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Cryptocaryon irritans, a holotrichous ciliate parasitic protozoan, can trigger marine white spot disease and cause substantial economic losses in mariculture. However, methods of preventing and curing the disease have negatively affect fish, human, other organisms, and the natural environment. The antiparasitic activity of some antimicrobial peptides (AMPs) has garnered extensive attention of scholars. In this study, we identified and characterised a novel antiparasitic peptide, named So-pis, from Sciaenops ocellatus. The sequence analysis, structural features, and tissue distribution suggested that So-pis is genetically related to the piscidins family. However, So-pis showed a relatively low overall conservation compared with other known piscidins. So-pis is abound in glycine residues (22.7 %) and it has a neutral isoelectric point, weak amphipathicity, relatively long α-helix, and high hydrophobicity. These key elements are responsible for its biological activity. Quantitative real-time polymerase chain reaction (qRT-PCR) data indicated that So-pis is a typically gill-expressed peptide. The expression of So-pis in the gill, skin, spleen, and head kidney could be regulated during C. irritans infection, thereby implicating a role of So-pis in immune defence against C. irritans. The synthetic So-pis had limited or no antimicrobial activity against bacterial and yeasts but exhibited potent antiparasitic activity against C. irritans in vitro. The activity of synthetic So-pis against erythrocytes was less potent than its antiparasitic activity against C. irritans. These results indicated that So-pis might be one of the crucial defence cytokines against C. irritans in the red drum. Cumulatively, our data suggested that So-pis might be a potential candidate for developing a novel, effective, and safe therapeutic agent against marine white spot disease.

Numerous studies have demonstrated that Cryptocaryon irritans can efficiently propagate in golden pompano (Trachinotus blochii), especially under intensive high-density culture, which can lead to large-scale infection, bacterial invasion, and major economic losses. By contrast, Siganus oramin is less susceptible to C. irritans infection. Here, we artificially infected S. oramin and T. blochii with C. irritans. We then used RNA-seq to characterize the expression of genes in the gills of S. oramin and T. blochii at different times after infection, conducted bioinformatics analysis of relevant pathways, and compared the differentially expressed genes in the two species. The aim of this study was to enhance our understanding of host-parasite interactions to aid the development of effective prevention and treatment strategies for C. irritans. Infection with C. irritans induced the differential expression of a large number of genes in the gills of S. oramin, indicating that S. oramin may respond to C. irritans infection by modifying the expression of genes at the transcriptional level. Our research showed that the Toll-like receptor signaling pathway, Antigen processing and presentation, Complement and coagulation cascades, and Cytosolic DNA-sensing pathway are involved in the immune response of S. oramin and T. blochii to C. irritans infection. However, T. blochii has a weak ability to mobilize neutrophils to participate in defense against C. irritans infection and differs from S. oramin in its ability to induce specific immune responses. Because of gill tissue damage during infection, dissolved oxygen intake is reduced, which increases physiological and metabolic stress. The metabolic pathways of S. oramin and T. blochii significantly differed; specifically, the main pathways in S. oramin were related to glucose and lipid metabolism, and the main pathways in T. blochii were related to amino acid metabolism. This may reduce the efficiency of ATP biosynthesis in T. blochii and result in dysfunctional energy metabolism. Therefore, differential immune and metabolic responses underlie differences in the resistance of S. oramin and T. blochii to C. irritans.

Cryptocaryonosis is the greatest threat to most teleost species among all parasitic diseases, causing mass loss to the marine aquaculture industry. Epidemiological investigation of teleost susceptibility to Cryptocaryon irritans infection revealed that yellow drum (Nibea albiflora) is highly resistant. In order to further understand the activation of the immune system in the gill, which is one of the main mucosal-associated lymphoid tissues and a target of parasites, transcriptome analysis of the yellow drum gill was performed. Gill samples were collected from fish challenged after 24 hr and 72 hr with theronts at a median death rate (2050 theronts per gram fish). Gene expression profiles showed that TLR5 was the only receptor that activated the downstream immune response. The infection activated complement cascade through alternative pathway and increased the expression of C5a anaphylatoxin chemotactic receptor 1. In addition, possible antimicrobial molecules, including lipoprotein and haptoglobin, which are responsible for trypanolysis in humans, were among the top significantly upregulated genes at 24 hr. After 72 hr, the expression of secreted immunoglobulin T-related genes was induced. These results suggested a rapid innate and adaptive immune response at the mucosal level. In conclusion, the results provide new perspectives on mucosal immune resistance in yellow drum against cryptocaryonosis and provide the possibility of mining resistance genes for future therapy.

As an economically important marine fish, the large yellow croaker Larimichthys crocea suffered from marine white spot disease caused by the ectoparasite Cryptocaryon irritans in recent years. This disease not only could result in physiological damage, but also lead to secondary bacterial invasion. Reports indicated some AMPs (antimicrobial peptides) were of antiparasitic activity to C. irritans. Hepcidin-like (Lc-HepL) was one of the significant differential expression genes excavated from the transcriptome following a challenge with C. irritans. In this study, we characterized this AMP’s bioactivity based on the levels of mRNA and protein. After challenged by C. irritans, qRT-PCR showed Lc-HepL was significantly upregulated in six tissues, including gill, muscle, liver, head kidney and spleen during theront infection, trophont falling off, and secondary bacterial invasion stages, which implicated a role Lc-HepL played in the immune defense against C. irritans and secondary bacterial infection. Recombinant Lc-HepL (rLc-HepL) was induced and purified successfully. rLc-HepL exhibited antibacterial activity to certain bacteria in a dose- and time-dependent manners. Anti-C. irritans activity was explored for the first time and found it could cause the theronts membrane rupture and contents leakage. These results provided the first evidence that Lc-HepL had strong antiparasitic activity against marine fish ectoparasites C. irritans theronts. Together, data indicated that Lc-HepL might be an important component in the innate immune system against C. irritans and has the potential to be employed in future drug development.

Cryptocaryon irritans is an extremely harmful ciliated obligate parasite that is responsible for large economic losses in aquaculture. C. irritans infection can cause an insect-resistant immune response in fish, and many immune cells can be observed in the local infection site. However, it is unclear whether macrophages are involved in the host defense against C. irritans infection. The Mpeg1 protein can form pores and destroy the cell membrane of invading pathogens, and is also used as a macrophage-specific marker in mammals. Therefore, a polyclonal antibody against grouper recombinant Mpeg1a was produced to mark macrophages in this study, which could recognize both isoforms of Mpeg1 (Mpeg1a/b). Immunofluorescence revealed that EcMpeg1 positive cells were mostly distributed in the head kidney and spleen in healthy grouper. Immunofluorescence and immunohistochemistry showed that the number of EcMpeg1 positive cells increased in the gills after infection with C. irritans, implying that EcMpeg1 positive cells may be involved in the process of grouper resistance against C. irritans infection.

