Clinico-histopathological and phylogenetic analysis of protozoan epibiont Epistylis wuhanensis associated with crustacean parasite Lernaea cyprinacea from ornamental fish in Iran

Parasitic infestations are one of the most concerning problems limiting ornamental fish farming. In addition to the direct economic losses due to the major mortalities, parasites may significantly negatively impact the body shape, coloration, growth rate, and health condition of the fish. The results of the study highlight the importance of comprehensive parasitological analysis in the diagnosis and treatment of parasitic infections in ornamental fish farms. The presence of multiple parasites in the affected fish emphasizes the need for effective biosecurity measures, such as extending the quarantine period for newly imported fish, closely monitoring fish populations, and implementing isolation units to prevent the spread of infections. By implementing these preventative measures, ornamental fish farmers can reduce the risk of parasitic infections and ensure the health and well-being of their fish populations. This, in turn, can lead to increased profitability and sustainability for their business. Overall, the current study aimed to conduct a clinical, histopathological, and phylogenetic analysis of the epibiont ciliated protozoan Epistylis wuhanensis and the copepod crustacean Lernaea cyprinacea in a freshwater ornamental fish farm in Iran. Furthermore, it provides valuable insights into the prevalence and impact of parasitic infections in ornamental fish farms and underscores the need for continued research and the development of effective preventative measures to address this issue. A total of 60 symptomatic freshwater ornamental fish, including 30 guppy (Poecilia reticulata) and 30 sailfin molly (Poecilia latipinna), were packed in polyethylene bags filled with oxygenated pond water and transported to the Ornamental Fish Clinic, Faculty of Veterinary Medicine, University of Tehran, for parasitological analysis. Following the clinical examination, histopathological analysis was performed on 10% NBF (neutral buffered formalin)-fixed samples from affected tissues, including the skin, skeletal muscle, and liver, to identify any pathological changes associated with the parasitic infections. Furthermore, the DNA was extracted from the 99% ethanol-fixed samples using a commercial DNA extraction tissue kit (SinaPure DNA, Iran), and PCR was performed using Peri18S-F1 (5′-ACC TGG TTG ATC CTG CCA GT-3′) and Peri18S-R1 (5′-TGC AGG TTC ACC TAC GGA AA-3′) (first reaction), and Peri18S-F2 (5′-CCG CGG TAA TTC CAG CTC-3′) and Peri18S-R2 (5′-GAT CCC CTA ACT TTC GTT CTT GA-3′) (second round) primers for the identified parasites. Finally, the PCR products were sequenced using Sanger dideoxy sequencing methods, and the resulting sequences were compared to sequences in the BLAST search program to provide a comprehensive picture of the current parasite-based disorder. The crustacean L. cyprinacea and the epibiont sessilid E. wuhanensis were identified in the examined ornamental guppy (6/30) and sailfin molly (6/30), with an overall parasitic prevalence of 20.00% (12/60). Ciliates were found in all tissue lesions but not in fish without lesions. A great number of the ciliated protozoan E. wuhanensis were found attached to the integumentary area of L. cyprinacea. Microscopically, oval to round granulomatous lesions were observed in cutaneous and skeletal muscles. Lymphoplasmacytic dermatitis and myositis were also observed. The crustacean L. cyprinacea serves as a mechanical vector for E. wuhanensis infection and spreads the disease in ornamental fish farming operations. For the first time in Iran, we successfully presented diagnostic morphological and molecular data on sessilids isolated from L. cyprinacea. Based on the findings of the current study, such parasitic infections may cause significant economic losses following invasion of the integument area of fish, eventually leading to death, if treatment is neglected or inadequate. Furthermore, the findings of the analysis were used to develop effective diagnostic approaches for the affected fish, as well as recommendations for improved health conditions to prevent future outbreaks of parasitic infections. However, further research is needed to determine the precise mechanisms of crustacean attachment and host-crustacean-peritrich protozoan interactions. Furthermore, the direct and indirect effects of various environmental factors on the emergence and spread of the current disease should be considered.

