Catechol compounds as dual-targeting agents for fish protection against Ichthyophthirius multifiliis infections.

Aquaculture is one of the fastest growing sectors in global food production, recognized as a significant contributor to poverty alleviation, food security, and income generation. However, the frequent occurrence of diseases caused by pathogen infections result in reduced yields and economic losses, posing a substantial constraint to the sustainable development of aquaculture. Here, our study identified that four catechol compounds, quercetin, luteolin, caffeic acid, and chlorogenic acid, exhibited potent antiparasitic effects against Ichthyophthirius multifiliis in both, in vitro and in vivo. The parasite is recognized as one of the most pathogenic to fish worldwide. Using a combination of in silico methods, the dipeptidyl peptidase (DPP) was identified as a critical target for catechol compounds. The two hydroxyl radicals of the catechol group were essential for its binding to and interacting with the DPP protein. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses indicated that catechol compounds disrupt pathways associated with the metabolism and growth of I. multifiliis, thereby exerting antiparasitic effects. Furthermore, these compounds attenuated the expression of proinflammatory cytokines in vivo in fish and promoted macrophage polarization toward M2 phenotype by inhibiting the STAT1 signaling pathway. The dual activity of catechol compounds, acting as both direct antiparasitic and anti-inflammatory agents in fish, offers a promising therapeutic approach for combating I. multifiliis infections in aquaculture.

Identification of Philaster apodigitiformis in aquaculture and functional characterization of its ß-PKA gene and expression analysis of infected Poecilia reticulata.

Cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) is a distinctive member of the serine­threonine protein AGC kinase family and an effective kinase for cAMP signal transduction. In recent years, scuticociliate has caused a lot of losses in domestic fishery farming, therefore, we have carried out morphological and molecular biological studies. In this study, diseased guppies (Poecilia reticulata) were collected from an ornamental fish market, and scuticociliate Philaster apodigitiformis Miao et al., 2009 was isolated. In our prior transcriptome sequencing research, we discovered significant expression of the ß-PKA gene in P. apodigitiformis during its infection process, leading us to speculate its involvement in pathogenesis. A complete sequence of the ß-PKA gene was cloned, and quantified by quantitative reverse transcription-polymerase chain reaction to analyse or to evaluate the functional characteristics of the ß-PKA gene. Morphological identification and phylogenetic analysis based on small subunit rRNA sequence, infection experiments and haematoxylin­eosin staining method were also carried out, in order to study the pathological characteristics and infection mechanism of scuticociliate. The present results showed that: (1) our results revealed that ß-PKA is a crucial gene involved in P. apodigitiformis infection in guppies, and the findings provide valuable insights for future studies on scuticociliatosis; (2) we characterized a complete gene, ß-PKA, that is generally expressed in parasitic organisms during infection stage and (3) the present study indicates that PKA plays a critical role in scuticociliate when infection occurs by controlling essential steps such as cell growth, development and regulating the activity of the sensory body structures and the irritability system.

Northern coral triangle coral ciliates diseases and disease prevention: A first record.

This study is the first to report ciliate infection on soft corals in the Northern Coral Triangle. Infected Briareum violacea will undergo tissue ulceration and death within a short period of time. This ciliate was identified as Scuticociliatia sp. through 18S rRNA gene identification. In this study, the body length of the ciliate was approximately 80-85 µm before parasitizing the B. violacea. After being parasitizing, the body length was approximately 200-250 µm, and the body width was 50 µm. Body size increased three times after parasitism. According to observations, ciliates will first parasitize the coral endoderm in the early stage of infection, and no ciliates were found in the ectoderm. Preliminary judgment suggests that it may invade the coral endoderm through the mouth for parasitism. After parasitism, the ciliate eats the coral tissue and zooxanthellae. The antioxidant enzymes SOD, CAT, and MDA of infected corals were significantly increased, which also means that the corals are in a stress response. Ciliates will eat the zooxanthellae in the coral body, resulting in a significant reduction in the number of zooxanthellae and chlorophyll a. To effectively prevent and treat this disease, Combretum indicum extract was used in this study. It is a tropical plant commonly used medicinally to treat roundworms, pinworms and parasitic diseases. The results showed that at a concentration of 1500-2500 ppm, Combretum indicum extract can be used to treat ciliates and can applied via medicinal bath therapy for long periods without causing coral stress reactions. The results of this study regarding coral disease prevention are in line with SDG 14 and promote the practical application of coral reef ecological sustainability and large-scale coral aquaculture.

