Huang et al. Parasites Vectors (2021) 14:318 https://doi.org/10.1186/s13071-021-04821-3 Parasites & Vectors RESEARCH Open Access Comparison of mitochondrial genome and development of specifc PCR primers for identifying two scuticociliates, Pseudocohnilembus persalinus and Uronema marinum Yu‑Xi Huang1, Sen Wang1, Yan‑Qi Gao1, Jie‑Hu Chen2, Xiu‑Li Wang1 and Rui‑Jun Li1* Abstract Background: Pseudocohnilembus persalinus and Uronema marinum (Ciliophora, Scuticociliatia), as parasitic scutico‑ ciliatid ciliates, were isolated from Scophthalmus maximus and Takifugu rubripes, respectively, in our previous studies. These ciliates are morphologically very similar; hence, it is difcult to identify specifc scuticociliate species using traditional classifcation methods for performing taxonomic research and disease control studies. Methods: We annotated the mitochondrial genomes of these two scuticociliates on the basis of previous sequenc‑ ing, including analyses of nucleotide composition, codon usage, Ka/Ks, and p‑distance. We also compared the nucleo‑ tide and amino acid similarity of the mitochondrial genomes of P. persalinus, U. marinum, and other 12 related ciliates, and a phylogenetic tree was constructed using 16 common genes. We chose the nad4 and nad7 genes to design specifc PCR primers for identifcation. Results: P. persalinus and U. marinum were found to have a close evolutionary relationship. Although codon prefer‑ ences were similar, diferences were observed in the usage of codons such as CGA, CGC, and GTC. Both Ka/Ks and p‑distance were less than 1. Except for yejR, ymf57, ymf67, and ymf75, the amino acid sequence similarity between P. persalinus and U. marinum was greater than 50%. Conclusions: The mitochondrial genomes of P. persalinus and U. marinum were thoroughly compared to provide a reference for disease prevention and control. The specifc PCR primers enabled us to identify P. persalinus and U. mari- num rapidly and accurately at the molecular level, thus providing a basis for classifcation and identifcation. Keywords: Pseudocohnilembus persalinus, Uronema marinum, Genome comparison, Specifc PCR primers Background In recent years, there has been an increase in the occur- rence and extent of damage due to diseases of mari- culture animals caused by ciliates. Scuticociliatosis, a *Correspondence: [email protected] 1 Liaoning Key Laboratory of Marine Animal Immunology, Dalian Key disease caused by a type of ciliates known as scuticocili- Laboratory of Marine Animal Disease Control and Prevention, College ates, occurs globally and has led to a high fatality rate of of Fisheries and Life Science, Dalian Ocean University, Dalian, Liaoning fshes [1, 2]. Scuticociliates include approximately 20 spe- 116023, People’s Republic of China Full list of author information is available at the end of the article cies that cause scuticociliatosis, including Miamiensis © The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Huang et al. Parasites Vectors (2021) 14:318 Page 2 of 10 avidus, Uronema marinum, Uronema nigricans, Metan- level. Te use of PCR primers to identify scuticociliates ophrys orientalis, Pseudocohnilembus persalinus, and provided the basis for the classifcation and identifcation Pseudocohnilembus hargisi [3–7]. Te species of para- of scuticociliates. sitic scuticociliates difer according to the diferent liv- ing environments of the host, but the symptoms caused Methods by them are very similar [8]. Scuticociliates can not only Whole mitochondrial genomes of P. persalinus and U. invade the body surface, skin, fn, and muscle of fshes marinum but also their abdominal cavity, kidney, pancreas, and Te whole mitochondrial genome sequences of P. persa- even brain. Scuticociliates cause blackening of fsh body linus and U. marinum were deposited in NCBI (https:// color, tissue and organ bleeding, ulceration, and other www. ncbi. nlm. nih. gov/) with accession numbers pathological changes, leading to the death of Paralich- MH608212 and MG272262, respectively. thys olivaceus, Tunnus maccoyii, Scophthalmus maxi- mus, and Dicentrachus labrax [4, 9–12]. Tus, to prevent Annotation of the mitochondrial genome and control scuticociliatosis efectively and rapidly, it is Te MITOS (http:// mitos. bioinf. uni- leipz