemination, and
proliferation [86]. A cysteine protease inhibitor recently characterized in T. kitauei is suggested
to play a role in blocking host proteases and interfering with immune recognition [93]. T. kitauei
further shows massive expansion of serine protease inhibitors (114 homologs) when compared
with other myxozoan species (one to seven homologs) [94]. This may be related to host invasion
via the host gut, thereby deactivating host digestive enzymes or modulating mucosal immunity.

Apart from proteases and their inhibitors, lipases and low-density lipoprotein receptor class A
domain-containing proteins have been found to show important expression levels in myxozoans
[23,37]. This is strongly indicative of host cholesterol and acyglycerol exploitation and was sug-
gested to be a major contributory factor to disease pathogenesis mediated by T. bryosalmonae
[23]. As for proteases, viable pharmacological approaches exist that interfere with parasite utiliza-
tion of host lipids, which may be of relevance to the future control of myxozoan parasites [95,96].

In conclusion, recent genomic and transcriptomic studies have highlighted the first candidate
molecules that could be targeted in the design of antimyxozoan therapeutic and prophylactic
strategies, and it is now timely to embark on functional characterization, including the implemen-
tation of RNAi and protein-interaction studies, as well as vaccination trials based on target
molecules or their RNA precursors. Such applied approaches are still scarce [93,97], likely due
to the lack of laboratory models for most myxozoan species and resulting difficulties with data
reproducibility. Emphasis should also be placed on investigating parasite molecules involved in
B cell responses and their variation during the intrapiscine life cycle, as antibody production
can have a major effect on the course of infections and disease [43]. Finally, vaccine developments
based on novel candidates are urgently required for myxozoans, including optimization of vaccine
delivery and tests evaluating the duration of protection, as global warming predicts myxozoan
disease outbreaks in the near future [8,98,99].

Concluding Remarks
Genomic sequence data of myxozoan parasites have proven highly difficult to generate due to
the skewed quantities of parasite and host DNA, 10–50× smaller genome sizes, subsequent
assembly complications, and contamination issues with filtration databases. These challenges
could be overcome by performing laboratory studies with isolated parasite lineages in specific
pathogen-free fish, by developing new parasite separation protocols, and by bioinformatic
analyses using multiple filtration steps and databases. Innovative methodological solutions and
the use of novel sequencing platforms, such as nanopore and single-cell sequencing technologies,
could contribute considerably to generating improved genomic datasets of these organisms. The

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 Trends in Parasitology

few existing datasets have demonstrated extraordinary characteristics of myxozoan genomes. To

gain a better understanding of the biology, diversity, evolution, and host–parasite interaction of
myxozoans (see Outstanding Questions) it is imperative that we continue to sequence genomes,
metagenomes, and transcriptomes of a wide range of myxozoans from different phylogenetic
clades, but also the genome of the cnidarian sister lineage, P. hydriforme. Future avenues for
research include myxozoan comparative genomics, which provides exciting perspectives and
will revolutionize our understanding of the evolutionary history of cnidarians and basal metazoan
parasites in general. Comparative transcriptomic and proteomic approaches of different species
and life cycle stages will allow us to further pinpoint gene groups of major importance to parasite
propagation and host immune evasion. Downstream functional analyses of these molecules re-
quire the development of methods to test gene function, for example by RNAi, to localize proteins
using highly specific antibodies or GFP (green fluorescent protein) fusion proteins, that can be used
as reporters of expression levels and would allow visualization of targets in life cells throughout the
intrapiscine development. Such methods represent a solid base for targeted disease interventions
and the design of vaccines, which are essential for the future of aquaculture in the light of climate
change and emerging myxozoan diseases.

Acknowledgments
We acknowledge funding by the Czech Science Foundation (grant number 19-28399X to A.S.H. and G.A.-B.). Drawings
and figures were prepared by M. Hajdušková (biographix).

Declaration of Interests
The authors have no interests to declare.

Supplemental Information
Supplemental information associated with this article can be found online at https://doi.org/10.1016/j.pt.2021.01.010.

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