Lipid changes and molecular mechanism inducing cuproptosis in Cryptocaryon irritans after copper-zinc alloy exposure.

Cryptocaryons irritans is a ciliate parasite responsible for cryptocaryoniasis, leading to considerable economic losses in aquaculture. It is typically managed using a copper-zinc alloy (CZA), effectively diminishing C. irritans infection rates while ensuring the safety of aquatic organisms. Nevertheless, the precise mechanism underlying cuproptosis induced C. irritans mortality following exposure to CZA remains enigmatic. Therefore, this study delves into assessing the efficacy of CZA, investigate cuproptosis as a potential mechanism of CZA action against C. irritans, and determine the alterations in antioxidant enzymes, peroxidation, and lipid metabolism. The mRNA expression of dihydrolipoamide S-acetyltransferase was upregulated after 40 and 70 min, while aconitase 1 was implicated in cuproptosis following 70 min of CZA exposure. Furthermore, the relative mRNA levels of glutathione reductase experienced a significant increase after 40 and 70 min of CZA exposure. In contrast, the relative mRNA levels of glutathione S-transferase and phospholipid-hydroperoxide glutathione peroxidase were significantly decreased after 70 min, suggesting a disruption in antioxidant defense and an imbalance in copper ions. Lipidomics results also unveiled an elevation in glycerophospholipids metabolism and the involvement of the lipoic acid pathway, predominantly contributing to cuproptosis. In summary, exposure to CZA induces cuproptosis in C. irritans, impacts glutathione-related enzymes, and alters glycerophospholipids, consequently triggering lipid oxidation.

Vibrio harveyi co-infected with Cryptocaryon irritans to orange-spotted groupers Epinephelus coioides.

The orange-spotted grouper (Epinephelus coioides) is a high economic value aquacultural fish in China, however, it often suffers from the outbreak of parasitic ciliate Cryptocaryon irritans as well as bacterium Vibrio harveyi which bring great loss in grouper farming. In the present study, we established a high dose C. irritans local-infected model which caused the mortality of groupers which showed low vitality and histopathological analysis demonstrated inflammatory response and degeneration in infected skin, gill and liver. In addition, gene expression of inflammatory cytokines was detected to assist the estimate of inflammatory response. Furthermore, we also found that the activity of Na+/K+ ATPase in gill was decreased in groupers infected C. irritans and the concentration of Na+/Cl- in blood were varied. Base on the morbidity symptom occurring in noninfected organs, we hypothesized that the result of morbidity and mortality were due to secondary bacterial infection post parasitism of C. irritans. Moreover, four strains of bacteria were isolated from the infected site skin and liver of local-infected groupers which were identified as V. harveyi in accordance of phenotypic traits, biochemical characterization and molecular analysis of 16S rDNA genes, housekeeping genes (gyrB and cpn60) and species-specific gene Vhhp2. Regression tests of injecting the isolated strain V. harveyi has showed high pathogenicity to groupers. In conclusion, these findings provide the evidence of coinfections with C. irritans and V. harveyi in orange-spotted grouper.

Transcriptome analysis of Cryptocaryon irritans tomont responding to Bacillus licheniformis treatment.

Cryptocaryon irritans is a ciliated obligate parasite that causes cryptocaryonosis (white spot disease) and poses great threat to marine fish farming. In recent years, the use of probiotics protects fish from pathogens, which has been identified as the sustainable and environmentally friendly tool to maintain the health and well-being of the host. Accordingly, Cryptocaryon irritans tomont and probiotic Bacillus strain (B.licheniformis, previously isolated from aquaculture water) were co-cultured to detect whether B. licheniformis has anti-C. irritants effect. The result showed that during 4-day incubation, B. licheniformi with 1 × 107 CFU/mL and 1 × 108 CFU/mL concentration effectively inhibited the incubation of C. irritans tomont, indicating that B. licheniformi could inhibit the transformation from reproductive tomont to infective theront of C. irritans. Later, C. irritans samples in the control (without B. licheniformi supplementation) and 1 × 107 CFU/mL B. licheniformi treatment group were sent for transcriptome analysis. Compare with the control group, a total of 3237 differentially expressed genes were identified, among which 626 genes were up-regulated and 2611 genes were down-regulated in 1 × 107 CFU/mL B. licheniformi group. Further Kyoto Encyclopedia of Genes and Genomes pathways analysis showed that anti-C. irritans mechanism of B. licheniformi was mainly involved in the energy metabolism (carbon metabolism, oxidative phosphorylation, biosynthesis of amino acids), transcription and translation (Ribosomes, spliceosomes, RNA transport, etc), lysosome-based degradation (lysosome, phagosome, protein processing in endoplasmic reticulum) and PI3K-Akt pathways. Our study findings raised the possibility of using marine microorganism B. licheniformi in handling aquaculture associated pathogen C. irritans, and preliminarily clarified the molecular mechanism.

