Epidemiology and identification of two species of Chilodonella affecting farmed fishes in China

Veterinary Parasitology 264 (2018) 8–17

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Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar

Research paper

Epidemiology and identification of two species of Chilodonella affecting farmed fishes in China

T

Ming Lia, Runqiu Wanga, Giana Bastos Gomesb, Hong Zoua, Wen-xiang Lia, Shan-gong Wua, ⁎ ⁎ Gui-tang Wanga, , Francisco Ponce-Gordoc, a Key Laboratory of Aquaculture Disease Control, Ministry of Agriculture, and State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China b Tropical Research Institute, James Cook University Singapore, Singapore 387380, Singapore c Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad Complutense de Madrid, Plaza Ramón y Cajal s/n, 28040 Madrid, Spain

A R T I C LE I N FO

A B S T R A C T

Keywords: Chilodonella hexasticha Chilodonella piscicola Morphology rDNA genes Geographic distribution Species identification China

The genus Chilodonella includes free-living ciliated protozoa as well as pathogenic species for freshwater fish, with Chilodonella hexasticha and Chilodonella piscicola being the most important ones. These parasites cause outbreaks with high mortalities among farmed freshwater fishes with great economic losses. There are few reports of these species in China, and their identification has been based mostly on their morphological characteristics. In the present work, the parasites causing five outbreaks occurring in China between 2014 and 2017 have been identified by morphological and genetic analysis. We provide the first records of Ctenopharingodon idella and Siniperca chuatsi as hosts of C. hexasticha, and of Procypris rabaudi and Schizothorax wangchiachii as hosts of C. piscicola. There are no differences in the gross pathological findings produced by C. hexasticha and C. piscicola, consisting in desquamation and necrosis of epithelial cells in the skin and gills and in severe fusion of gill lamellae. However, both species differ in their geographic distribution: C. piscicola was found in farms located at altitudes over 1500 m above sea level and with a water temperature ≤18 °C, while C. hexasticha was found in farms located at altitudes under 50 m above sea level and with a water temperature ≥21 °C. Present results confirm that C. hexasticha and C. piscicola are two different species that can be differenced by their morphology; however, their biological variability may lead to erroneous identifications and the diagnosis should be preferably based in genetic analysis including nuclear LSU rDNA and mitochondrial SSU rDNA sequences.

1. Introduction Chilodonellosis is a serious disease of freshwater fish and accounts for great economic losses in affected farms (Leibovitz, 1980; Mitra and Haldar, 2004; Bastos Gomes et al., 2017a). It can result in rapid epizootic events on fish farms with mortalities within 2 or 3 days of infection (Paperna and Van As, 1983; Karvonen et al., 2010; Bastos Gomes et al., 2017b). Chilodonellosis is caused by pathogenic species of the genus Chilodonella (Phyllopharyngea: Chilodonellidae), mainly Chilodonella hexasticha and Chilodonella piscicola (syn. C. cyprini, see Shulman and Jankosvski, 1984). Chilodonella species are widely distributed and they can infest a wide range of freshwater fishes without host specificity. Some of the clinical signs of chilodonellosis is anorexia, lethargy, skin depigmentation, ulceration, scale loss, excessive mucus excretion and gill damage (Hoffman et al., 1979; Paperna and Van As, 1983; Langdon et al., 1985; Pádua et al., 2013; Bowater and

⁎

O’Donoghue, 2014; Bastos Gomes et al., 2017a). There are very few reports of chilodonellids infecting fish in China. Up to date, the species C. hexasticha has been described from Carassius auratus and Pelteobragus fluvidraco in the Sichuan province (Hu, 2012, 2015); and the species C. piscicola has been described in Schizothorax o’connori and Oxygymnocypris stewartii in Tibet (Deng et al., 2015) and (as Chilodonella cyprini) in several freshwater fish species in Wuhan and Sichuan (Xiao and Li, 1995). All these identifications were based on the morphology of the cells; Deng et al. (2015) also did genetic characterization of their isolates, but no comparative analysis were made. Except for the greater cell size in the descriptions by Hu (2012) and Deng et al. (2015), there are no significant morphological differences between these reports despite different species identified. This arises the issue of misidentifications or that both species are in fact the same (as proposed by Bastos Gomes et al., 2017b). In the present paper an epidemiological study has been carried out by characterizing and

Corresponding authors. E-mail addresses: [email protected] (G.-t. Wang), [email protected] (F. Ponce-Gordo).

https://doi.org/10.1016/j.vetpar.2018.10.009 Received 12 July 2018; Received in revised form 8 October 2018; Accepted 9 October 2018 0304-4017/ © 2018 Elsevier B.V. All rights reserved.

Veterinary Parasitology 264 (2018) 8–17

comparing the species causing several infections over several years in different fish farms located in Southern, Central and Eastern China.