Pearl gentian grouper is a new aquacultural hybrid resulted from breeding of female tiger grouper (Epinephelus fuscogutattus) and male giant grouper (E. lanceolatus). Our preliminary study found that pearl gentian grouper exhibits less susceptible to the primary infection of Cryptocaryon irritans, which is an important parasitic ciliate in marine aquaculture, indicated that pearl gentian grouper might own a strong innate immune system. Complement system play key roles in innate immunity, whether pearl gentian grouper's complement component contribute for the defensing against the C. irritans infection remain unclear. In the present study, we found that C. irritans can be immobilized by untreated serum but not heat-treated serum from pearl gentian grouper, suggested that the heat-labile components in serum are responsible for the immobilization of C. irritans. Moreover, we cloned and characterized the encoding sequence of pearl gentian grouper complement C3 (PGC3), a key component in complement system. We also found that the expression level of PGC3 was increased in infected grouper serum when compared with that of control grouper. Furthermore, the binding of PGC3 on the surface of C. irritans trophonts located on the grouper skin was detected. These data suggested that pearl gentian grouper's complement system indeed play roles in the immune response against the C. irritans infection.

Nibea albiflora (Yellow drum) was discovered to be highly resistant to the infection of Cryptocaryon irritans a lethal ciliate protozoa parasite during disease outbreak. Investigation of miRNAs which post-transcriptionally regulate genes will deeper our understanding of regulation of immune response against infection. In this study, we challenged N. albiflora with C. irritans at median lethal concentration (2050 theronts/g fish). Skins from infected and controlled fish were sampled at 24 h and 72 h for miRNA sequencing. In total, we identified 180 known and 71 novel miRNAs, in which 30 miRNAs were significantly affected by infection, 19 were influenced by time and 16 significantly affected by the interaction between time and infection. Totally, 7396 genes were predicted as targeted regulated by infection differential expressed genes. The genes were significantly enriched in C-type lectin receptor pathway, MAPK signaling pathway and mTOR signaling pathway which closely related to antigen recognition, presentation and downstream signal transduction for immuno-activation. Overall, our data suggest that most of miRNAs were intensively regulating gene expression involved in C-type lectin receptor pathway, MAPK signaling pathway and mTOR signaling pathway rather than directly involve in immunological factor regulation during immune response against parasite infection.

The adoption of new investigative strategies based on environmental DNA (eDNA) can be used to monitor parasites, associated bacterial microbiomes, and physical-chemical parameters in fish farms. In this study, we used the economically important and globally distributed fish ciliate parasite Cryptocaryon irritans as a model to understand the parasite abundance and potential drivers of its presence in marine fish farms. Environmental (rainfall) and physical-chemical (temperature, oxygen, salinity, pH) data collected from a marine fish farm in Hong Kong were analyzed together with the eDNA approach targeting C. irritans abundance based on digital droplet PCR and 16S metagenomics to determine associations and triggers between parasites and specific bacterial groups. Rainfall and temperature demonstrated positive associations with high abundance of C. irritans (eDNA) at the studied marine fish cage farm. However, rainfall was the only parameter tested that demonstrated a significant association with parasite eDNA, indicating that the raining season is a risky period for fish farmers in Hong Kong. Coraliomargarita was the bacterial genus with the most significant relationship with low abundance of C. irritans in water. Understanding the environmental triggers of ciliate parasites propagation and associated bacterial microbiome could elucidate new insights into environmental control, microbial management, and promote the reduction of chemical use in marine fish farms.

Ciliates are fundamental components of microzooplankton, with important ecological roles. However, ciliate communities are particularly difficult to monitor using conventional morphological approaches. New molecular tools, such as DNA metabarcoding, can facilitate the study of these communities. This study used high-throughput sequencing to examine the diversity and seasonal dynamics of ciliate communities in four estuarine ecosystems in the South China Sea from June 2019 to March 2020. The amplification of the V4 region of 18S rDNA using ciliate-specific primers identified a total of 1645 amplicon sequence variants (ASVs), corresponding to 13 ciliate classes, 97 families, and 157 genera. The dominant species across all four sampling stations were spirotrichs (including choreotrichs, oligotrichs, and stichotrichs), oligohymenophorean scuticociliates, litostomateans Didinium, and prostomateans Cryptocaryon. Significant differences in ciliate diversity and community composition in the four stations were mainly due to differences in rare, rather than abundant, ASVs. Analysis of the ciliate communities and seasonal patterns in their composition revealed that variations in habitat and environmental conditions have a greater effect than seasonal changes on community composition.

Eukaryotic microorganisms are ubiquitous in the marine environment, and have a wide variety of ecosystem functions. Shenzhen is one of the most developed cities in South China, but the eukaryotic communities in the water along its coastlines remain poorly understood. The study applied 18S rRNA gene ITS (internal transcribed spacer) sequencing to identify the eukaryotic community from twenty sites of Shenzhen coast water. The alpha-diversity of the samples between these sites were significantly different, and the seawater of eastern coast had higher alpha-diversity compared to that of the western coast. The abundance of Chlorophyta was notably higher in the seawater of western coast, but Picozoa was relatively depleted. Specifically, Cryptocaryon, Pseudovorticella, and Cyclotella were significantly higher in the water of western coast, while Guinardia, Minutocellus, and Amoebophrya were increased in eastern samples. The spatially variations of eukaryotic microorganism community in the seawater of Shenzhen coast were associated with the water quality. The results have important significance for the understanding of coastal eukaryotic community, their interaction network, and build a foundation for future management and protection of coastal water quality.

Large-scale transcription studies have revealed numerous lncRNAs (long non-coding RNAs). lncRNAs have been proposed to participate in the regulation of a diverse range of biological processes, including transcriptional regulation. Although lncRNAs have attracted increasing attention, the studies in large yellow croaker (Larimichthys crocea) are still rare, and they lack systematic analysis. In this study, 101 RNA-seq datasets varied in ages, sexes, and tissues were retrieved from the NCBI database to generate a comprehensive catalog of large yellow croaker transcriptome database. A set of 14,599 high-confidence lncRNAs from 13,673 loci were identified and characterized. Furthermore, RNA-seq datasets obtained from the infection of C. irritans were employed to investigate the differential expression pattern of lncRNAs and analyze potential biological functions. A total of 77 differentially expressed lncRNAs targeting to 567 protein-coding genes were identified by using expression analysis. Several immune genes, including TLR5, CD2AP, and MMP9, were highlighted. With GO enrichment and KEGG pathway analysis, the immune-related terms or pathways were enriched. This study created a comprehensive dataset of lncRNAs for large yellow croaker, which would be helpful for the researches of functional roles of lncRNAs in large yellow croaker.

