Draft macronuclear genome sequence of the marine ciliate Miamiensis avidus causing flatfish scuticociliatosis

TL;DR

This paper presents the draft genome of a marine ciliate parasite that causes a serious disease in farmed fish and crustaceans.

Contribution

The novel contribution is the first draft macronuclear genome sequence of Miamiensis avidus, a scuticociliate parasite.

Findings

The draft genome will help understand the somatic functions of scuticociliates.

It provides insights into the physiology and pathology of the parasite in aquaculture.

Abstract

Scuticociliatosis is a severe and challenging protozoal disease affecting farmed flatfish and crustaceans. This draft genome assembly of the macronuclear (MAC) genome of Miamiensis avidus, a scuticociliate parasite, will be valuable for understanding the somatic functions, physiology, and pathology of scuticociliates in aquaculture worldwide.

Tables

TABLE 1. Comparison of different ciliate macronuclear genomes with M. avidus MAC genome

Parameters	Miamiensis avidus	Pseudocohnilembus persalinus	Paramecium bursaria	Paramecium caudatum	Paramecium tetraurelia	Tetrahymena thermophila	Oxytricha trifallax
Genome size (Mb)	75.8	55.5	26.8	30.5	72.07	103.01	55.4
Sequencing platform	PacBio/Illumina	Illumina	PacBio/Illumina	Illumina	Sanger/Illumina	Sanger/Illumina	PacBio/Illumina
No. of contigs	213	288	515	1,202	697	1,158	19,152
Average GC content (%)	23.5	19	28.8	28.2	28.0	22.0	31.0
N50 (Kb)	754	368	100	312.9	413	520	3.5
Gene number	10,723	13,186	15,101	18,673	40,460	26,996	18,400

Full text

ANNOUNCEMENT

Miamiensis avidus is a free-living marine protozoan belonging to the order Philasterida, subclass Scuticociliatida, class Oligohymenophorea, and phylum Ciliophora. It has caused severe disease outbreaks and mass mortalities in cultured marine fish such as turbot (1), sea bass (2), and olive flounder (Paralichthys olivaceus) (3), but an effective chemical treatment for this disease has not yet been discovered. Therefore, we sequenced the macronuclear (MAC) genome of a survey for pandemic serotypes from M. avidus to help us understand its genomic and biological features and the development of an effective vaccine against parasitic disease.

M. avidus strain MagSerotype2-33 (M. avidus Serotype 2, SCU-MA2) was isolated from olive flounder P. olivaceus on a local farm in Pohang, Korea, in 2008. To obtain macronuclear DNA, M. avidus strain SCU-MA2 was cultured in the CHSE-214 cell line (ATCC CRL 1691) at 20°C in Eagle’s MEM with 10% heat-inactivated fetal bovine serum (FBS). Macronuclei were isolated from the cell lysate using a Whatman 25 mm Nuclepore Polycarbonate filter membrane (pore size: 5 µm), as previously reported (4). M. avidus MAC DNA was extracted using a HiGene Genomic DNA Prep Kit (Biofact, Korea). The sample was used to prepare the SMRTbell library with an SMRTbell Template Prep Kit. Sequencing was performed on the PacBio sequencing platform (Pacific Biosciences, USA). Additionally, MiSeq sequencing data previously reported and used for the assembly of the complete mitochondrial genome of this species were also used to polish the M. avidus genome sequence (5).

The sequencing of the PacBio SMRT cell produced 22.3 Gbp of raw reads at 293.6-fold coverage of the genome. The reads were assembled using CANU v. 1.6 with default parameters (6). To enhance the accuracy of the initial contig assembly, clean Illumina short reads were aligned using BWA-MEM (7), followed by polishing with Pilon version 1.23 (8), both executed with default parameters. The resulting assembly was searched for putative telomeric repeats (5′-CCCCAA)n using BioEdit and identified 119 contigs with telomeric repeat sequences on both ends and 49 with only a single telomere found. The final draft M. avidus MAC genome was assembled into 213 contigs with a total sequence length of 75,782,695 bp and a contig N50 of 754,125 bp (L50: 30, 23.52% GC content). The annotation information derived from the draft genome contig sequences, along with the corresponding transcript and protein sequences, has been deposited in “https://doi.org/10.6084/m9.figshare.26866936.v2.” By combining the information and comparisons between the M. avidus MAC genome and those of six other ciliate species, M. avidus was found to be closely related to Pseudocohnilembus persalinus as a member of the Order Philasterida (Table 1). The use of the draft MAC genome could have widespread implications for gaining a better understanding of the pathology of scuticociliatosis, as well as for developing effective treatment strategies and fish vaccines.