2.1. Specimen collection Chilodonella spp. analyzed in this study were collected from five farms located alongside the course of the Yantze river or affluents (Table 1, Fig. 1). All hosts were juvenile or fingerling. In each farm, several infected fish were randomly sampled, examined and the number of parasites in their gills counted. Ciliates were scraped from the gills and skin mucus using Pasteur micropipettes with the aid of a stereoscopic microscope Stemi SV6/AxioCam MRc5 (Zeiss, Oberkochen, Germany). The Australian C. hexasticha used in this study was isolated originally from Asian sea bass (Lates calcarifer) from a commercial fish farm from North Queensland, Australia (Bastos Gomes et al., 2017b). 2.2. Morphological identification For the morphological identification, silver nitrate staining was performed on dry smears (Wellborn, 1967), while some other specimens were fixed in saturated HgCl2 solution and stained with protargol (Wilbert, 1975). The two methods were used to reveal different parts of the cells: silver nitrate was better for revealing the infraciliature while protargol was mainly used for the inner organelles such as the cyrtos and the nuclei. Thirty cells per sample were measured at 400× magnification and photographed using Axioplan 2 imaging and Axiophot 2 (Zeiss, Oberkochen, Germany). Moreover, to confirm the presence of the ciliates in the lesions, the first gill arch of the diseased fish was collected and fixed in a 10% buffered formalin solution for gross histopathological analysis. Gills were embedded in paraffin and sliced into 5 μm thick sections and stained with hematoxylin and eosin (HE) (Pádua et al., 2013). Slides 2014W021–026, 2015W010–018, 2016W001–015, 2017W013–019 of silver nitrate staining specimens and 2014W027–030, 2015W019–022, 2016W016–025, 2017W020–024 of protargol stained specimens have been deposited in the Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China.

15 °C Indoor Rock carp (Procypris rabaudi)

2. Materials and methods

2.3. Scanning electron microscope (SEM) For SEM, washed specimens were fixed in 2.5% glutaraldehyde in 0.2 M phosphate buffered saline (PBS, pH 7.4) on clean glass slides (1 cm × 1 cm). Glass slides were previously treated with 0.1% poly-Llysine and dried completely in air at room temperature (RT). After being washed with PBS three times, cells were post-fixed in 1% osmium tetroxide at 4 °C for 1 h, followed by serial dehydration in acetone and critical point drying using a Hitachi critical point dryer HCP-2 (Hitachi Science Systems, Ibaraki, Japan). Then glass slides were mounted on an aluminum stub using double-sided adhesive tape and sputter-coated with a thin layer of gold in an IB-3 ion coater (Eiko Engineering, Ibaraki, Japan) before observing and photographing with a Quanta 200 SEM (FEI, Amsterdam, Netherlands).

Liangshan City, Sichuan Province

May 2017

Outdoor May 2016

Crucian carp (Carassius auratus)

23 °C

Mortality ∼50%; fishes in the leeward side of the ponds; fish gills filled with chilodonellids. Outbreak after a 3-days raining; mortality ∼40%; fishes in the leeward side of the ponds; fish gills filled with chilodonellids. 10 tanks of fishes, mortality ∼50%. 24 °C

Hanchuan City, Hubei Province Jiangsu Province

2.4. DNA extraction, PCR and sequencing For each isolate, genomic DNA was extracted from 100 parasite cells using the REDExtract-N-Amp Tissue PCR Kit (Sigma, St. Louis, USA). PCR amplifications and sequencing were performed with primers indicated in Table 2. An EDC-810 DNA Engine (EastWin Bio., Co., Ltd.) was used to control the cycling conditions: denaturation for 4 min at 94 °C, followed by 40 cycles of denaturation for 30 s at 94 °C, primer annealing for 30 s at 54 °C (for SSU rDNA and ITS genes), 55 °C (for LSU rDNA gene) or 56 °C (for mtSSU rDNA gene), and extension for 2 min (SSU rDNA and mtSSU rDNA genes), 1 min (ITS gene) or 4 min (LSU

Jinping Hydropower Station

Hongze Lake Fish Hatchery

Ludila Fish Breeding And Releasing Station Diaocha Lake Fish Hatchery

August 2015

May 2016

Outdoor

Mortality reaching 100%; fish gills filled with chilodonellids. 18 °C Indoor

Ray-finned fish (Schizothorax wangchiachii) Chinese perch (Siniperca chuatsi)

Eight tanks of fishes, mortality ∼60%; fish gills filled with chilodonellids. Indoor Grass carp (Ctenopharyngodon idella) October 2014 Guanqiao Fish Hatchery

Wuhan City, Hubei Province Dali City, Yunnan Province

21 °C

Pond location Fish species Year Location Farm

Table 1 Data on the fish farms considered in the present study.