Silencing of heat shock protein 90 (hsp90): Effect on development and infectivity of Ichthyophthirius multifiliis.

BACKGROUND: Recently, an increasing number of ichthyophthiriasis outbreaks has been reported, leading to high economic losses in fisheries and aquaculture. Although several strategies, including chemotherapeutics and immunoprophylaxis, have been implemented to control the parasite, no effective method is available. Hence, it is crucial to discover novel drug targets and vaccine candidates against Ichthyophthirius multifiliis. For this reason, understanding the parasite stage biology, host-pathogen interactions, molecular factors, regulation of major aspects during the invasion, and signaling pathways of the parasite can promote further prospects for disease management. Unfortunately, functional studies have been hampered in this ciliate due to the lack of robust methods for efficient nucleic acid delivery and genetic manipulation. In the current study, we used antisense technology to investigate the effects of targeted gene knockdown on the development and infectivity of I. multifiliis. Antisense oligonucleotides (ASOs) and their gold nanoconjugates were used to silence the heat shock protein 90 (hsp90) of I. multifiliis. Parasite stages were monitored for motility and development. In addition, the ability of the treated parasites to infect fish and cause disease was evaluated. RESULTS: We demonstrated that ASOs were rapidly internalized by I. multifiliis and distributed diffusely throughout the cytosol. Knocking down of I. multifiliis hsp90 dramatically limited the growth and development of the parasite. In vivo exposure of common carp (Cyprinus carpio) showed reduced infectivity of ASO-treated theronts compared with the control group. No mortalities were recorded in the fish groups exposed to theronts pre-treated with ASOs compared with the 100% mortality observed in the non-treated control fish. CONCLUSION: This study presents a gene regulation approach for investigating gene function in I. multifiliis in vitro. In addition, we provide genetic evidence for the crucial role of hsp90 in the growth and development of the parasite, suggesting hsp90 as a novel therapeutic target for successful disease management. Further, this study introduces a useful tool and provides a significant contribution to the assessing and understanding of gene function in I. multifiliis.

Effect of copper sulphate on Cryptocaryon irritans based on metabolome analysis.

Cryptocaryon irritans is one of the most harmful marine parasites in mariculture. Copper sulphate is often used to kill parasites and the influence of copper sulphate on the tomont stage of C. irritans was explored in this study. The results showed that excystment rate was not significantly affected when tomonts were exposed to 5 mg/L (76.7%) and 10 mg/L (78.9%) of copper sulphate for 3 h. However, excystment rate was significantly inhibited when exposed to 15 mg/L (33.3%) for 3 h and 5 mg/L (28.9%), 10 mg/L (33.3%) and 15 mg/L (33.3%) for 6 h. After treatment with high concentrations of copper sulphate, the interior of the tomonts was fuzzy under the microscope, and the division process could not be observed. Metabolomic results combined with preliminary transcriptome analysis results showed that the tomonts were induced to produce linoleate, riboflavin, inositol and other substances under the stress of Cu2+ , which affected the antioxidant mechanism of the body. Using MDA content determination and antioxidant enzyme activity analysis, copper sulphate was found to cause oxidative damage to tomonts by affecting the generation of metabolites, leading to the death of tomonts.

Antiparasitic effect of copper alloy mesh on tomont stage of Cryptocaryon irritans in aquaculture.