Ornamental fish farming is one of the industries that has grown significantly in recent years and benefits from a high annual turnover [1][2][3] .According to recent statistics on the top exporter continents of ornamental fish, Asia ranked first (57% of total world exports), as well as Singapore, Japan, the Czech Republic, Thailand, Malaysia, Indonesia, Israel, Brazil, Sri Lanka, and Columbia, which were announced as the top 10 exporters of ornamental fish.On the other hand, the United States, United Kingdom, Germany, Singapore, Japan, China, France, the Netherlands, Italy, and Malaysia were announced as the top 10 importers of ornamental fish 4 .Although a large portion of imported ornamental fish was previously allocated to East Asian countries 5 , the trade of ornamental fish has significantly improved in recent decades and has evolved into an economically profitable industry among Iranian enthusiasts.Iran has long been a major importer of ornamental fish, and the majority of its imports come from Japan, Singapore, Thailand, and Indonesia.However, in the last 20 years, the cultivation and breeding of ornamental fish have increased dramatically [6][7][8][9] .
Parasitic infestations are one of the most concerning issues limiting ornamental fish farming and, as a result, affecting the global aquaculture industry 6,7,10,11 .Essentially, transferring freshwater ornamental fish from the wild to size-limited environments may increase the risk of parasitic epidemics in ornamental fish farms.Furthermore, failure to adhere to hygienic protocols during the temporary maintenance period of imported fish in wholesalers' and importers units may be regarded as a booster for the occurrence of parasitic diseases 12,13 .Some parasites only infiltrate the outer surfaces of the various organs.Other parasites, on the other hand, can penetrate the parenchyma of the host's various tissues 14 .Parasites may have a significant negative impact on the body shape, body weight, coloration, growth rate 15,16 , health condition 17 , and reproductive function of the fish, in addition to direct economic losses due to major mortalities 16,18 .
Copepod crustaceans are one of the most significant parasitic pathogens 17,19 found in ornamental fish farms and natural habitats 17,20,21 .Lernaea cyprinacea Linnaeus, 1758, also known as the anchor worm, is a highly modified and widely distributed copepod crustacean in the Lernaeidae family, which includes more than 14 genera and 110 species 22 .As a relatively immobile parasite, L. cyprinacea is capable of attaching to the gills as well as the skin 23 , causing tissue lesions such as epithelial hyperplasia, telangiectasis, hemorrhage 24 , and gill epithelium necrosis and disruption, which can lead to death in both marine and freshwater ornamental fish 22,23 .Because of the increasing prevalence of this parasite in various species of ornamental fish all over the world, the economic importance of the crustacean L. cyprinacea has been increasingly considered 25 .Lernaea cyprinacea 17,[26][27][28][29] and other parasitic crustaceans [30][31][32] may also provide substrate for epibionts such as Epistylis.The ability to be passively transported with a clear increase in feeding, improvement of food capturing efficiency 33 and feeding rate 34 , and a decrease in predation 35,36 are the main benefits for epibionts.
Despite the occasional reports of parasitic infections caused by peritrich ciliate attachment to the anchor worm, in a wide range of freshwater fish species in different geographical regions of the world (Table 1), the present study aimed to fill the knowledge gap regarding parasitic infections caused by the attachment of peritrich ciliates to L. cyprinacea in ornamental fish farms in the Middle East, specifically in Iran.The study used a combination of clinical, histopathological, and phylogenetic analyses to identify and characterize the parasites in question.By doing so, the study aimed to provide a better understanding of the prevalence, pathology, and genetic relatedness of these parasites in ornamental fish farms in the region.

Methods
Fish and sampling.During the period from October 2020 to September 2021, listlessness, anorexia, weakness, and the flashing of guppy (Poecilia reticulata) and sailfin molly (Poecilia latipinna) were accompanied by mass mortalities (31% and 42%, respectively), leading to significant economic losses in an ornamental fish farm located in the Esfahan province of Iran (33.3250°N, 53.3906° E) (Fig. 1).A total of 60 symptomatic freshwater ornamental fish, including 30 guppy and 30 sailfin molly (a length of 2-2.5 cm), were packed in polyethylene bags filled with oxygenated pond water and transported to the Ornamental Fish Clinic, Faculty of Veterinary Parasitological analysis.A thorough macroscopic examination of the body surface and fins performed at the Ornamental Fish Clinic to verify any parasites, lesions, or alterations revealed the clear attachment of hookworms to the external surfaces of the fish (Fig. 2a,d).Following the owner's consent, fish were anaesthetized in 100 ppm PI222 (the major active ingredients of which are eugenol, carvacrol, and eugenol acetate) (Pars Imen Daru, Iran), and wet mounts of scrapings (of the body surface and dorsal, pectoral, ventral, and caudal Squalidus gracilis gracilis Peritrich ciliates -Japan Nagasawa et al. 41

