DNA Methylation Analysis Reveals Potential Mechanism in Takifugu rubripes Against Cryptocaryon irritans Infection.

Takifugu rubripes (T. rubripes) is a valuable commercial fish, and Cryptocaryon irritans (C. irritans) has a significant impact on its aquaculture productivity. DNA methylation is one of the earliest discovered ways of gene epigenetic modification and also an important form of modification, as well as an essential type of alteration that regulates gene expression, including immune response. To further explore the anti-infection mechanism of T. rubripes in inhibiting this disease, we determined genome-wide DNA methylation profiles in the gill of T. rubripes using whole-genome bisulfite sequencing (WGBS) and combined with RNA sequence (RNA-seq). A total of 4659 differentially methylated genes (DMGs) in the gene body and 1546 DMGs in the promoter between the infection and control group were identified. And we identified 2501 differentially expressed genes (DEGs), including 1100 upregulated and 1401 downregulated genes. After enrichment analysis, we identified DMGs and DEGs of immune-related pathways including MAPK, Wnt, ErbB, and VEGF signaling pathways, as well as node genes prkcb, myca, tp53, and map2k2a. Based on the RNA-Seq results, we plotted a network graph to demonstrate the relationship between immune pathways and functional related genes, in addition to gene methylation and expression levels. At the same time, we predicted the CpG island and transcription factor of four immune-related key genes prkcb and mapped the gene structure. These unique discoveries could be helpful in the understanding of C. irritans pathogenesis, and the candidate genes screened may serve as optimum methylation-based biomarkers that can be utilized for the correct diagnosis and therapy T. rubripes in the development of the ability to resist C. irritans infection.

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.

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.

Transcriptomic analysis of large yellow croaker (Larimichthys crocea) reveals the suppression of the inflammatory response from Cryptocaryon irritans infection.

Large yellow croaker (Larimichthys crocea) is the most productive marine fish in China. Cryptocaryon irritans is an extremely destructive parasite that causes great economic losses in large yellow croaker aquaculture industry. Therefore, it is very necessary to study the immune response of large yellow croaker in response to C. irritans infection. In this study, the transcriptomic profiles of large yellow croaker were sequenced and analyzed in the brain and head kidney at 72 h after C. irritans infection. Cytokines and chemokines related terms were significantly enriched based on the GO enrichment of down-regulated differentially expressed genes (DEGs) from the head kidney. Meanwhile, cytokine-cytokine receptor interaction was significantly enriched based on the KEGG enrichment of up-regulated DEGs from the brain and down-regulated DEGs from the head kidney, respectively. Moreover, the majority of inflammation-related DEGs were significantly up-regulated in the brain, but distinctly down-regulated in the head kidney. These results showed that the brain and head kidney might play different roles against C. irritans infection, and the inflammatory response of large yellow croaker may be restrained during C. irritans infection. Taken together, the transcriptomic analyses will be helpful to more comprehensively understand the immune mechanism of teleost against C. irritans infection, and provide a theoretical basis for the prevention and treatment of Cryptosporidiosis.

Effect of light exposure on circadian rhythm in theront excystment in Cryptocaryon irritans.

We examined the effects of light exposure on the theront excystment circadian rhythm in Cryptocaryon irritans using a newly invented apparatus, which enabled us to examine the excystment rhythms of theronts from tomonts with low labor. Using the apparatus, we examined the timings of theront excystment from tomonts exposed directly to light and from tomonts exposed to light-exposed seawater by counting the number of excysted theronts. We found that the theront excystment time changed only when tomonts were directly exposed to light, indicating that light reception is essential for circadian rhythm entrainment. When tomonts were exposed to light only once for 12 h, either on Day 1, Day 2, or Day 3 after leaving host and being encysted, the circadian rhythm was entrained according to the photoperiod given on tomonts. Tomonts exposed to a low light (1 lx) with 12L:12D photoperiod daily showed a circadian rhythm similar to that in tomonts exposed to an intense light (500 lx) under the same photoperiod. When tomonts were incubated at 22 °C, 25 °C, or 28 °C under the same photoperiod, almost the same circadian rhythm was developed, suggesting temperature has little effect on the circadian rhythm entrainment between the range, even though the date of excystment was delayed in lower temperatures. These results suggest the circadian rhythm of theront excystment can be entrained in tomonts on the seabed of inner bays where net-cage aquaculture is conducted, and be involved in the outbreaks of cryptocaryoniasis there.