Large yellow croaker is an important marine culture species in China. Recently, the large yellow croaker industry is threatened by various disease problems, especially for the white spot disease, which is caused by parasite Cryptocaryon irritans. In the current study, we conducted a genome-wide association study (GWAS) for C. irritans resistance in two large yellow croaker populations (n = 264 and n = 480, respectively). We identified 15 QTL with explained genetic variance ranging from 1 to 8% in the two populations. One QTL on chromosome 23 was shared by the two populations, and three QTL had been reported in the previous study. We identified a lot of biological pathways associated with C. irritans resistance, such as hormone transport, response to bacterium, apoptotic process, acute inflammatory response to antigenic stimulus, and NF-kappa B signaling pathway. The genes casp8 and traf6 involved in regulatory network for apoptosis and inflammation were identified to be candidate genes for C. irritans resistance. Our results showed the complex polygenic architecture of resistance of large yellow croaker against C. irritans. These results would be helpful for the researches of the molecular mechanism of C. irritans resistance and genome-assisted breeding of large yellow croaker.

Artificial breeding of small yellow croaker (Larimichthys polyactis) was recently achieved, providing a bright future for its commercial farming. In May 2019, a disease outbreak occurred among small yellow croakers in an aquaculture farm near Xiangshan Bay, charactering by white spots spotted on the surface of fish skin, gills and fins. The parasite was preliminarily identified as Cryptocaryon irritans based on morphological feature of the parasite and the symptoms on fish. However, the previously published specific primer pairs failed to confirm the existence of C. iriitans. Six nucleotides mismatches were discovered after mapping specific forward primer back to targeted gene. Therefore, an updated PCR specific primer was developed within the 9th highly variable region of 18S rRNA gene and conserved in all C. irritans sequences available in GenBank database. The specificity was verified in silico by Primer-BLAST against GenBank nucleotide. Laboratory cultured ciliates (Mesanophrys, Pseudokeronopsis and Uronema) as well as natural microbial community samples collected from sea water and river water was used as negative control to verify the specificity of the primer in situ. Besides, tank transfer method was used to evaluate the treatment of the parasite infection. By tank transfer method, 2.00 ± 0.61 out of 10 fish that already sever infected were successfully survived after 8 days treatment, meanwhile the control group died out at d 6. More loss to the treatment group during first five days was observed and may attribute to the combined effect from infection and stress the recent domesticated fish suffered during rotation. Therefore, tank transfer method was also effective to prevent small yellow croaker from further infection, however the loss of the small yellow croaker suffered from stress during rotation also needs to be carefully concerned. In conclusion, this study reported the first diagnose of C. irritans infection on small yellow croaker, provided updated specific primer to detect C. irritans infection on fish body and reported the effect of tank transfer on small yellow croaker treatment.

Takifugu rubripes and Dicentrarchus labrax are important commercial fish in China that are under serious threat from Cryptocaryon irritans. C. irritans is a ciliated obligate parasite that causes marine white spot disease and leads to heavy economic losses. We analysed the transcriptome in the gills of T. rubripes and D. labrax to compare differentially expressed genes (DEGs) and pathways during infection with C. irritans. In total, we identified 6,901 and 35,736 DEGs from T. rubripes and D. labrax, respectively. All DEGs were annotated into GO terms; 6,901 DEGs from T. rubripes were assigned into 991 sub-categories, and 35,736 DEGs from D. labrax were assigned into 8,517 sub-categories. We mapped DEGs to the KEGG database and obtained 153 and 350 KEGG signalling pathways from T. rubripes and D. labrax, respectively. Immune-related categories included Toll-like receptors, MAPK, lysosome, C-type lectin receptor and NOD-like receptor signalling pathways were significantly enriched pathways. In immune-related signalling pathways, we found that AP-1, P38, IL-1β, HSP90 and PLA were significantly up-regulated DEGs in T. rubripes, but P38 and PLA were significantly down-regulated in D. labrax. In this study, transcriptome was used to analyse the difference between scaly and non-scaly fish infection by C. irritans, which not only provided a theoretical basis for the infection mechanism of C. irritans, but also laid a foundation for effectively inhibiting the occurrence of this disease. Our work provides further insight into the immune response of host resistance to C. irritans.

A cross-sectional survey was used to estimate the prevalence of infections with the Infectious spleen and kidney necrosis virus (ISKNV, Megalocytivirus), nervous necrosis virus (NNV, Betanodavirus), and infestations with ectoparasites during the rainy season in juvenile grouper (Epinephelus spp.) farmed in Aceh, Indonesia. The survey was intended to detect aquatic pathogens present at 10% prevalence with 95% confidence, assuming 100% sensitivity and specificity using a sample size of 30 for each diagnostic test. Eight populations of grouper from seven farms were sampled. Additional targeted sampling was conducted for populations experiencing high mortality. Infection with NNV was detected at all farms with seven of the eight populations being positive. The apparent prevalence for NNV ranged from 0% (95% CI: 0&ndash, 12) to 73% (95% CI: 54&ndash, 88). All of the fish tested from the targeted samples (Populations 9 and 10) were positive for NNV and all had vacuolation of the brain and retina consistent with viral nervous necrosis (VNN). Coinfections with ISKNV were detected in five populations, with the highest apparent prevalence being 13% (95% CI: 4&ndash, 31%). Trichodina sp., Cryptocaryon irritans and Gyrodactylus sp. were detected at three farms, with 66% to 100% of fish being infested. Hybrid grouper sourced from a hatchery were 5.4 and 24.9 times more likely to have a NNV infection and a higher parasite load compared to orange-spotted grouper collected from the wild (p &lt, 0.001). This study found that VNN remains a high-impact disease in grouper nurseries in Aceh, Indonesia.