Water temp

Remarks

M. Li et al.

9

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Fig. 1. Location and data of the fish farms where samples were taken for the present study. Table 2 Primers used for PCR reactions and sequencing (F – forward; R – reverse). Primer

Sequence

Usage

Reference

MedlinA (F) MedlinB (R) P1 (F) NC2 (R) 28S-1F (F) XG28SF0 (F) XG28SF3 (F) XB28SR1 (R) 28S-4R (R) mtF (F) mtR (R)

5′-AACCTGGTTGATCC TGCCAGT 5′-TGATCCTTCTGCAGGTTCACCTAC 5′-GTTCCCCTTGAACGAGGAATTC 5′-TTAGTTTCTTTTCCTCCGCT 5′-ACCCGCTGAATTTAAGCAT 5′-GGAGTGTCTTCAAACTCACC 5′-CAGGGATAACTGACTTGTGGC 5′-AMCGGTTCCTCTCGT ACTRAG 5′-TTCTGACTTAGAGGCGTTCAG 5′-TGTGCCAGCAGCCGCGGTAA 5′-CCCCTACCGGTACCTTGTG T

SSU rDNA amplification and sequencing SSU rDNA amplification and sequencing ITS amplification and sequencing ITS amplification and sequencing LSU rDNA amplification and sequencing LSU rDNA sequencing LSU rDNA sequencing LSU rDNA sequencing LSU rDNA amplification and sequencing mtSSU rDNA amplification and sequencing mtSSU rDNA amplification and sequencing

Medlin et al. (1988) Medlin et al. (1988) Diggles and Adlard (1995) Sun et al. (2006) Moreira et al. (2007) This study This study This study Moreira et al. (2007) Dunthorn et al. (2014) Dunthorn et al. (2014)

rDNA gene) at 72 °C; and a final extension of 10 min at 72 °C. PCR products were isolated using 1% agarose gel electrophoresis and purified using the Agarose Gel DNA Purification Kit Ver. 2.0 (TaKaRa Biotechnology, Dalian). The amplified fragments were cloned into pMD-18T vector (TaKaRa Biotechnology, Dalian, China) and four positive clones were chosen for sequencing in both directions. Sequencing was made on an ABI PRISM® 3730 DNA Sequencer (Applied Biosystems, CA, USA).

region (SSU rDNA, ITS1-5.8S rDNA-ITS2, LSU rDNA, and mtSSU rDNA). For the identification of each region, sequences of the isolate from S. chuatsi were assembled into one single contig with ChromasPro ver. 2.01 (Technelysium Pty Ltd). The boundaries of the SSU rDNA-ITS15.8S rDNA-ITS2-LSU rDNA regions were identified by using the Rfam database (http://rfam.xfam.org/) (Kalvari et al., 2017). The boundaries of the 5.8S rDNA-ITS2-LSU rDNA regions were confirmed by identifying the ITS2 proximal stem (a hybridized 5.8S-28S rRNA fragment forming a characteristic ∼15 bp imperfect helix; Keller et al., 2009) by using the ITS2 Annotation feature (Keller et al., 2009) of the ITS2 database (http://its2.bioapps.biozentrum.uni-wuerzburg.de/) (Ankerbrand et al., 2015). Once the limits of each region were identified in the sequences of the isolate from S. chuatsi, they were also identified (by comparison) in the sequences from the other Chilodonella isolates obtained in this study and in the sequences retrieved from GenBank. The fragments corresponding to each region were aligned by using the ClustalW algorithm (Thompson et al., 1994) implemented in ClustalX 2.1 for Windows (Larkin et al., 2007), and alignments were refined by considering the secondary structures of the sequences. These structures were constructed by using those proposed for other protozoa (Cannone et al., 2002) as templates; for the variable and the non-homologous regions, the folding was predicted using the RNAfold WebServer (http://rna.tbi.

2.5. Sequence analysis and comparisons Sequences obtained in this study have been deposited in the GenBank/EMBL/DDBJ databases under the accession numbers MH341591, MH342041–MH342043, MH342045, MH341624, MH341631–MH341633 and MH356260–MH356271 (Table 3). Sequences of other Chilodonella species used in this study for comparison were retrieved from GenBank and indicated in Table 3. Sequence FJ873805 identified as Chilodonella cyprini (a synonym of C. piscicola) is considered unreliable (Deng et al., 2015) and it has been not included in this study. The number and length of sequences available in GenBank from other Chilodonella species vary depending on the gene considered (Table 3); therefore, analyses were performed separately for each 10

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Table 3 Chilodonella species sequences used in the present study. Species SSU-rDNA Chilodonella acuta Chilodonella hexasticha

Chilodonella parauncinata Chilodonella piscicola

Chilodonella uncinata

ITS1 – 5.8S-DNA – ITS2 Chilodonella hexasticha

Chilodonella piscicola

Chilodonella sp.