Transcriptomic Analysis Reveals Functional Interaction of mRNA-lncRNA-miRNA in Trachinotus ovatus Infected by Cryptocaryon irritans.

The skin of Trachinotus ovatus is a crucial component of the mucosal immune system and serves as the primary site of infection by Cryptocaryon irritans. In order to investigate the significant role of skin in C. irritans infection, a comprehensive transcriptome analysis was conducted on skin tissues from the infection group, infection-adjacent group, and infection group compared with the infection-adjacent group (ATT_vs_PER, ADJ_vs_PER, ATT_vs_ADJ). This study identified differentially expressed long non-coding RNAs (DE lncRNAs), microRNAs (DE miRNAs), and differentially expressed genes (DEGs). The prediction of lncRNA target genes was accomplished by utilizing positional relationship (co-location) and expression correlation (co-expression) with protein-coding genes. Subsequently, functional enrichment analysis was conducted on the target genes of differentially expressed lncRNAs, revealing their involvement in signaling pathways such as tight junction, MAPK, and cell adhesion molecules. This study describes the regulatory network of lncRNA-miRNA-mRNA in T. ovatus skin tissue infected with C. irritans. Functional prediction analysis showed that differentially expressed lncRNA and miRNA may regulate the expression of immune genes such as interleukin-8 (il8) to resist the infection of C. irritans. Conducting additional research on these non-coding RNAs will facilitate a deeper understanding of their immune regulatory function in T. ovatus during C. irritans infection. The study of non-coding RNA in this study laid a foundation for revealing the molecular mechanism of the immune system of T. ovatus to respond to the infection of C. irritans. It provided a choice for the molecular breeding of Trachinotus ovatus against C. irritans.

Transcriptomic Variation of Amphiprion Percula (Lacepède, 1802) in Response to Infection with Cryptocaryon Irritans Brown, 1951.

Cryptocaryon irritans (Brown 1951) frequently infect the Pomacentridae fishes causing severe economic losses. However, the anti-C. irritans' molecular mechanism in these fishes remains largely unknown. To address this issue, we conducted RNA-Seq for C. irrtians-infected gills of the clownfish Amphiprion percula (Lacepède 1802) at the early (day 1) and late (day 3) stages of infection. A total of 1655 differentially expressed genes (DEGs) were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of DEGs showed a vast genetic variation related to the following aspects: ECM-receptor interaction, P13K-Akt signalling, cytokine-cytokine receptor interaction, and endocytosis. During the early phase of infection, key genes involved in ATP production, energy homeostasis, and stress control were abruptly increased. In the late phase, however, acute response molecules of the peripheral nervous system (synaptic transmission and local immunity), metabolic system triggering glycogen synthesis, energy maintenance, and osmoregulation were found to be critical. The highest number of upregulated genes (URGs) recovered during the early phase was included under the 'biological process' category, which primarily functions as response to stimuli, signalling, and biological regulation. In the late phase, most of the URGs were related to gene regulation and immune system processes under 'molecular function' category. The immune-related URGs of early infection include major histocompatibility complex (MHC) class-II molecules apparently triggering CD4+ T-cell-activated Th responses, and that of late infection include MHC class-1 molecules for the possible culmination of CD8+ T-cell triggered cytotoxicity. The high level of genic single nucleotide polymorphisms (SNPs) identified during the late phase of infection is likely to influence their susceptibility to secondary infection. In summary, the identified DEGs and their related metabolic and immune-related pathways and the SNPs may provide new insights into coordinating the immunological events and improving resistance in Pomacentridae fishes against C. irritans.

Identification of Genes Related to Resistance to Ichthyophthirius multifiliis Based on Co-expression Network Analysis in Grass Carp.