(Plectropomus leopardus) or grouper coral trout is one kind of commodities that have high economic value. As marine fisheries commodities are much in demand for large-scale farmers or household level will require broodstock who have qualified to produce quality eggs and a good seed. qualified to produce quality eggs and a good seed. The purpose of research is to gain knowledge in maintenance activities of the broodstock grouper coral trout on a concrete tank by comparing theory and application in the field. Maintenance engineering of coral trout grouper broodstock includes the preparation of concrete tanks, broodstock selection, water quality management, feeding, spawning and harvesting of eggs, and disease. Research in Balai Besar Penelitian dan Pengembangan Budidaya Laut (BBPPBL) Gondol. Penyabangan Village, Subdistrict Gerokgak, Buleleng, Bali. On 18th January – 18th February 2016. Methods of work used in this research is descriptive data retrieval methods include primary data and secondary data. Data retrieval is done by means of active participation, observation, interviews and literature study. Results of Field Work Practice is the broodstock of grouper coral trout are maintained as much 217 with an average weight of 512.9 kg and an average length of 56.1 cm. Maintenance of broodstock grouper coral trout performed in container maintenance the round-shaped sized 100 m3 with a depth of 3 m with inlet and outlet Water management is performed by using a system is running water that flowing water as much as 300% -400%, the quality of sea water showing the value of 34.9 ppt salinity, DO 6.0 ppm, pH 8, and the temperature of 29 ˚C. Feed given in the form trash fish as much as 7-10 kg a day. Grouper coral trout spawning is done naturally by the male and female parent ratio of 1: 2, which produce eggs an average 2,252,292 items. During maintenance activities parasites invade have been found. Parasites invade the broodstock of grouper coral trout is Benedenia sp., Cryptocaryon sp., And viral infections Viral Nervous Necrosis (VNN)

Cryptocaryon irritans, a marine fish ectoparasite, causes mass economic loss in the mariculture and ornamental fish industries. It is important to find effective alternatives that may be both affordable and environmentally acceptable to control the damage of C. irritans. In this study, honokiol from Magnolia officinalis was screened from 36 phytochemical compounds for its significant antiparasitic activity against C. irritans theronts. Honokiol at a concentration of 1.00 μg/mL resulted in 100% mortality of C. irritans theronts within 30 min of exposure and >88.6% inhibition of tomont and protomont transformations post 8-h exposure to 1.00 and 4.00 μg/mL, respectively. Moreover, after exposure to 0.80, 1.00, and 4.00 μg/mL of honokiol solution for 30 min, 8 h, and 8 h, abnormal morphologies of theronts, tomonts, and trophonts were observed, respectively. After consecutively fed a diet containing 400 mg/kg of honokiol for 7 d, pompano (Trachinotus ovatus) were exposed to C. irritans theronts and the results showed that the number of C. irritans trophonts on the pompanos' gills was significantly lower than that in the control group 3 days later, while a similar efficacy was observed at doses above 100 mg/kg 8 days later. In addition, a dose of 200 mg/kg honokiol significantly reduced the chemotaxis of C. irritans theronts to the sera; meanwhile, the chemotaxis of C. irritans theronts to dorsal trunk muscle solutions was significantly reduced at doses above 100 mg/kg. Compared to the control group, a dose of 400 mg/kg honokiol increased the survival rate of the infected pompano by 65 ± 5% (mean ± SE). The results of hemolysis assay showed that the EC1 of honokiol to pompano erythrocytes was 6.36 μg/mL, 6.36 times the concentration that allowed honokiol to completely stop the movement of 100% C. irritans theronts in 30 min. Therefore, honokiol has the potential to be a safe and effective candidate, and could also serve as a leading compound in the development of a commercial drug to control C. irritans in mariculture.

Immobilization antigens (i-antigens) are surface membrane proteins that are widely recognized to be the ideal candidates as vaccines antigens for immunization against Cryptocaryon irritans. In this study, we cloned a putative i-antigen gene from C. irritans, which was expressed in all three stages of the C. irritans life-cycle, and localized primarily to the cell surface. The recombinant GDCI3 i-antigen was expressed and purified using the free-living ciliate, Tetrahymena thermophila as an expression system. The purified recombinant protein was recognized by rabbit anti-C. irritans antiserum and was capable of eliciting immobilizing antibodies in rabbits and fish suggesting that the antigen itself was correctly folded. Following immunization and parasite challenge, groupers vaccinated with, recombinant GDCI3 i-antigen had a 25% cumulative percent survival rate compared to 8.3% for controls. Both non-specific and parasite-specific IgMs were generated in fish following immunization, with the levels of both increasing following challenge. Parasite-specific IgM in mucus could only be elicited after challenge of the GDCI3 i-antigen vaccinated groupers. To our knowledge, this is the first report using the Tetrahymena expression system to generate C. irritans i-antigens and investigate their use for fish vaccination.

Cryptocaryon irritans is an obligate parasitic ciliate protozoan that can infect various commercially important mariculture teleosts and cause high lethality and economic loss, especially Larimichthys crocea. Current methods of controlling or preventing this parasite with chemicals or antibiotics are widely considered to be environmentally harmful. The antiparasitic activity of some antimicrobial peptides (AMPs) attracted extensive attention of scholars. In the study, a novel piscidin 5-like type 4 (termed Lc-P5L4) excavated from comparative transcriptome of C. irritans - immuned L. crocea was identified and characterized. Sequence analysis shows the full-length cDNA of Lc-P5L4 is 539 bp containing an open reading frame (ORF) of 198 bp which encodes a peptide of 65 amino acid residues. The genome consists of three exons and two introns which exist in its ORF, and all the exon-intron boundaries are in accordance with classical GT-AG rule (GT/intron/AG). Multiple alignments indicate the signal peptides share highly conserved identity, while mature peptides are more diverse. Phylogenetic analysis displays Lc-P5L4 clusters together with other members of piscidin 5-like family. Next, quantitative Real-time PCR (qRT-PCR) detection found C. irritans infection could upregulate Lc-P5L4 expression level in all tested tissues significantly, it appeared earliest upregulation in the theronts infection stage in the head kidney; the expression contents reached to maximum level in the intestine, gill and muscle during trophonts falling off stage; while it was just upregulated during secondary bacterial infection stage in the liver and spleen. The data showed Lc-P5L4 upregulation time points were in accordance with different infection stages. With recombinant Lc-P5L4 (rLc-P5L4) obtained through Escherichia coli system, in vitro assay showed rLc-P5L4 could cause cilia deactivation, cell bodiesclumping and sticking to each other, then cell membrane rupture and contents leakage. The data illustrated Lc-P5L4 played critical roles in the immune defense against C. irritans infection, and provided another proof that piscidins exhibit multiple anti- C. irritans features.

Cryptocaryon irritans can cause cryptocaryonosis (white spot disease) in marine fish but the pathogenesis of the disease is unclear. In this work, we used high-throughput proteomics to identify differentially expressed proteins in the serum of Takifugu rubripes challenged with C. irritans. By using quantitative proteomic assays combined with Tandem Mass Tag-labeled quantitative proteomic analysis, we identified a total of 2088 differentially abundant proteins (1706 proteins were quantified, p

Garlic (Allium sativum) has been shown to possess antimicrobial properties against a range of disease‐causing agents, including fish parasites. Our study aimed to investigate the potential use of garlic as a treatment against Cryptocaryon irritans infection, using guppies (Poecilia reticulata) as the fish model. Garlic was freeze‐dried, powdered, and used as it is or as an aqueous extract. The content of allicin, its main active ingredient, was 1.25 mg/g in garlic powder and 0.82 mg/ml in the aqueous extract, as determined by HPLC analysis. Aqueous garlic extract fully immobilized C. irritans theronts and protomonts within 20 and 40 min, respectively, following exposure to 5 µl/ml. Treatment trials were performed, in which infected fish were fed with garlic powder‐supplemented feeds (5%, 10%, or 20% supplementation), bathed in garlic aqueous extract (250 or 500 µl/L) and subjected to a combined treatment which included both feeding and bathing. Results revealed that the tested treatments failed to cure the infection, though reduction in infection intensity on the caudal fin, but not on the gills, was evident following dietary supplementation with 20% of powdered garlic in one of the trials.