Chilodonella uncinata LSU-rDNA Chilodonella hexasticha Chilodonella piscicola mtSSU-rDNA Chilodonella acuta Chilodonella hexasticha

Chilodonella parauncinata Chilodonella piscicola

Chilodonella uncinata

Host/origin of the sample

Procedence

Sequence length

%GC

GenBank acc. number

Reference

Environment (soil) Lates calcarifer Carassius auratus Ctenopharyngodon idella Siniperca chuatsi Lates calcarifer Envirorment (freswater lake)

Saudi Arabia Australia China China China Australia China

1548 965 1709 1709 1709 1709 1709

45.5 46.0 44.9 44.9 44.9 44.9 44.7

KJ452458 KY508252 MH341591 MH342042 MH342041 MH342045 KJ509197

Fan et al. (2014) Bastos Gomes et al. (2017b) This study This study This study This study Qu et al. (2015)

Maccullochella peelii Schizothorax o’connori & Oxygymnocypris stewartii Procypris rabaudi Schizothorax wangchiachii Scenedesmus sp. culture

Australia Tibet

965 1592

45.8 44.7

KY508253 KR005625

Bastos Gomes et al. (2017b) Deng et al. (2015)

China China China

1709 1709 1704

44.9 44.9 44.7

MH342043 MH341624 KY476314

Culture contaminant Culture contaminant Culture contaminant Culture contaminant Culture contaminant Freshwater pond Freshwater pond Freshwater pond

ATCC:50194 ATCC:50194 ATCC:50194 ATCC:50194 Poland USA USA USA

1662 1662 1659 1612 1638 605 1047 1660

44.7 44.9 44.8 45.0 44.6 43.5 42.9 44.5

AF300281 AF300282 AF300283 AF300284 JN111976 JN111977 JN111978 JN111979

This study This study Wang, Zhan, Hu and Gong, unpublished Riley and Katz (2001) Riley and Katz (2001) Riley and Katz (2001) Riley and Katz (2001) Katz et al. (2011) Katz et al. (2011) Katz et al. (2011) Katz et al. (2011)

Lates calcarifer Carassius auratus Siniperca chuatsi Ctenopharyngodon idella Lates calcarifer Schizothorax o’connori & Oxygymnocypris stewartii Maccullochella peelii Maccullochella peelii Procypris rabaudi Schizothorax wangchiachii Freshwater sources

Australia China China China Australia Tibet

271 390 390 390 390 390

41.0 40.8 40.8 40.8 40.8 40.8

KY508248 MH356260 MH356264 MH356262 MH356261 KR005626

Bastos Gomes et al. (2017b) This study This study This study This study Deng et al. (2015)

Australia Australia China China South Africa

271 271 390 390 180

41.0 41.0 40.8 41.0 41.1

KY508249 KY508250 MH356263 MH356270 KC215927

Freshwater sources

South Africa

173

40.5

KC216026

Culture contaminant Lates calcarifer

ATCC:50194 Australia

390 271

41.0 41.0

EU047813 KY508251

Bastos Gomes et al. (2017b) Bastos Gomes et al. (2017b) This study This study Jordaan and Bezuidenhout, unpublished Jordaan and Bezuidenhout, unpublished Robinson and Katz (2007) Bastos Gomes et al. (2017b)

Siniperca chuatsi Lates calcarifer Schizothorax wangchiachii

China Australia China

3084 3082 3082

47.9 48.2 48.2

MH341631 MH341633 MH341632

This study This study This study

Not available Lates calcarifer Lates calcarifer Carassius auratus Siniperca chuatsi Ctenopharyngodon idella Lates calcarifer Not available

China Australia Australia China China China Australia China

894 467 470 944 944 944 944 892

24.9 28.5 28.9 28.1 28.1 28.2 28.1 29.0

KX302683 KY508247 KY508243 MH356265 MH356271 MH356266 MH356269 KX302672

Wang et al. (2017) Bastos Gomes et al. (2017b) Bastos Gomes et al. (2017b) This study This study This study This study Wang et al. (2017)

Maccullochella peelii Maccullochella peelii Procypris rabaudi Schizothorax wangchiachii Freshwater lake

Australia Australia China China Poland (ATCC:PRA256) USA ATCC:50194 USA USA Australia

470 470 944 943 894

28.9 28.9 28.3 28.3 25.5

KY508244 KY508245 MH356268 MH356267 HM246404

Bastos Gomes et al. (2017b) Bastos Gomes et al. (2017b) This study This study Dunthorn et al. (2011)

894 895 895 902 469

25.4 26.4 26.5 24.7 28.8

JN111980 JN111981 JN111982 JN111983 KY508246

Katz et al. (2011) Katz et al. (2011) Katz et al. (2011) Katz et al. (2011) Bastos Gomes et al. (2017b)