Copper alloy sheets have been shown to prevent cryptocaryoniasis. Therefore, we studied the potential efficiency of copper alloy mesh (CAM) in aquaculture tanks to prevent cryptocaryoniasis outbreaks. The effectivenesses of CAM against the tomont stage of Cryptocaryon irritans and in protecting fish from cryptocaryoniasis were tested both in vitro and in vivo. The mortality rate of C. irritans tomonts increased as the contact time with CAM rose and peaked at 70 min (100% of mortality). Morphological changes were observed such as the shrinking of the protoplasm of the treated tomonts, resulting in a larger gap between the cytoplasm and the cyst wall. Mitochondrial dysfunction due to shrinkage in the inner portion, outer and inner mitochondrial membrane damage and cytoplasmic vacuolation was revealed by ultrastructural analysis. The use of CAM effectively preventing reinfection was also provided. In comparison with group B (infected fish without CAM), both groups A (uninfected fish as a control group) and C (infected fish treated with CAM) had a 100% survival rate until the end of the trial. CAM has the same anticryptocaryoniasis effect as copper alloy sheets but is more advantageous due to its lightweight, reduced labor cost and lower purchase cost. It is noticeable that CAM exposure also prevents the excessive accumulation of copper ions in aquaculture sea water.

Effects of autophagy inhibition by 3-methyladenine on encystation, morphology, and metabolites of Cryptocaryon irritans.

Encystment is crucial for defense and reproduction in Cryptocaryon irritans. Therefore, understanding the encystment-related events in the protomont stage can help prevent and control C. irritans. Autophagy promotes protozoan parasite encystation. However, 3MA can inhibit autophagy. In this study, the effects of autophagy inhibition on encystation, survival rate, ultrastructural features, and metabolomic profiles of C. irritans, were evaluated using protomonts treated with 3MA (20 mM). The treatment with 3MA for about 4 h significantly lowered survival and encystation rates of protomonts to about 86.44% and 76.08%, respectively. Microstructural observations showed that the 3MA-treated protomonts showed deformed cell membranes and the cytoplasmic content spill. Furthermore, observation of the ultrastructure of 3MA-treated protomonts showed the destruction of organelles (Golgi bodies and mucocyst) and a lack of autophagosomes. However, no abnormality was observed in the control experiments. Furthermore, the metabolic analysis revealed suppression of metabolites, such as lipids, amino acids, and carbohydrates. These results demonstrate that 3MA can inhibit autophagy in C. irritans, thus hindering encystation, suppressing the metabolism of metabolites, and altering morphological ultrastructure in these parasites.

pH Regulates the Formation and Hatching of Cryptocaryon irritans Tomonts, Which Affects Cryptocaryoniasis Occurrence in Larimichthys crocea Aquaculture.

Cryptocaryon irritans are the main pathogens of white spot disease in marine teleost. However, the occurrence of cryptocaryoniasis is influenced by several abiotic factors including the pH. To explore the effect of pH on the life cycle of C. irritans (encystment, cleavage, and hatchability), protomonts and tomonts of C. irritans were incubated in seawater of 10 different pH levels (2-11). pH 8 was used as the control. The change in morphology and infectivity of theronts that hatched from tomonts against Larimichthys crocea were then recorded. We found that pH 6-9 had no significant effect on the encystment, cleavage, and hatching of the parasites. However, pH beyond this limit decreased the cleavage and hatching of the tomonts. Furthermore, extreme pH decreased the number of theronts hatched by each tomont and the pathogenicity of the theronts, but increased the aspect ratio of the theronts. Infectivity experiments further revealed that extreme pH significantly decreased the infectivity of C. irritans against L. crocea. In conclusion, the C. irritans can survive in pH of 5 to 10, but pH 6-9 is the optimal range for the reproduction and infectivity of C. irritans. However, extreme pH negatively affects these aspects. IMPORTANCECryptocaryon irritans is a ciliate parasite that causes "white spot disease" in marine teleosts. The disease outbreak is influenced by hosts and a range of abiotic factors, such as temperature, salinity, and pH. Studies have shown that change in pH of seawater affects the structure (diversity and abundance of marine organisms) of marine ecosystem. However, how pH affects the life cycle and survival of C. irritans, and how future ocean acidification will affect the occurrence of cryptocaryoniasis, are not well understood. In this study, we explored the effect of pH on the formation and hatching of C. irritans tomonts. The findings of this study provide the foundation of the environmental adaptation of C. irritans, the occurrence of cryptocaryoniasis, and better management of marine fish culture.