Cryptocaryon irritans is a type of marine ectoparasitic ciliate that infects teleost fishes. To illustrate the susceptibility and innate immune mechanism of fishes to C. irritans, four species of marine perciform fishes were selected in Fujian Province, a high-prevalence area of cryptocaryoniasis in China. The survival, diameter/number of tomonts, and infection ratio among Larimichthys crocea, Lateolabrax japonicus, Pagrus major, and Nibea albiflora were compared after artificial infection. Meanwhile, the immobilization titers of four fish species with no C. irritans infection were detected. Results showed that survival and serum immobilization titer of N. albiflora were significantly higher than those of the other three fish species. A strong negative linear correlation was found between the survival/serum immobilization titer and the mean tomont diameter. In addition, the smallest C. irritans infection ratio was found in N. albiflora, implying that the serum of fishes especially that of N. albiflora, inhibited the development of parasitic C. irritans cells, and the smallest tomont size was directly related to the number of infective theronts corresponding to the highest survival of fish. Moreover, complement activity inhibition assays suggested that the alternative complement pathway might play a major role in C. irritans resistance.

Larimichthys crocea, the special marine economy fish, owns the largest annual yield for a single species in China. One of the most significant factors affecting large yellow croaker culture is the diseases, especially the threat of marine white spot disease which caused by a protozoan Cryptocaryon irritans. Antimicrobial peptides (AMPs) have been demonstrated to be active against bacterium, fungi and parasites, showing their potential usefulness in aquaculture as substitutes for antibiotics. Many researches have been carried out about the AMPs concentrating on the activity resist on C. irritans, and piscidin-like of L. crocea owning widely antibacterial spectrum and strong activity against C. irritans was screened in our team. In the paper, taking advantage of the large yellow croaker hepatic comparison transcriptome in response to C. irritans at 3d post infection, seven kinds of AMPs have been excavated from the differently expressed genes, including LEAP2 like, LEAP-2A, hepcidin, hepcidin-like, piscidin-5-like, piscidin-5-like type 4 and bactericidal permeability increasing protein (BPI). Hepcidin, hepcidin-like, piscidin-5-like, piscidin-5-like type4 and BPI were up-regulated to protect large yellow croaker from being damaged by C. irritans infection; while LEAP2 like and LEAP-2A were down-regulated, they might be as a negative-feedback regulation factor or some other regulatory mechanisms to adjust the immune response in the process of C. irritans infection. The differential expression changes were verified with quantitative real-time PCR (qRT-PCR) to illustrate the reliability of the sequenced data. Hearteningly, piscidin-5-like type 4 was a novel type which was high similar to other piscidin-5-like types. Interestingly, the infection may well cause alternative splicing of LEAP-2A mRNA, which was a surprised phenomenon and finding after C. irritans infection, but more further study was needed to be conducted. Therefore, the data showed that these AMPs were involved in the immune response to the C. irritans infection. In all, these results implied that the immune response of AMPs to C. irritans infection was a complex and sophisticated regulatory process.

Infection by Cryptocaryon irritans, a ciliated protozoa parasite of marine fish, causes mass mortality and economic loss to the mariculture industry. In this study, plate and mesh galvanized zinc were embedded on the bottom of seawater to evaluate the protection to susceptible host Trachinotus ovatus from C. irritans reinfection at various seawater exchange rates and dosages. Furthermore, an evaluation of the zinc ion released from galvanized materials was carried out through detection of the zinc ion content in seawater and muscle tissue, and the observed pathological changes of liver. The results showed that, at all flow exchange rates (0, 50, or 1200%), the parasite abundance of infected T. ovatus were significantly decreased with galvanized materials. Survival rates were 100% at 14 days post infection when 56.25% of the tank was covered with galvanized iron plates or galvanized iron mesh at a dose of 0.2 m2/m3. The zinc ion concentrations in seawater after using galvanized materials increased continuously. However, residual zinc ion in the muscle of T. ovatus were not significantly different to the control group. Furthermore, no obvious pathological change was observed in the liver of T. ovatus. Consequently, using galvanized materials can control the reinfection of C. irritans and poses no threat to protected fish. Therefore, the use of galvanized materials could be a promising strategy to control cryptocaryoniasis.

Cryptocaryoniasis, caused by Cryptocaryon irritans, is a major threat to marine cage culture in tropical and subtropical waters; however, controlling the disease remains challenging. In this study, we constructed DNA vaccines encoding a cysteine protease of C. irritans (pcDNA3.1-cp2-full-myc and pcDNA3.1-cp2-partial-myc) and examined the protective efficacy of the vaccines. The results of the challenge experiment showed that the number of parasites recovered from fish immunized with the DNA vaccines was lower than that recovered from control fish (phosphate-buffered saline-injected and mock vector-injected groups); this difference was statistically significant when pcDNA3.1-cp2-full-myc was used for vaccination (p

Cryptocaryon irritans is a pathogenic ectoparasitic ciliate that infects marine teleosts, causing massive mortality and economic losses in aquaculture. Ultraviolet light (UV) and ozone (O3) are commonly used for disinfection in factory aquaculture; however, the efficacy of neither method against C. irritans had been evaluated. This study aimed to evaluate the efficacy of UV and ozone in destroying C. irritans. UV or ozone treatment changed or ruptured the shape of C. irritans theronts, although C. irritans tomonts showed some resistance. The minimum in vitro lethal doses of UV and ozone against theronts were 2.88× 105 μWs/cm2 and 0.15 mg/L, respectively. In vivo, UV and ozone-treated theronts lost their ability to infect pompano (Trachinotus ovatus) when exposed to doses above 1.84 × 105 μWs/cm2 and 0.10 mg/L, respectively. Unexpectedly, exposure to low ozone levels (

Cryptocaryon irritans, an important protozoan ciliate, infects almost all species of marine teleosts and causes heavy economic losses in aquaculture. In industrial aquaculture, culture container rotation is an effective method to control cryptocaryoniasis. However, the accurate frequency and times of container rotation for different temperatures or fish species has not been reported but is very important information for the application of the method. In the present study, we investigated the duration of C. irritans trophont residence and theront production in three fish species at temperatures from 18 °C to 32 °C with 2 °C intervals. The results indicate that duration of trophont residence and theront excystment was shorter with rises in temperature, especially from 18 °C to 22 °C. In different fish species, the length of trophont residence varied whereas the length of theront excystment was very similar at the same temperature. In line with these data, we determined the frequency and times that tomonts were removed, and results demonstrated that all the tomonts can be completely removed with 2 or 3 days interval for 2 or 3 times (from 22 °C to 32 °C), and the removal of tomonts was identical in three fish species at the same temperature. At 18 °C and 20 °C, however, the interval of tomont removal was > 4 days. Lastly, we verified the removal of tomonts with Trachinotus ovatus as host at 22 °C and 30 °C by a placemat removal method. In conclusion, these data provide an important basis for defense against C. irritans infection by the interruption of its life cycle in an intensive aquaculture system.