Freshwater pond Culture contaminant Freshwater pond Freshwater pond Lates calcarifer

sequence relationships, unrooted phylogenetic trees were constructed for each gene by maximum likelihood (ML) and maximum parsimony (MP) methods in MEGA-X (Kumar et al., 2018). For ML analysis, the best model of nucleotide evolution was selected based on the Akaike Information Criterion (Akaike, 1974) and the Bayesian Information

univie.ac.at//cgi-bin/RNAWebSuite/RNAfold.cgi) (Gruber et al., 2008) with the default options. Sequence-structure edition and alignment, and comparisons among Chilodonella sequences were made with the 4SALE program (Seibel et al., 2008). For esthetic purposes, structure diagrams have been drawn with RNAviz 2.0 (De Rijk et al., 2003). For analysis of 11

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Fig. 2. Light microscope images of Chilodonella hexasticha from crucian carp (Carassius auratus) as representative of the general morphology of Chilodonella sp. (A) Living specimens, to show a large presence of chilodonellids rotating forward rapidly in the water. Scale bar in A = 50 μm. (B) Living specimens, to show a chilodonellid creeping slowly on the gill. Contractile vacuole (CV) and macronucleus (Ma) are visible. Scale bar in B = 10 μm. (C) Specimens stained with protargol, to show the general morphology of chilodonellids, right/short (R) and left/ long (L) kinety bands and Cytostome (Cy). Scale bar in C = 10 μm. (D). Specimens stained with silver nitrate, to show the infraciliature of chilodonellids, consisting of preoral kinety (P), outer (OC) and inner (IC) circumoral kinety, and two parts of somatic kineties positioned on right (R) and left (L) respectively. The left part of kineties (arrow) are divided earlier than the right ones (double arrow), with its inner kinetofragments forming the new circumoral kineties (arrowhead). Scale bar = 10 μm.

The single macronucleus is oval in shape and centro-posteriorly located (Fig. 2C). The number of kinety rows present in the left and right bands is a crucial characteristic that distinguishes C. hexasticha from C. piscicola. Chilodonella samples collected in the present study have been identified as follows: chilodonellids isolated from C. idella in late October 2014, from S. chuatsi in early May 2016 and from C. auratus in late May 2016 were identified as C. hexasticha, possessing seven right kineties (ranging from 6 to 7) and nine left kineties (ranging from 8 to 10) (Fig. 5A and C); chilodonellids isolated from S. wangchiachii in midAugust 2015 and from P. rabaudi in late May 2017 were identified as C. piscicola, having more numerous and more closely spaced kineties, thus ten right kineties (ranging from 8 to 11) and twelve left kineties (ranging from 10 to 15) (Fig. 5B and D). The C. hexasticha isolates have been found in fish grown in both indoor and outdoor facilities at farms located at low altitude, under 30 m above sea level and with a water temperature of 21–24 °C, while the C. piscicola isolates have been found in fishes grown in indoor facilities at farms located at altitudes above 1500 m and with a water temperature of 15–18 °C (Table 1). In the analysis of the SSU rDNA gene, no differences were found between sequences of C. piscicola retrieved from GenBank and those of the isolates from the S. wangchiachii and P. rabaudi (morphologically identified as C. piscicola). In addition, there were no differences between C. hexasticha retrieved from S. chuatshi, C. idella and the Australian L. calcarifer (morphologically identified as C. hexasticha). The complete SSU rDNA gene was sequenced (for C. piscicola and C. hexasticha), and it comprises 1709 nucleotides in length. The GC content was 44.9%, which is within the range observed for the sequences retrieved from GenBank (42.9–46.0%) (Table 3). In general, all Chilodonella sequences have a high similarity; in the alignment (1717 positions) we have found 1632 conserved positions, this meaning 95.1% of the total. Between C. hexasticha and C. piscicola, the complete sequences have only 5 base differences (99.7% similarity) (Suppl. Fig. 1). In the phylogenetic trees (for ML: T92 + G + I, G = 0.1686, I = 47.64% sites), the sequences were placed into two main groups, one including the C. piscicola and C. hexasticha sequences and the other including the C. uncinata sequences; those of C. acuta and C. parauncinata are placed apart (Fig. 6). Although the sequences of C. piscicola and C. hexasticha