Oryzias latipes
Peritrich ciliates -Japan Nagasawa et al. 42 Peritrich ciliates -Japan Nagasawa et al. 43 Peritrich ciliates -Japan Nagasawa et al. 44 Peritrich-like ciliates -Japan Beatte et al. 45 Peritrich-like ciliates -Japan Nagasawa et al. 46 Poecilia  fins) were prepared and used for microscopic observation of parasites under a light microscope (Nikon E600, Japan) (Figs. 2c and 3a-d) and a trinocular stereomicroscope (Olympus SZ60, Japan) (Fig. 2a,b).A microscopic examination of fish revealed some peritrich ciliated protozoans that were firmly attached to copepod crustaceans.The use of fresh fish samples may aid in the visualization of peritrich parasite motivation 37,38 .Copepod crustaceans were clarified with lactophenol and identified according to keys described by Damaree 39 , and Lester and Hayward 23 .In addition, the Plustek OpticLab H850 slide scanner was used to scan carmen-stained sections of the crustacean L. cyprinacea and attached E. wuhanensis (Fig. 4a-c).

Histopathological analysis.
For histological examinations, integumentary lesions of fish were dissected and fixed in 10% neutral buffered formalin, dehydrated in an ethanol series, and embedded in paraffin with a paraffin tissue processor and paraffin dispenser.Several sections were cut at 4 µm, and stained with haematoxylin-eosin (H&E).Sections were examined by light microscopy (Nikon E600, Japan), and representative images were taken using an IDS UI-2250 microscope camera (IDS imaging).
The cycling conditions included an initial denaturation at 94 °C for 5 min, followed by 35 cycles at 94 °C for 30 s, 54 °C for 30 s, 72 °C for 60 s, and 72 °C for 5 min as the final extension.The amplification products (5 µl) were resolved by electrophoresis on a 1.5% agarose gel in 1X TBE buffer for 1 h at 100 V. Afterwards, the agarose gel was stained with 1 µg/ml ethidium bromide (CinnaGen, Iran) to visualize PCR Products under UV light.The gel was screened using a UV-transilluminator (BIORAD, UK) to visualize the DNA fragments.The presence of the DNA was confirmed by comparing the size of the PCR products with a DNA ladder of known sizes run alongside the PCR products on the gel.
Phylogeny and sequence analysis.The phylogenetic analysis was performed to determine the evolutionary relationships between the strains of L. cyprinacea and Epistylis wuhanensis isolated from guppy (Poecilia reticulata) and sailfin molly (Poecilia latipinna).The strains were sequenced by the Macrogen Company in South Korea, and Sanger dideoxy sequencing methods were used to obtain these sequences.Analyzed individually using BioEdit version 7 was used for gene annotation and sequence trimming 42 .High-quality sequences (determined by the size of the fluorescent signal and the clarity of the peak) obtained from the study, were analyzed using the BLAST search program (https:// blast.ncbi.nlm.nih.gov/ Blast.cgi), and submitted to BankIt.Bootstrap values were calculated in MEGA7 based on 1000 replicates to assess the statistical support for each node.The accession numbers are OP175983 for L. cyprinacea isolate RS13 and OP175994 for E. wuhanensis isolate RS14.Each of the gene sequences of Sessilids was retrieved from GenBank, and a FASTA dataset was generated.Multiple sequence alignments were generated by ClustalW and then used to generate distance matrices using the General Time Reversible (GTR) model implemented in MEGA software version 7 43,44 .Finally, the Maximum Likelihood (ML) trees were plotted by MEGA7 utilizing a 1,000-fold bootstrap approach, as prescribed by Kumar et al. 43 and Nei and Kumar 44 .
Ethics approval and consent to participate.In the current study, clinical records were provided following owner consent, and the data were securely stored.In addition, ethical approval for this study was granted by the University of Tehran Veterinary Ethical Review Committee.All methods were performed in accordance with the guidelines and regulations of the University of Tehran Veterinary Ethical Review Committee.Also, the study is reported in accordance with ARRIVE guidelines.Written informed consent was obtained from the owner for the participation of the animal in the study.