We incubated tomonts of Cryptocaryon irritans in a hypoxic seawater environment (1.4–1.7 mg/L O2) (low dissolved oxygen; DO) and examined their development using a acetocarmine whole-mount staining method we developed for nuclear staining. They showed little development and stayed in the dormant phase in the hypoxic environment. When transferred into the hypoxic environment after incubation in an oxic environment (air-saturated, 8.7–8.9 mg/L O2) for 1–4 days, their development stopped in 1 day. However, when dormant tomonts generated in the hypoxic environment were transferred to the oxic environment, they resumed development and released theronts. These results indicate that tomonts can become dormant when exposed to a hypoxic environment, but can resume development when exposed to an oxic environment at any developmental stage. When exposed to the oxic environment, tomonts recovered from 1-month dormancy and released as many theronts as control tomonts constantly incubated in the oxic environment. The infectivity of theronts from the recovered tomonts was similar to the control tomonts. Thermoclines prevent oxygen-rich surface seawater from reaching the bottom of water column and create a hypoxic sea floor environment in summer; these thermoclines are broken down in autumn or after typhoons. The long-term viability of dormant tomonts in hypoxic environments may be a key factor in the autumn outbreaks of cryptocaryoniasis in floating net cages in temperate waters.

Previous studies have found that copper alloy sheets can kill the protozoan Cryptocaryon irritans effectively as the tomonts of this particular pathogen adhere to the hard surface of tanks. This study explored the potential use of copper alloy particles incorporated into coatings to be applied to the bottom of aquaculture tanks, in order to control cryptocaryoniasis outbreaks. Results showed that the number of trophonts on Trachinotus ovatus exposed to such copper coated surfaces was significantly lower than that in the control and blank groups 5 days later and effectively blocked the reinfection of C. irritans, thereby decreasing the mortality rate of T. ovatus. In in vitro assays, the mortality rate of protomonts and tomonts was up to 100% at 6th h and 24th h of post contacting with the copper coated surfaces, respectively. Safety assessments found that copper concentration was not significantly high in flowing seawater, but it showed an upward trend with longer immersion time, increasing significantly from 0.56 to 5.90 μg/L within a 30-day period. When exposed to the copper alloy coating, the bottom-crawling fish Epinephelus coioides, did not accumulate an excessive copper concentration in the serum, liver or muscle tissues. This study provides a safe and effective strategy to control cryptocaryoniasis in marine factory farming.

The parasite Cryptocaryon irritans causes massive losses in the marine fish culture industry and is one of the most threatening pathogens affecting teleost species. The acute death of infected fish is primarily caused by the destruction of gill cells, resulting in osmotic imbalance and respiratory stress. C. irritans has wide host specificity; however, the yellow drum Nibea albiflora is highly resistant to this parasite. Metabolomic approaches in combination with transcriptomic analysis were used to characterize the host immune reaction and metabolic changes in yellow drum in response to C. irritans infection and to identify the key genes and compounds in the gills that have the strongest contribution to disease resistance. The yellow drum was challenged with theronts at a median death rate (2050 theronts per gram fish). The samples were collected from the gills 24 h and 72 h after the infection (hpi). The results of metabolomic analysis indicated that metabolites involved in energy metabolism were predominantly downregulated. In contrast, a compensatory increase in the expression of the genes involved in the citric acid cycle and glycolysis was detected 24 hpi. The suppression of metabolites was alleviated after feed intake recovery 72 hpi. The levels of amino acids were decreased, and the expression of aminoacyl-tRNA was increased. Additionally, elevated levels of arachidonic acid derivatives, primarily prostaglandins, were responsible for anti-inflammatory, osmotic, and hypoxia regulations. Purine metabolism was also involved in the immune response via generation of reactive oxygen species catalyzed by xanthine oxidase. A significant increase in the generation of retinoic acid, which could enhance mucosal adaptive immunity by stimulating the synthesis of antibodies and accelerating the restoration of epithelial integrity, was observed at 72 hpi. This result was consistent with high expression of the genes related to secreted immunoglobulin T 72 hpi. In conclusion, the present study comprehensively described the key compounds and genes related to C. irritans infection in yellow drum gills. Biomarkers that were significantly changed during the infection may represent future targets for nutritional intervention to enhance host immunity against C. irritans infection and to accelerate disease recovery.

Vibrio harveyi, known as a pathogenic bacterium caused severe secondary bacterial infections of the large yellow croaker Larimichthys crocea, was identified as an endosymbiont in the marine parasitic ciliate protozoan Cryptocaryon irritans. Meta 16S sequencing method was used to identify the bacterial flora in C. irritans, and V. harveyi was isolated via culture-dependent method. Vibrio harveyi was observed in cytoplasm of C. irritans at the stage of tomont both by transmission electron microscopy and by Fluorescence in situ hybridization; no signal, however, was detected in nucleus area. The relationship between V. harveyi and C. irritans and the role of endosymbiotic V. harveyi in C. irritans merit further investigation.

Among other functions, CCL25/CCR9 has an important role in regulating the trafficking of developing T cells in the thymus, and in homing memory T cells to the small intestine. The function of this chemokine-receptor complex is not well studied in fish. We identified a CCL25-like (EcCCL25, 108 aa) and two CCR9-like sequences (EcCCR9aa 373 aa; and EcCCR9b, 375 aa) from a transcriptome database of orange-spotted grouper (Epinephelus coioides). EcCCL25, EcCCR9a, and EcCCR9b shared conserved structural features with homologs from mammals and from other fish, and a consistent relationship with phylogenetic trees and sequence identities. In healthy grouper, EcCCL25, EcCCR9a, and EcCCR9b were highly expressed in the thymus, and the gills, were expressed at lower levels in the stomach, and had different expression levels in other tissues. After infection with Cryptocaryon irritans, EcCCL25 expression was up-regulated at early time points in the spleen and head kidney, and in the skin, and gills at later time points; EcCCR9a expression was increased in the gill, spleen, and head kidney. After infection with C. irritans, EcCCR9b expression was reduced in all tissues tested. These results suggested that grouper CCL25/CCR9a complex may be involved in host defense against C. irritans infection.