Criterion (Schwarz, 1978) calculated in MEGA-X; in all cases, the tamura 3-parameter model (Tamura, 1992) with a discrete Gamma distribution and a proportion of invariable sites (T92 + G + I) were selected. In both methods (ML and MP), data were bootstrap resampled 1000 times to estimate the relative branch support. 3. Results In all outbreaks, the affected animals presented dark colored back and anorexia, lack of balance and a gasping behavior at the water surface. Fish mortality varied depending on the farm, from 40% to almost 100% (Table 1). Microscopical examination revealed large presence of chilodonellids, rotating forward rapidly in the water (Fig. 2A) or creeping slowly on the gill, body surface and fins (Figs. 2B and 3A ). The clinical and pathological findings in the infected fish were similar in all cases: host fry exhibited skin ulceration, excessive mucus production and gill lesions characteristic of necrosis. Numerous chilodonellids were scattered over gill filaments (Fig. 4A and B) and located between or at the tips of gill lamellae (Fig. 4C and D), resulting in severe fusion of lamellae and desquamation of epithelium cells (Fig. 4B). Necrosis of the epithelium between gill lamellae was evident in some areas (Fig. 4D). All Chilodonella specimens identified in this study were dorsally vaulted, ventrally flattened, oval in outline, left margin somewhat straight while right margin cambered (Figs. 2B–D, 3B and 5A and B). Terminal fragment containing 16–19 kinetosomes, positioned on top left of dorsal side (Fig. 3C). Cytostome subapically located and transverse ellipse-shaped. Cyrtos hook-like, composed of 9–11 toothed nematodesmal rods and curved in a circle posteriorly (Figs. 2C and 5D ). Two circumoral kineties parallel to each other, with outer one slightly longer than inner one (Figs. 2D and 5C and D). One preoral kinety encircling circumoral kineties and extending to anterior-left of cell (Figs. 2D and 5C and D). The infraciliature of chilodonellids usually consists of two parts of somatic kineties, positioned on right and left side, respectively (Figs. 2C and D and 3D). Left part of kineties are divided earlier than the right ones (Fig. 2C), with its inner kinetofragments forming the new circumoral kineties and preoral kinety (Fig. 2D). 12

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Fig. 3. SEM images of Chilodonella hexasticha from crucian carp (Carassius auratus) as representative of the general ultrastructure of Chilodonella sp. (A) to show a chilodonellid (arrow) attached to the gill surface. Scale bar = 50 μm. (B) Overview of the dorsal side, showing the shape as oval in outline and dorsally vaulted, with left margin somewhat straight and right margin cambered. Scale bar = 5 μm. (C) Selective magnification of figure B, showing the terminal fragment kinety (dorsal bristles). Scale bar = 2.5 μm. (D) Overview of the ventral side, showing the body ciliary rows. Scale bar = 5 μm.

isolates of C. hexasticha were identical to that of C. piscicola from P. rabaudi and to those previously published of C. hexasticha, C. piscicola and C. uncinata (except sequence EU047813, which had one difference in the ITS1 region). The sequence of C. piscicola from S. wangchiachii had one base difference in an unpaired position of the 5.8S rDNA fragment (Suppl. Fig. 2). The main differences were found in the sequences of Chilodonella sp. (KC215927 and KC2160269) which showed

were shown in the trees as two separate subgroups, only the C. piscicola subgroup demonstrated bootstrap support (ML: 39%; MP: 96%); the bootstrap values for the C. hexasticha subgroup were very low (ML: the C. hexasticha sequences did not form a clear group; MP: 64%). The ITS region was 390 bases in length (ITS1, 73 bases; 5.8S rDNA, 158 bases; and ITS2, 159 bases) and the similarity among the different species was almost 100%. All sequences obtained in this study from the

Fig. 4. Histopathological sections of crucian carp (Carassius auratus) gills stained by hematoxylin and eosin (HE) to show the findings found in all outbreaks. Chilodonellids were scattered over gill filaments (A and B, arrows) and located between or at the tips of lamellae (C and D, arrows), resulting in severe fusion of lamellae (B and C, yellow double arrow) and desquamation of epithelium cells (B, arrowhead). Necrosis of the epithelium between gill lamellae was evident in some areas (D, asterisk). Scale bars in A = 200 μm, in B = 100 μm, in C = 50 μm, in D = 50 μm.

13

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Fig. 5. Light microscopy images of Chilodonella hexasticha (from Carassius auratus) and C. piscicola (from Schizothorax wangchiachii), to show their morphological differences. Chilodonella hexasticha (A: living specimen) is somewhat smaller than C. piscicola (B: living specimen). Chilodonella hexasticha has seven kineties in the right band of the ciliature and nine in the left band (C: silver-nitrate stained specimen), while C. piscicola has 10 or 11 kineties on the right side and 12 or 13 on the left (D: protargol stained specimen). Moreover, the kineties of C. hexasticha are more loosely arranged when compared with C. piscicola in which they form regular rows close to each other. Arrowheads in figure C and D mean preoral kinety and arrow in figure D means the cyrtos composed of nematodesmal rods. Scale bars = 10 μm.

hexasticha were identical, 944 bases in length, except one base difference in the sequence of the isolate from C. idella. There were 36 base differences (3.8%) between sequences of C. piscicola and C. hexasticha, mainly located in helices 23m1 (ES6), 29 (ES7), 44 (ES9) and 49 (ES12) (Suppl. Fig. 3). Sequences of C. piscicola available in GenBank (KY508244 and KY508245) were identical to those of C. hexasticha from the present study. When comparing the mtSSU rDNA sequences between different species, a high variability was found and there were only 658 conserved positions (68.7%) in the alignment (958 positions it total). Most of the variable sites were located in the helices 23m1 (ES6), 29 (ES7), 37, 37m1 (ES8), 43, 44 (ES9), 46 (ES10) and 49 (ES12) (data not shown). GC content was similar among sequences, varying in the range 24.7–29.0% (Table 3). In the phylogenetic trees (ML: T92 + G + I, G = 0.7773, I = 50.04% sites), C. piscicola and C. hexasticha were within the same branch but as different subgroups with high support (100% in ML and MP); C. uncinata sequences were placed in a sister branch, and C. acuta and C. parauncinata were placed separately (Fig. 6).

