Taxonomy, diversity and diagnosis of Tetrahymenosis, and its recent identification measures in aquaculture inferred from infections in Northeastern China

Background Tetrahymenosis caused by about ten parasitic Tetrahymena species belonging to the Phylum Ciliophora has been recognized as an emerging problem inflicting significant economic loss in aquaculture industry in the world. Increasing knowledge and identification of Tetrahymenosis are important. Methods Four parasitic Tetrahymena species were collected from eight commercially farmed fishes in Harbin, northeastern China. Specific oligonucleotide probe and fluorescence in situ hybridization staining was designed and tested. Hoechst33342 staining methods, gene sequencing and phylogenetic analyses were also conducted. Results Tetrahymena pyriformis, T. vorax, T. chironomi and T. bergeri are the four species responsible for Tetrahymenosis in various fishes in Harbin. Taxonomy, diagnosis, diversity, pathogenicity, and histopathology of Tetrahymenosis were supplemented, analyzed and summaried, based on the present and previous work. The term Tetrahymenosis is diagnosised as diseases affecting a number of fishes, crustaceans, mollus, beetle, dragonfly, salmon, slug, midge larvae and freshwater mussel species that caused by ciliates of the genus Tetrahymena which constitutes an abundant group inhabit various aquaculture and natural habitats. Improved classification of parasitic Tetrahymena was provided. Phylogeny of parasitic Tetrahymena species was studied, and an SSU-rDNA targeted oligonucleotide probe labeled with a fluorochrome was designed, and the FISH protocol was optimized for identification of Tetrahymena species, Conclusions The manifestations of histopathology in fish typically include lesions on the body surface, and affected organs include the skin, musculature, viscera, eye socket and spinal cord; masses of ciliates can be detected in copious amount of mucus and between spaces in the damaged tissues. Improved classification of parasitic Tetrahymena is provided: facultatively parasitic forms ( T. pyriformis , T. rostrate , T. bergeri and T. vorax

), facultatively free-living forms ( T. chironomi , T. corlissi, T. rotunda, T. glochidiophila and T. papula ) and parasitic forms ( T. stegomyiae and T. limacis ).The phylogenic results indicate that Tetrahymena spp. belonging to 'borealis' group have a greater probability to become parasitism. the method of which can be used for quick and early detection of Tetrahymenosis.
While morphological method is the routine way to identify Tetrahymena spp., it has the limitations as follows: 1) the commonly used silver staining methods for reliable identification need considerable experience and staining failure is often the case, making species identification difficult, 2) parasitic Tetrahymena species are hard to control when massive infection occurs. Thus, early detection, quantification, and control of these infectious pathogens are important in aquaculture [1,26]. FISH with specific fluorochrome-labeled oligonucleotide probes is a staining technique that allows molecular identification of targeted organisms in mixed assemblages by means of a fluorescence microscopy, this method can overcome the drawbacks of the morphological method and yield prompt detection, and has been applied successfully to pathogenic ciliates (e. g. Pseudocohnilembus persalinus, Boveria labialis and B. subcylindrica) [27][28][29][30]. However, to date, there are no general fluorochrome-labeled oligonucleotide probes for parasitic Tetrahymena spp.

Methods
Ciliates isolation, cultivation and morphological identification.

Taxonomy
The term Tetrahymenosis covers diseases affecting a number of fishes, crustaceans, mollus, beetle, dragonfly, salmon, slug, midge larvae and freshwater mussel species that caused by ciliates of the genus Tetrahymena which constitutes an abundant group inhabit various aquaculture and natural habitats (  [2-5, 13, 19]. Tetrahymena is generally pyriform-shaped, uniformly covered with meridional kineties, the buccal cavity is equipped with one curved undulating membrane with paired basal bodies organized in zigzag pattern (i.e. stichodyad), located on right edge of the buccal cavity, and three parallel membranelles.
Within species, size variations depend on culture medium, physiological state and life stage (Corliss, 1973). The life cycle of Tetrahymena species is typified by the following developmental stages: theront, trophont, tomont and tomite.