P38 mitogen-activated protein kinases (MAPKs) are one of the most important central regulatory proteins response to extra environmental stresses. In this study, two novel p38 MAPKs, Ec-P38γ and Ec-P38δ, were identified from Epinephelus coioides, an economically important cultured fish in China and Southeast Asian counties. Both of Ec-p38γ and Ec-p38δ sequences contain a serine/threonine protein kinase (S_TKc) domain and a highly conserved Thr-Gly-Tyr (TGY) motif. Analysis of phylogenetic relationships illustrated that p38 amino acid sequences were conserved between different species indicating that the functions may be similar. The four subtypes of p38 (α, β, γ, and δ) mRNA can be detected in all thirteen tissues examined, but the expression level is different in these tissues. The expression patterns of the four Ec-p38 subtypes in E. coioides were also detected response to Cryptocaryon irritans infection, one of the most important protozoan pathogens of marine fish. The expression of four p38 subtypes was up-regulated in the tissues examined, with the highest expressions of Ec-p38α (5.2 times) and Ec-p38δ (4.2 times) occurring in the skin, while Ec-p38β (24.8 times) and γ (16.6 times) occurred in the spleen. There was no significantly correlation between the expression of Ec-p38γ/Ec-p38δ and the expression of nuclear factor kappaB (NF-kB). The results indicated the sequences and the characters of Ec-p38γ and Ec-p38δ were conserved, the p38 subtypes showed tissue-specific expression patterns in healthy grouper, and their expressions were significantly up-regulated post C. irritans infection, suggesting these p38 MAPKs may play important roles in these tissues during pathogen-caused inflammation.

The ciliate protozoan Cryptocaryon irritans parasitizes marine fish and causes lethal white spot disease. Sporadic infections as well as large-scale outbreaks have been reported globally and the parasite's broad host range poses particular threat to the aquaculture and ornamental fish markets. In order to better understand C. irritans' population structure, we sequenced and compared mitochondrial cox-1, SSU rRNA, and ITS-1 sequences from 8 new isolates of C. irritans collected in China, Japan, and Taiwan. We detected two SSU rRNA haplotypes, which differ at three positions, separating the isolates into two main groups (I and II). Cox-1 sequences also support the division into two groups, and the cox-1 divergence between these two groups is unexpectedly high (9.28% for 1582 nucleotide positions). The divergence is much greater than that detected in Ichthyophthirius multifiliis, the ciliate protozoan causing freshwater white spot disease in fish, where intraspecies divergence on cox-1 sequence is only 1.95%. ITS-1 sequences derived from these eight isolates and from all other C. irritans isolates (deposited in the GenBank) not only support the two groups, but further suggest the presence of a third group with even greater sequence divergence. Finally, a small Ka/Ks ratio estimated from cox-1 sequences suggests that this gene in C. irritans remains under strong purifying selection. Taken together, the C. irritans species may consists of many subspecies and/or syngens. Further work is needed to determine if there is reproductive isolation between the groups we have defined.

To explore the resistance mechanism of locally infected skin of yellow drum (Nibea albiflora) against Cryptocaryon irritans infection, N. albiflora were infected with C. irritans at a median lethal concentration of 2050 theronts/g fish. Then, the skin of the infected group (24 hT and 72 hT) and the control group (24 hC and 72 hC) were sampled at 24 h and 72 h for quantitative proteomics analysis. A total of 643 proteins were identified, of which 61 proteins were significantly affected by interaction between time and infection, 83 and 119 proteins were significantly affected by the infection and time, respectively. In addition, 17, 61, 81 and 45 differentially expressed proteins (DEPs) were obtained from pairwise comparison (24 hT vs 24 hC, 72 hT vs 72 hC, 72 hT vs 24 hT and 72 hC vs 24 hC), respectively. DEPs in 24 hT vs 24 hC and 72 hT vs 72 hC were mainly enriched in Gene Ontology terms (transferase activity, protein folding and isomerase activity) and Kyoto Encyclopedia of Genes and Genomes pathways (biosynthesis of antibiotics, carbon metabolism and Citrate cycle). Among them, enriched DEPs were malate dehydrogenase 2 (MDH2), malate dehydrogenase 1 ab (MDH 1 ab), citrate synthase, etc. Immune-related DEPs such as complement component C3 and Cell division cycle 42 were involved in response to stimulus and signal transduction, etc. Also, DEPs such as collagen, heat shock protein 75 and MDH2 play a role in helping fish skin wounds to heal and provide energy. Furthermore, protein-protein interaction analysis indicated that 18 proteins such as MDH2, MDH 1 ab, complement C3 and collagen were interrelated. In conclusion, this study found that many proteins in N. albiflora contribute to resist against C. irritans and promote fish recovery.

Several researches reported that piscidin members of teleosts owned strong antiparasitic activity. Cryptocaryon irritans, a type of ectoparasite, could infect most of the marine teleosts. Larimichthys crocea could severely suffer from marine white spot disease caused by C. irritans, and their mortality rate was significantly high. Concentrating on this problem, we have done many related works. Piscidin 5 like (termed Lc-P5L) was another piscidin member isolated from a comparative transcriptome of C. irritans-immuned L. crocea. In the paper, quantitative Real-time PCR (qRT-PCR) showed Lc-P5L was upregulated in examined tissues, including gill, head kidney, muscle, liver, spleen and intestine after challenged by C. irritans, the significant upregulation time was in accordance to key developmental stages of C. irritans, which implied different infection stages could result in host immune response. Furthermore, using microscope techniques, we observed theronts or trophonts became weakly motile, cilia became detached, cells were out of shape, membranes eventually lysed in different cell positions and cytoplasmic contents leaked. Laser confocal scanning microscope (LCSM) observed theronts macronucleus grew swell and depolymerized after treated by recombinant Lc-P5L (rLc-P5L). Data suggested rLc-P5L was significantly lethal to C. irritans, and the death state of the parasite incubated with rLc-P5L was remarkably similar to other piscidin members or other antiparasitic peptides (APPs). Thus, these data provided new insights into L. crocea immunity against C. irritans and potential of rLc-P5L as a therapeutic agent against pathogen invasion.