ig. de/ index. important to accurately detect scuticociliates. However, py) [18] and ORF Finder (https:// www. ncbi. nlm. nih. the morphological forms of the subclass Scuticociliatia gov/ orf nder/) tools were used to preliminarily annotate are very similar. Te body of scuticociliates is 20–45 μm mitochondrial genome sequences. BLASTp and BLAST long, melon seed-shaped, and covered with cilia. Tere- (https:// blast. ncbi. nlm. nih. gov/ Blast. cgi) were used to fore, it is difcult to distinguish scuticociliates using a compare the preliminary results of annotation with light microscope. Previously, the species of scuticociliates encoded protein and rRNA sequences belonging to the could only be identifed through repeated verifcation by reported related species in order to verify the accuracy of a series of methods such as scanning electron micros- the results and make corrections. Te schematic diagram copy, protein silver staining, and silver impregnation. was drawn using OGDRAW (https:// chlor obox. mpimp- Although the morphological analysis of scuticociliates is golm. mpg. de/ OGDraw. html). a useful approach to identify them, it is time-consuming and laborious, and is thus not suitable for wider use prac- Analysis of codon usage tically in treating scuticociliatosis. Moreover, morpholog- Te codon usage bias of P. persalinus and U. marinum ical analysis is based on staining techniques, which may was compared. Te RSCU was calculated using the for- obscure subtle changes, leading to identifcation errors mula described in the study by Sharp and Li [19]. [13]. Te ribosomal small subunit rDNA (SSU-rDNA) sequence analysis method is a universal tool for parasite Analysis of Ka/Ks and p‑distance classifcation. However, the SSU-rDNAs of related para- Ka/Ks analysis was performed on 25 common encoding site species show few diferences, which limits the identi- genes of P. persalinus and U. marinum. MUSCLE v3.8.31 fcation of closely related species [14]. (http:// www. drive5. com/ muscle/) software was used to In a preliminary study, we isolated P. persalinus and compare the selected 25 common encoding genes of P. U. marinum from Scophthalmus maximus and Takifugu persalinus and U. marinum [20]. Te KaKs_Calculator rubripes, respectively [15, 16]. P. persalinus and U. mari- 2.0 was then used to calculate the Ka/Ks of all compared num are dominant scuticociliate pathogens that are dis- sequences [21], and MEGA6 software was used to calcu- tributed in the north of China marine aquaculture area late p-distance [22]. [17]. In our previous studies, we sequenced the whole mitochondrial genomes of P. persalinus and U. marinum Phylogenetic analysis [15, 16]. In the present study, we analyzed interspecifc Te complete mitochondrial sequences of 12 ciliates diferences in the mitochondrial genome between P. per- related to P. persalinus and U. marinum were down- salinus and U. marinum by comparing codon usage, Ka/ loaded from NCBI for phylogenetic tree construction. Ks, and p-distance. We also revealed evolutionary rela- Te sequences used for comparison and their GenBank tionships and mitochondrial genome diferences between accession numbers were as follows: Tetrahymena para- these species and 12 related ciliates by constructing a vorax (Accession Number: NC_008338.1), Tetrahy- phylogenetic tree and comparing nucleotide and amino mena pyriformis (Accession Number: NC_000862.1), acid similarity of the mitochondrial genomes. Specifc Tetrahymena thermophila (Accession Number: PCR primers for identifcation were designed according NC_003029.1), Paramecium caudatum (Accession Num- to the specifc genes in the mitochondrial genomes of P. ber: NC_014262.1), Tetrahymena pigmentosa (Acces- persalinus and U. marinum. Tese primers enable easy, sion Number: NC_008339.1), Tetrahymena malaccensis rapid, and accurate species identifcation at the molecular (Accession Number: NC_008337.1), Laurentiella strenua Huang et al. Parasites Vectors (2021) 14:318 Page 3 of 10 (Accession Number: KX529838.1), Turicola similis nad7. Primers P2-F (5ʹ-GTT ATG GTT ACA ATG TTT (Accession Number: MW221262), Oxytricha trifallax GGT GTT AT-3ʹ) and P2-R (5ʹ-TAT AGT ACC AAC ATG (Accession Number: JN383843), Halteria grandinella TTT TCT CAT CA-3ʹ) were designed based on