Characterization of the complete mitochondrial genome of Miamiensis avidus causing flatfish scuticociliatosis.

Miamiensis avidus is a parasitic pathogen that causes the disease scuticociliatosis in teleost fish species. It is a ciliate and a free-living marine protozoan belonging to the order Philasterida, subclass Scuticociliatida, class Oligohymenophorea, and phylum Ciliophora. The complete mt-genome of M. avidus was linear and 38,695 bp in length with 47 genes, including 40 protein-coding genes, two ribosomal RNA (rRNA) genes, and five transfer RNA (tRNA) genes. Of these, 20 genes typically belong to the clusters of orthologous groups, playing roles in energy production and conversion, translation, ribosomal structure and biogenesis, and defense mechanisms. This is the first report of sequencing and characterization of the mt-genome of M. avidus, which was observed to be linear and possessing the typical ciliate mitochondrial genome organization and phylogenetic relationships. Remarkable differences were observed between M. avidus and other ciliates in the mitochondrially encoded rRNAs, extensive gene loss in ribosomal genes and tRNAs, terminal repeat sequences, and stop codon usage. A comparative and phylogenetic analysis of M. avidus and Uronema marinum of the order Hymenostomatida, which is most closely related to the order Philasterida, signified the promise of the mitogenome data of M. avidus as a valuable genetic marker in species detection and taxonomic research. The present study has potential applications in epidemiological studies and host-parasite interaction investigations facilitating disease control.

Differential immune and metabolic responses underlie differences in the resistance of Siganus oramin and Trachinotus blochii to Cryptocaryon irritans infection.

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.

Molecular characterization of a profilin gene from a parasitic ciliate Cryptocaryon irritans.

Profilin, known as one of the core actin-binding proteins, is an integral part of actin-based cytoskeleton involved in cell motility, cytokinesis, neuronal differentiation, and synaptic plasticity. In this study, a putative profilin gene designated as CiProfilin (GenBank accession number: JX987286) was screened out from a cDNA library of Cryptocaryon irritans trophonts. The full-length cDNA of CiProfilin gene is 582 bp, containing an open reading frame (ORF) of 471 bp, which encodes a polypeptide consisting of 156 amino acids with a predicted molecular weight of 17.3 kDa. Quantification of CiProfilin mRNA expression by real-time PCR suggested that CiProfilin was expressed in all stages of C. irritans life cycle with a significantly higher level in trophonts. Five non-universal codons (TAAs) coding glutamines (Gln) were found in the ORF and mutated to CAAs (universal codons for Gln) by site-directed mutagenesis. Then the modified ORF was inserted into the plasmid pGEX-4T-1, the recombinant plasmid was subsequently transformed into Escherichia coli. The bacteria were subsequently induced to express the recombinant CiProfilin protein fused with glutathione S transferase (G-rCiProfilin), which was then purified with glutathione sepharose 4B and thrombin cleavage. The molecular weight and the antigenicity of rCiProfilin were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. The native CiProfilin was found abundant in the peripheral area beneath the cell membrane and around the cytostomes of theronts, suggesting its vital roles in food uptake, stomatogenesis, and parasitic invasion. Co-precipitation assay also revealed the activity of rCiProfilin in actin binding. This study will help further elucidate the specific roles of CiProfilin on the growth of C. irritans and the preliminary mechanism of its invasion to hosts.

Quantitative detection of parasitic ciliate Cryptocaryon irritans in seawater with an optimized sample processing method.

The protozoan Cryptocaryon irritans is one of the most important ectoparasites of marine fish, causing 'white spot disease' and mass mortality in aquaculture. To accurately predict disease outbreaks and develop prevention strategies, improved detection methods are required that are sensitive, convenient and rapid. In this study, a pair of specific primers based on the C. irritans 18S rRNA gene was developed and used in a real-time PCR (qPCR) assay. This assay was able to detect five theronts in 1 L of natural seawater. Furthermore, a linear model was established to analyse the log of Ct value and parasite abundance in seawater (y = -2.9623x + 24.2930), and the coefficient of determination (R2 ) value was 0.979. A lysis buffer was optimized for theront DNA extraction and used for storage sample. This method was superior to the commercial water DNA kit, and there was no significant degradation of DNA at room temperature for 24-96 hr. A dilution method was developed to manage qPCR inhibitors and used to investigate natural seawater samples in a net cage farm with diseased fish, and the findings were consistent with the actual situation. This study provides a valuable tool for assisting in the early monitoring and control of cryptocaryoniasis in aquaculture.