4 and 10 base differences, respectively, located in the ITS1 region (only one base difference in their 5.8S rDNA region). The GC content varied from 40.5 to 41.1%; in the sequences obtained in this study, the GC content was 40.8% (Table 3). Due to the high similarity between sequences, the phylogenetic trees (ML: T92 + G + I, G = 200.0000, I = 0.00% sites) were not informative (Fig. 6). Up to the present study, there were no sequences of the LSU rDNA available in GenBank. Numerous attempts with different primer combinations were made until we successfully amplified this region with the primers indicated in material and methods; due to non-sufficient DNA extract from the isolates of C. idella, C. auratus (corresponding to C. hexasticha) and P. rabaudi (corresponding to C. piscicola), LSU rDNA sequences were not obtained for these isolates. The sequence of the isolate from S. wangchiachii (corresponding to C. piscicola) was 3082 bases in length, while those corresponding to C. hexasticha have 3082 (from the Australian isolate) and 3084 bases (from S. chuatshi). There were 14 differences (0.45%, including one indel) between the sequences of C. hexasticha, scattered thorough the complete sequence and not related to variable regions. Apart from these differences, the sequence of C. piscicola had 22 base differences with C. hexasticha, half of which are located in the helix E21e1 (this belonging to the high variable expansion segment 27, ES27) (Suppl. Fig. 2). The GC content varied from 47.9% (in C. hexasticha from S. chuatshi) to 48.2% (in C. piscicola and the Australian isolate of C. hexasticha) (Table 3). As only three sequences from LSU rDNA region were available, phylogenetic analysis was not performed. The mtSSU rDNA sequences corresponding to isolates identified as C. piscicola were almost identical (one indel in a poly-T fragment), being of 943–944 bases in length (for the isolates from S. wangchiachii and P. rabaudi, respectively). All sequences of the isolates identified as C.

4. Discussion In the present study, five chilodonellosis outbreaks occurred in different Chinese fish farms in the last years have been investigated and a comparative analysis of the species involved have been done by considering their morphological features and their macronuclear and mitochondrial ribosomal genes. Present results confirm that C. hexasticha and C. piscicola are different species; although the clinical signs experienced by fish affected by these ciliates are similar, each species is related to different water temperatures. For their genetic differentiation, novel data about the LSU rDNA genes are provided; also, the 14

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Ichthyophthirius), the correct diagnosis should be based on the identification of the etiological agent. Once the parasite has been identified as belonging to the genus Chilodonella, the correct identification of the species (C. hexasticha or C. piscicola) is not important for the chemical treatment of the infected fish (both species are treated the same way, although the chemicals used may have a low effectiveness and present adverse effects; Bastos Gomes et al., 2017a); however, it is important for epidemiological studies. It appears to exist a relationship between water temperature and the Chilodonella species involved in the infection (Hoffman et al., 1979; Sharif, 1984; Rintamäki et al., 1994; Bastos Gomes et al., 2017a). Our present results also support this consideration, as C. piscicola has been found only in the fish farms located at higher altitudes and with cooler water. However, the water temperature could be considered as suggestive for a given species but is not a definitive differential criterion, as the range for both species is wide (greater in C. piscicola) and overlap (Kazubski and Migala, 1974; Rintamäki et al., 1994; Bastos Gomes et al., 2017a). Usually, the Chilodonella species infecting fishes have been identified by their morphology (see Bastos Gomes et al., 2017a). The main differential characters are the cell size and, especially, the number of ventral kineties (Kazubski and Migala, 1974). The cell size is highly variable (31–76 μm in length and 25–72 μm in width for C. hexasticha, 41–79 μm in length and 28–66 μm in width for C. piscicola; Rintamäki et al., 1994) although in general, the C. hexasticha cells are smaller. Then, the criterion commonly used is the number of body kineties: between 5–9 in the right side and 6–11 in the left side for C. hexasticha (Kazubski and Migala, 1974; Langdon et al., 1985; Wiles et al., 1985; Rintamäki et al., 1994; Bastos Gomes et al., 2017b; present results) and between 6–14 in the right side and 8–16 in the left side for C. piscicola (Krascheninikow, 1953; Kazubski and Migala, 1974; Wiles et al., 1985; Rintamäki et al., 1994; Deng et al., 2015; present results). The kineties of C. hexasticha are more loosely arranged when compared with C. piscicola, in which they form regular rows close to each other (see Fig. 3C and D in the present study). As the ranges of cell size and number of kineties overlap between both species, mistakes may occur especially if the identifications are based on a low number of cells. In China, both C. hexasticha (Hu, 2012, 2015) and C. piscicola (=C. cyprini), (Xiao and Li, 1995; Deng et al., 2015) have been previously recorded; however, the identifications made by Hu (2012) (as C. hexasticha) and Xiao and Li (1995) (as C. piscicola = C. cyprini) are controversial, as the number of kineties are in the overlapping ranges but the cell sizes are clearly different of the normal ranges for each species: Hu (2012) gave values of 60.5–91.6 × 53.9–77.8 μm (mean, 75.5 × 62.2 μm) for C. hexasticha, and Xiao and Li (1995) gave values of 27–38 × 23–31 μm (mean 32.8 × 27.0 μm) for C. piscicola. The genetic analysis would allow overcoming the problems related to the morphological variability within the species of Chilodonella and possible misidentifications. This approach has shown the variability within the morphospecies C. uncinata and the possible existence of cryptic species (Katz et al., 2011). However, the genetic data available about the species infecting fishes is very scarce and limited to those provided by Deng et al. (2015) and Bastos Gomes et al. (2017b). Our present genetic results coincide with those of Deng et al. (2015) and partially with those by Bastos Gomes et al. (2017b); discrepancies with these latter authors are related to the results for C. piscicola. We have found clear genetic differences that allow the distinction between C. piscicola and C. hexasticha and support their consideration as distinct species, while Bastos Gomes et al. (2017b) proposed they are synonyms. This discrepancy is probably due to an incorrect morphological identification of their C. piscicola isolate, being in fact C. hexasticha (their identification was based on the analysis of only three cells and both the cell size and number of kineties fall within the range for C. hexasticha). In relation to the genetic markers that have included in this study, the two genes usually considered for the identification of species (the SSU rDNA and the ITS) have very little or no variability between C.