Results
Clinical findings and laboratory examinations.The crustacean L. cyprinacea and the epibiont sessilid ciliate E. wuhanensis were identified in the examined ornamental guppy (6/30) and sailfin molly (6/30), with an overall parasitic prevalence of 20.00% (12/60) (Fig. 2a,b,d).Ciliates were found in all tissue lesions but not in fish without lesions.These sessile ciliates were identified and assigned to the subclass Peritrichia based on morphological characteristics such as the absence of feeding tentacles and the presence of a cytopharynx.They were also classified as Epistylididae due to the presence of a contractile body, a non-contractile stalk, the absence of aboral arms, and the absence of a stalk on the peristomal disc (Fig. 3d).They were assigned to Epistylis due to their colonial nature and three-turning oral cilia (Fig. 3d).A great number of the ciliated protozoan E. wuhanensis were found attached to the integumentary area of L. cyprinacea (Figs.2c and 3a,b).
Histopathological findings.Tissue lesions of variable severity were present in all parasitized fish.Microscopically, oval to round granulomatous lesions were observed in cutaneous and skeletal muscles.These lesions caused pressure atrophy of the adjacent skeletal muscle fibers.Lymphoplasmacytic dermatitis and myositis were also observed (Fig. 5a,b).In granulomatous structures, cross sections of parasite and necrotic cell debris were seen in the central part.The parasite was surrounded by a dense zone of inflammatory cells.There were macrophages, lymphocytes, plasma cells, and eosinophils in the inner area (Figs.5c and 6a).The outermost area surrounding the granuloma consisted of fibroblasts (Fig. 5c).Hemorrhage was also observed in skeletal muscle around the granulomas (Fig. 5d).Finally, a chronic and parasitic eosinophilic granulomatous inflammation was diagnosed.Adhesion of E. wuhanensis and algae in the cephalothorax and trunk of the crustacean L. cyprinacea was observed in histological sections (Fig. 6b-d).

Molecular findings.
The results of the BLAST search conducted on the newly obtained partial 18S rDNA sequences (OP175994 and OP175983) confirm the identification of the epibiont ciliate E. wuhanensis and the copepod crustacean L. cyprinacea in the examined ornamental fish.The sequences were found to be identical, by original description, to the reference sequences KU869709 and KY435939, respectively, with 100% coverage (Figs. 7 and 8; Tables 2 and 3).
The analysis of L. cyprinacea isolates RS13 and OP175994 revealed high identity (up to 95%) with the reference sequences KP235363.1,DQ107557.1,KY435939.1,and DQ107556.1,which are all identified as L. cyprinacea.This indicates that the identified L. cyprinacea parasites in the examined ornamental fish are closely related to other L. cyprinacea strains and are distinct from other Lernaea species.

Discussion
Based on the importance of parasitic infections as one of the most concerning problems affecting ornamental fish 10,45,46 , attempting to gain a better understanding of parasitic infestations affecting ornamental fish may play an important role in the success of the ornamental fish industry 47 .Although the overall prevalence of parasitized fish in the visited ornamental fish farm was not particularly high (20.00%), the presence of detectable parasites at any level is of significance because the majority of parasites recovered from freshwater fish in Iran have the potential to induce significant mortalities among captive and wild stocks, particularly those parasites that do not typically require an intermediate host, such as ciliated protozoans and copepod crustaceans.Therefore, proper diagnosis, treatment, and prevention of parasitic infections are essential for maintaining the health and wellbeing of ornamental fish in captivity.This includes regular monitoring and screening for parasites, implementing effective biosecurity measures, and providing appropriate water quality and nutrition to reduce stress and susceptibility to parasitic infections.By doing so, ornamental fish farmers can ensure the long-term sustainability and profitability of their business while also promoting the welfare of the fish under their care.The parasitism of peritrich ciliates has previously been reported as a cause of mortality in North American 48 and European fish 49 , particularly when combined with opportunistic infections 50,51 .The epistylidid ciliate genus Epistylis Ehrenberg, which was described in 1830, contains over 260 nominal species 52 .It is believed that a particular number of species are free-living, but the majority are thought to be epibionts on aquatic metazoans such as aquatic plants, insects, and crustaceans that live in marine and freshwater environments 53 .These epibiont species can occasionally infect fish hosts with diseases such as red-sore disorders 28,54 .On the other hand, crustaceans are important mechanical vectors of various pathogenic agents such as bacteria, viruses, and parasites 55 , and their spread in aquatic environments may have a significant negative impact on commercial fish farming 17,56 .There are only a few reports that show a vectorial relationship between the isopod Gnathiid and the haemoprotozoan Haemogregarina 57 , as well as the crustacean arthropod Argulus foliaceus and Rhabdovirus carpio 17,58 , Caligus rogercresseyi and ISAV (infectious salmon anemia virus) 59,60 , Ergasilus chelangulatus and Epistylis spp. 31 , Acartia bifilosa and Epistylis sp. 61, Dolops carvalhoi and Epistylis sp. 17,62, L. cyprinacea and E. wuhanensis 27,29 , and L. cyprinacea and Epistylis sp. 17 .Chronic stress and susceptibility to a wide range of infestations may be increased in fish exposed to crustaceans for an extended period of time due to physical and/or environmental immunosuppressive stressors 63,64 .This, in turn, may pose a significant challenge to the global aquaculture industry by affecting fish health.
Objective histopathological findings in our study confirmed that the invasion of the parasitic crustacean L. cyprinacea is capable of causing a wide range of microscopic lesions, including hemorrhage and the aggregation and infiltration of inflammatory cells in the host, which were consistent with the findings of Singh et al. 65 , Hemaprasanth et al. 66 , Pala et al. 17 , and Furtado et al. 67 .Furthermore, parasitic granulomatous inflammation was observed, which agreed with the findings of Furtado et al. 67 .However, alterations reported in the current parasitized fish, such as muscular necrosis and pressure atrophy of skeletal muscles, may be considered novel pathological findings.Because of intraspecific morphological variations, it is difficult to identify sessilids such as Epistylis using only clinical and histopathological evaluation without using molecular methods 68,69 .Sequences generated from E. wuhanensis specimens and L. cyprinacea samples from current study cases are either identical or recovered as sisters to sequences from Japan 29 , implying that they naturally range in or have potentially been transported between Iran and Japan.
In the current study, poor quarantine measures when importing new fish appear to be the main cause of infection caused by L. cyprinacea and E. wuhanensis.To prevent the spread of parasitic infections in ornamental fish farms, it is essential to implement effective biosecurity measures.This includes extending the quarantine period for newly imported fish and closely monitoring them during this period to detect any potential infections.Fish farmers should also consider dividing their farm into isolation units to prevent the spread of infections between fish populations.Using a specific-pathogen-free (SPF) water source can also help reduce the risk of introducing new parasites to the farm.Additionally, incorporating immunostimulant compounds such as probiotics and prebiotics into fish feed formulations can boost the immune system of the fish and reduce their susceptibility to parasitic infections.Overall, implementing these preventative measures can have significant impacts on disease prevention and control in ornamental fish farms.By reducing the risk of parasitic infections, fish farmers can ensure the health and well-being of their fish populations, as well as the long-term sustainability and profitability of their business.