Barcode sequence could be a good target for developing a species-specific anti-parasite agent based on CRISPR-Cas9.

Parasitic infections are a severe issue in many regions of the world. We assume that if a chemical can destroy a DNA barcode sequence, then this chemical could be developed as a species-specific parasiticidal agent. To test this hypothesis, we designed sgRNAs that target the sequences of both a DNA barcode (ITS-2) and a control (5.8S rDNA) in Cryptocaryon irritans. In in vivo tests, we found that exposure to Cas9 mRNA mixed with sgRNAs was able to significantly reduce the hatching rate of tomont and the survival rate of theront. Quantitative Real-time PCR demonstrated that the DNAs of tomont and theront exposed to sgRNAs and Cas9 mRNA were significantly disrupted, no matter whether they were exposed to a single sgRNA or a mixture of two sgRNAs. DNA sequencing also suggested the test group that was exposed to a single sgRNA mixed with Cas9-induced mutation at sgRNA targeted fragments and the test group exposed to two sgRNAs combined with Cas9-induced deletion of large pieces. The findings and principles provided by this study contribute to the development of novel nucleic acid therapeutic drugs for cryptocaryoniasis and other parasitic diseases and provide insight into the development of species-specific parasiticidal agents.

Updating specific PCR primer for detection of Cryptocaryon irritans from reared Larimichthys polyactis.

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.

The gill transcriptome reveals unique antimicrobial features that protect Nibea albiflora from Cryptocaryon irritans infection.

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.

Morphology, phylogenetics and pathology of "red sore disease" (coinfection by Epistylis cf. wuhanensis and Aeromonas hydrophila) on sportfishes from reservoirs in the South-Eastern United States.

The aetiological agents of red sore disease (RSD) reportedly comprise a taxonomically ambiguous stalked ciliate (a species of Epistylis) and Aeromonas hydrophila. The taxonomic identity of each pathogen remains provisional: using supra-specific morphological features for the ciliate and culture-based methods that cannot delineate bacterial strain. On 7 and 9 November 2017 and 28 May 2020, biologists and anglers reported a local epizootic (Hiwassee and Chattahoochee river basins; Georgia) wherein some moribund fish presented RSD-like lesions. The ciliates were assigned to Epistylis by morphology. The ciliate is regarded as Epistylis cf wuhanensis, as nucleotide sequences from its small subunit ribosomal DNA were identical to those of Epistylis wuhanensis. The bacterium was identified as Aeromonas hydrophila by phenotypic markers and nucleotide sequences from the DNA gyrase subunit B; our sequences comprised 3 strains and phylogenetically were recovered sister to strains of Eurasian origin. Histological sections of lesions revealed effacement or partial deterioration of the epithelium covering scales, scale loss, haemorrhaging, necrosis, oedema, and extensive inflammatory infiltrate in the dermis. This is the first nucleotide sequence information for the symbionts implicated in RSD.

Comparative proteomic analysis provides insight into the key proteins as potential targets underlying the effect of malachite green against Ichthyophthirius multifiliis.

Target identification is important for drug discovery. Unfortunately, no drug targets have been found in Ichthyophthirius multifiliis until now and further limited development of the novel drug for Ichthyophthiriasis. In this study, an iTRAQ-based quantitative proteomic analysis was used to find the target of malachite green (MG), exhibiting greater efficacy than the existing drugs, against I. multifiliis trophonts in situ. We also verified the proteomic results by RT-qPCR, TEM and cell apoptosis assay. Our results showed that major variations in protein abundance were found among many of the ribosome proteins, indicating ribosome might be a candidate target. Furthermore, GO and KEGG pathway analyses of differentially expressed proteins (DEPs) revealed that ribosome and PI3K-Akt signalling pathway were remarkably enriched. Taken together, the above DEPs were also verified by RT-qPCR and morphological observations. This study provides insights into the key proteins enriched in PI3K-Akt signal pathway and ribosome pathway as potential targets of MG killing I. multifiliis, which could be served as targets for other less toxic drugs and be tested as potential treatments for I. multifiliis.