Diversity of parasitic Tetrahymena
Tetrahymena species commonly exhibits a whole range of existence from the completely free-living state through stages of facultative parasitism to obligate endoparasitism [6]. Corliss (1971a) classified Tetrahymena parasitism into three types: facultatively parasitic forms (generally found free-living in nature but for which a parasitic existence is not uncommon), facultatively free-living forms (typically found in association with a host but capable in nature as well as experimentally) and parasitic forms (as endoparasites associated with specific hosts). To date, about ten Tetrahymena species (T. corlissi, T.

Detection and identification using FISH
The probe Tetr2020 evaluated with the probe match tool of the ARB software package and the GenBank BLAST tool showed that they are specific to Tetrahymena spp. The probe Tetr2020 (5 ' -TGTAGTAGCCGTTTCTCAG-3 ' ) had at least two mismatches to other closely related species like Uronema spp., Pseudocohnilembus spp. and Cyclidium spp. FISH with the probes Tetr2020 resulted in the presence of a red fluorescence signal each for T. pyriformis, T. vorax, T. chironomi and T. bergeri ( Figure 3D, E), clearly distinguishable from the faint autofluorescence signal achieved with negative-control hybridizations using the Tetr2020 probe to hybridize the untargeted ciliates Pseudocohnilembus persalinus, Uronema marinum and isolated from the same hosts ( Figure 3F). Thus, the probes Tetr2020 is also a general probe suitable for widely and rapidly detecting Tetrahymenosis. Corliss (1971a) classified Tetrahymena parasitism into facultatively parasitic forms (T. As a histophagous parasite, Tetrahymena disintegrates host tissue and feeds on cell debris. Infection is showed by whitish patches due to the masses of ciliates in copious amount of mucus, patches usually located on the dorso-lateral skin around the abdomen (Johnson, 1978). Typical gross lesions of Tetrahymena infection are characterized by protrusion of scales, swelling, ulcerative wounds on the skin, blindness, and protrusion of eyes (Johnson, 1978). Numerous Tetrahymena spp. may form a rim around the eye orbit (spectacled eye). The parasites route of entry into the body has not been reported. In histopathological analyses, the ciliates were found to be distributed between spaces in the damaged tissues, and extensive necrotic changes occur in the muscle and subdermal tissue. Necrosis of the epithelial cells, extending to the musculature, and disfigured dermal and subcutaneous tissue with edema and hemorrhage was also reported [24, 51].

Discussion
Compared with Scuticociliatosis, Tetrahymenosis can be found in a more wide-ranging hosts (e. g. slugs, hick embryos, dragonfly, helgramite, roach, cockroach and caterpillar), and Tetrahymenosis is easier to discovered in freshwater fish (vs. mostly in marine habitat fishes for Scuticociliatosis) (24, 46, 48, 52). However, their major clinico-pathological manifestations are similar: anemia, weight loss, dark coloration, enteritis, excessive body mucus, yellowish intestinal mucus, loss of scales, hemorrhagic and/or bleached spots on the skin, and dermal necrotic lesions that finally destroy tissues lead to high mortalities [8, 16, 50, 52].
As a test case using probes for the identification of pathogenic ciliates in aquaculture, the present study utilized the probe Tetr2020 to unambiguously detect Tetrahymenosis

Consent for publication Availability of data and materials
The datasets supporting the conclusions of this article are included within the article.
Representative sequences are submitted to the GenBank database (accession numbers are provided in Figure 4).

Competing interests
PXM and CY work at Harbin Normal University, Harbin, China. The study was conducted as part of a research program to investigate Tetrahymenosis in aquaculture in Northeastern China.

Funding
Natural Science Foundation of China (project numbers: 31970498), Heilongjiang Province Science Foundation for Youths (project number: QC2017017).

Authors' contributions
The study design, protocol and were prepared by ZSX, PMM, LWY, SM, WCN, LJL, WX and ZL, and report of the study and reviewed by PXM and CY. PXM and his team at Harbin Normal University were responsible for the animal phase and data collection. PXM were responsible for compiling the first draft of the manuscript. All authors revised and approved the final version.