Fig. 6. Unrooted phylogenetic trees obtained by the maximum parsimony (MP) analysis of the SSU rDNA, the ITS1-5.8S rDNA-ITS2, and the mtSSU rDNA of the Chilodonella sequences considered in this study. Bootstrap values obtained in the Maximum Likelihood and MP analyses are indicated, respectively, at branch nodes; values below 30 are marked as *.

secondary structure models of the macronuclear and mitochondrial rRNAs have been constructed to analyze the importance of the differences found and as a help for improving alignments and for comparative analysis between Chilodonella species. Chilodonellosis is a serious problem for the fish farming industry, and infections and outbreaks occur from time to time, causing high mortalities (Urawa and Yamao, 1992). The pathological findings found in the present work are similar to those already published for C. piscicola (Urawa and Yamao, 1992) and C. hexasticha (Paperna and Van As, 1983; Sharif, 1984; Langdon et al., 1985; Pádua et al., 2013). The damage caused by the parasite in the skin produces its color alteration, increased mucus secretion and scale loss, but the most severe lesions are those produce in the gills, where epithelial alteration, lamellae fusion and necrosis could produce the death of the infected fish (Urawa and Yamao, 1992; Pádua et al., 2013). It is probably that the gill damage and the consequent respiratory stress are responsible for the abnormal behavior of the animals (unbalanced swinging in the water surface, reduced growth and anorexia) (Lom and Dyková, 1992). As all these lesions and the associated symptoms are also presented in diseases produced by other organisms (i.e., Costia, Trichodina or 15

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hexasticha and C. piscicola. However, as shown by other authors, the mtSSU rRNA is a valid tool for species identification (Dunthorn et al., 2011, 2014; Yang et al., 2014) and it has been applied in taxonomic studies on Chilodonella (Katz et al., 2011; Bastos Gomes et al., 2017b); present results indicate that this gene is valid for an undoubtful identification of C. hexasticha and C. piscicola. In relation to the LSU rDNA, there are no previous data available for the use of this gene in this genus. The variability found in the LSU rDNA between Chinese and Australian isolates of C. hexasticha suggests the existence of subspecific variants (i.e., races or strains); the fact that the base differences are scattered thorough the gene (in both conserved and variable regions) and the lack of differences in other markers (especially in the mtSSU rDNA gene) and in the morphology support this consideration. Despite the variability found within C. hexasticha and the general low percentage of base differences in the entire LSU rDNA gene respect to C. piscicola (22 out of 3082 bases, 0.71%), this marker could be useful for species identification. Half of the base differences between the C. piscicola–C. hexasticha sequences are located in a same helix (E21e1), they account for 7.6% of the sites in the ES27 (10 out of 132 bases in this expansion segment) and there is a compensatory base change (a change in both bases of a pair, while the pair itself is maintained) between the sequence-structure of both species (1976C-2021G in C. hexasticha, 1976U-2021A in C. piscicola) (Suppl. Fig. 2). This fragment within the LSU rDNA gene could be used in epidemiological studies on these two species, but its validity in taxonomic studies within the genus Chilodonella should be confirmed by the analysis of isolates from other species.

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