Conclusions
The crustacean L. cyprinacea serves as a mechanical vector for E. wuhanensis infection and spreads the disease in ornamental fish farming operations.For the first time in Iran, we presented diagnostic morphological and molecular findings for sessilids isolated from L. cyprinacea.Based on the findings of the current study, such parasitic infections may cause significant economic losses following invasion of the integument area of fish, eventually leading to mass mortalities if treatment is neglected or inadequate.However, further research is needed to determine the precise mechanisms of crustacean attachment and interactions between hosts, crustaceans, and adhered peritrich protozoans.Furthermore, serious consideration should be given to the direct and indirect effects of various environmental factors on the emergence and spread of the current disorder.

Figure 2 .Figure 3 .
Figure 2. (a) A stereomicroscopic study of Poecilia reticulata that uses a stereoscope to do a more detailed inspection showing intense adhesion of E. wuhanensis (arrows) in the cephalothorax and trunk of the crustacean L. cyprinacea.Egg sacs of L. cyprinacea (arrowheads) are also visible (10X).(b) High-power view of the ciliated protozoan E. wuhanensis (arrows) adhered to L. cyprinacea on the body surface of P. reticulata.Note the algae (arrowheads) adhered to L. cyprinacea and E. wuhanensis (40X).(c) Wet mount preparation from P. reticulata showed infestation with a large number of the epibiont ciliated protozoan E. wuhanensis.(d) Macroscopic examination of Poecilia latipinna showed infestation with E. wuhanensis adhered to L. cyprinacea (arrows).

Figure 7 .Figure 8 .
Figure 7. Molecular phylogenetic analysis based on the nucleotide sequences of the partial 18 s gene using the maximum likelihood method based on the general time-reversible model44 .Evolutionary analyses were performed in MEGA743 .The bootstrap consensus tree derived from 1000 replicates represents the evolutionary history of the taxa analyzed.The percentage of trees in which the associated taxa were clustered together is indicated next to the branches.The tree is drawn to scale, with the length of branches measured in the number of substitutions per site.

Table 1 .
Records of peritrich ciliates attached to L. cyprinacea in different species of fish.

Table 2 .
The genetic homology between Epistylis wuhanensis and other selected Epistylididae was calculated using the maximum composite likelihood substitution model based on the partial 18 s gene.

Table 3 .
The genetic homology between the Lernaea cyprinacea isolate and other selected Lernaeidae was calculated using the maximum composite likelihood substitution model based on the partial 5.8 s rDNA gene.