The ciliated protozoan Cryptocaryon irritans infects a wide range of marine fish and causes the highly lethal white spot disease. This parasite possesses three morphologically and physiologically distinct life stages: an infectious theront, a parasitic trophont, and an asexually reproductive tomont. In the past few years, several attempts have been made to help elucidate how C. irritans transforms from one stage to another using transcriptomic or proteomic approaches. However, there has been no research studying changes in transcription profiles between different time points of a single C. irritans life stage-the development of this parasite. Here we use RNA-seq and compare gene expression profiles of theront cells collected by 1 and 10 hrs after they emerged from tomonts. It has been shown that infectivity of theront cells declines 6-8 hours post-emergence, and we used this characteristic as a physiological marker to confirm the aging of theront cells. We identified a total of 41 upregulated and 90 downregulated genes that were differentially expressed between young and aging theront cells. Using Blast2Go to further analyze functions of these genes, we show that genes related to energy production are downregulated, but quite surprisingly many genes involved in transcription/translation processes are upregulated. We also show that expression of all nine detectable agglutination/immobilization antigen genes, with great sequence divergence, is invariably downregulated. Functions of other differentially expressed genes and indications are also discussed in our study.

Large yellow croaker (Larimichthys crocea) is one of the most important mariculture fish in China. In the past decades, cryptocaryonosis caused by Cryptocryon irritans has led to huge economic losses, posing great threat to the healthy and sustainable development of L. crocea mariculture industry. As the largest immunologically active mucosal organ in fish, skin provides the first defense line against external pathogens. To better understand the gene expression dynamics, the large yellow croakers were artificially infected with C. irritans and their skin tissues were collected at 0 h, 24 h, 48 h, 72 h and 96 h post infection. The total RNA in the skin tissues were extracted and the transcriptome were sequenced. After sequencing, a total of 1,131, 311, 140 million high quality RNA-seq reads were collected. A set of 215, 473, 968, 1055 differentially expressed genes were identified at 24 h, 48 h, 72 h and 96 h post infection respectively. Further analysis clustered these DEGs into six profiles and 75 hub genes for six profiles were identified. Among these hub genes, 18 immune related genes including TLR5, TOPK, NFKBIZ, MAPK14A were identified post C. irritans infection. Cytokine-cytokine receptor interaction was the only pathway that significantly enriched at four timepoints post infection. This study provides an in-depth understanding of skin transcriptome variance of large yellow croaker after C. irritans infection, which would be helpful for further understanding of the molecular mechanism of L. crocea in response to C. irritans infection.

Cryptocaryon irritans infection could cause huge economic losses to the marine fish industry. Larimichthys crocea, a special economic species in China, suffered from the threat of serious infection, and L. crocea could enhance the level of piscidin 5-like to defense against the infection. This study set out to observe the main histopathological changes of some key tissues caused by infection, and determineed how an ectoparasite affected the expression of piscidin-5 like in its hosts. Pathological changes and immune response were assessed using histological and in situ hybridization (ISH) technologies. The infection induced inflammation occurring, especially in the gill where epithelium cells swell, hyperplasia, necrosis shedding adjacent to the parasites attachment sites. Infected hepatic cells grown big vacuoles in the cytoplasm. The boundary between red pulp and white pulp turned indistinct, splenic corpuscle lost the normal structure, the number and size of melano-macrophage centers increased apparently in the infected spleen. The whole structure of head kidney became loose. Immunostaining with RNA probes against piscidin 5-like showed subpopulations of mast cells (MCs) were positive. Piscidin 5-like-positive MCs existed mainly in the head kidney where they distributed around melano-macrophage center, followed in the gill located at different positions they also distributed in the margin of spleen, and randomly and sparsely existed in the liver. After being infected by C. irritans, the gill arch arose positive MCs groups, and they also migrated to spleen, while the positive staining deepen in other detected tissues. Therefore, organism enhanced the expression level through improving expression ability of positive MCs, or increasing the number of positive MCs.

Marine cultured fish in tropical and sub-tropical regions often suffer from white spot disease, caused by Cryptocaryon irritans infection. This can lead to mass mortality and economic loss. This study investigated the use of seawater-adapted red tilapias (Oreochromis niloticus × O. mossambicus) as scavengers for cleaning tomonts, to protect susceptible marine fish from C. irritans infection. The sensitivity of red tilapia to C. irritans infection, the effectiveness of red tilapia in consuming tomonts, and the efficacy for protecting the vulnerable Trachinotus ovatus from C. irritans infection were evaluated. The results showed that the susceptibility of red tilapia to C. irritans infection was significantly lower than T. ovatus and the rate of tomonts consumption by red tilapia was positively correlated with the number of tomonts in the seawater. The relative percent survival of T. ovatus infected with C. irritans and co-cultured with red tilapias (at a density of 19 red tilapias/m2) was 97.78% at the12 days post-infection. This study indicates that polyculture with red tilapia could be a potential alternative biocontrol strategy to eliminate tomonts, for controlling C. irritans reinfection.

In this study, we aimed to analyze the functions of highly expressed proteases at the theront (invasive stage) and trophont (parasitic stage) phases of Cryptocaryon irritans during the infection process using RNAi. We also analyzed the potential of these proteases as candidate antigens for vaccination against the parasite by infecting tiger pufferfish (Takifugu rubripes) with the recombinant proteases. Compared with the control group, RNAi against four proteases (including peptidases) genes reduced the number of parasites that infected and left the fish after complete development. The four recombinant proteases were then purified and evaluated as candidate vaccination antigens. The number of parasites recovered from fish immunized with the C. irritans recombinant proteins was lower than that recovered from control fish; this difference was statistically significant when a recombinant cysteine protease was used. These results suggest that the C. irritans proteases overexpressed at the theront and trophont stages are important for infection (including invasion, food uptake, digestion, and development) and can be targeted for the development of vaccines against cryptocaryoniasis.

SUMMARYNumerous studies have been conducted on the cellular morphology of Cryptocaryon irritans. However, details regarding the tomont stage of its life cycle remain lacking. In this study, we investigated the morphology of the tomont stage throughout encystment and cell division using light and electron microscopy. Results showed that there was no secretion of encystation-specific secretory vesicles or extrusomes during formation of the cyst wall. Instead, the synthesis and construction of the C. irritans cyst wall materials may involve molecular events at the pellicle. The somatic cilia and the cytostome were present during encystment and covered by the newly formed cyst wall. New somatic cilia were continuously created between old cilia and showed various lengths during cell division, a process that was similar to morphogenesis in many free-living ciliates. During cell division inside the tomont, dividing daughter cells formed temporary cell chains with no oral primordia before separating from each other into dissociative tomite precursors. The process of cell division may not be accompanied by stomatogenesis, and new oral primordia in offspring cells likely formed before the dividing cell chains split into dissociative spherical tomites. Mitochondrial autophagy was observed in encysting C. irritans cells. Numerous endosymbionts and Golgi structures were observed in the tomont cytoplasm. Cellular metabolic activity in the C. irritans tomont was quite high, with large amounts of materials or cellular organelles potentially being synthesized and prepared for the following infective theront stage.