The ciliate protozoan Ichthyophthirius multifiliis is an essential parasite causing white spot disease in grass carp, leading to significant economic losses. Understanding the molecular basis of grass carp's response to I. multifiliis has important scientific and environmental values. The transcriptional network analysis offers a valuable strategy to decipher the changes in gene expression in grass carp infected with I. multifiliis. Our goal was to screen the genes and pathways involved in resistance to I. multifiliis in grass carp. The different traits exhibited by grass carp infected with I. multifiliis may be caused by the differences in gene expression among grass carp individuals. Herein, to reveal those resistance-associated genes against I. multifiliis infection, we performed RNA sequencing using weighted gene co-expression network analysis (WGCNA). The biological function analysis and hub gene annotation for highly relevant modules revealed that different pathogen recognition and clearance responses resulted in different resistance to I. multifiliis infection. Furthermore, gene enrichment analysis revealed that I. multifiliis invasion in the disease-resistant group mainly activated immune pathways, including scavenger receptor activity and kappa B kinase/NF-kappa B signaling. By the annotation of the highly correlated module of the hub gene, we revealed that the apoptosis and ribosome biogenesis-related genes were enriched in the disease-resistant grass carp. The results of the dark grey module showed that several genes were mainly enriched in the two-component system (ko02020) and steroid biosynthesis (ko00100), suggesting that they are resistance-associated and energy metabolism-associated genes. In the disease resistance group, hub genes mainly included Nlrc3, fos, AAP8, HAP2, HAX, cho2, and zgc:113,036. This study revealed the gene network associated with disease resistance after I. multifiliis infection. The disease resistance-related pathways and central genes identified in this study are candidate references for breeders breeding disease-resistant. The results of this study may also provide some references for the development of drugs to antagonize I. multifiliis infection.

Tandem mass tag (TMT) quantitative proteomics and phosphoproteomic of Takifugu rubripes infected with Cryptocaryon irritans.

In this study, we identified the differentially expressed proteins in gills stimulated by infected ciliates and analyzed the immune mechanisms of T. rubripes infected with the ciliate Cryptocaryon irritans. Through liquid chromatography analysis, a total of 144 proteins were identified with significant differences, of which 58 were upregulated and 86 were downregulated. Among phosphorylated proteins, we identified a total of 167 significantly different phosphorylated proteins, of which 44 were upregulated, 123 were downregulated, 60 were upregulated, and 208 were downregulated. We analyzed the data of proteomics and Phosphorylated proteome quantification protein omics to finally identify three phosphorylated proteins (RPS27, eNOS and CaM) and two phosphorylated protein kinases(CaMKII and MAPK1) as potential biomarkers for T. rubripes immune responses. We finally identified three phosphorylated proteins (RPS27, eNOS and CaM) and two phosphorylated protein kinases (CaMKII and MAPK1) as potential biomarkers of immune response of T. rubripes. Our research findings provide new insights into the immune mechanism of T. rubripes, which may serve as an effective indicator of C. irritans infection in T. rubripes.

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.

Acute immune responses in zebrafish and evasive behavior of a parasite - who is winning?

The protozoan parasite Ichthyophthirius multifiliis is an economically important parasite for the aquaculture- and ornamental fish industry. The parasite is abundant worldwide and infects the skin, gills and fins of freshwater fish species. For approximately the last fifty years the innate and protective immune mechanisms induced by I. multifiliis have been in focus in different fish hosts. By utilizing transgenic zebrafish, new tools to investigate this have emerged. The aim of this study was therefore to elucidate early immune responses in zebrafish larvae by using gene expression and in vivo imaging of neutrophil and macrophage behavior during infection. For the first time, zebrafish larvae were infected with the parasite and infection dynamics, parasite size and host-parasite interactions were investigated. Results showed that the larvae responded with mild inflammation and that the 12 compared to 5 days post fertilization larvae were significantly less susceptible. It was furthermore observed that neutrophils and macrophages were attracted to the parasites and that neutrophils reacted with neutrophil extracellular traps (NETs) when fighting the parasite. The parasite was rotating vigorously, presumably to impede the neutrophils and macrophages from attaching to it but on rare occasions, neutrophils and macrophages were able to kill the parasite. Based on these observations, we concluded that the parasite uses the rotation as an immune evasive strategy and that the zebrafish larvae respond with high activity from neutrophils and macrophages locally but systemically only with mild inflammation.

Inhibiting thioredoxin glutathione reductase is a promising approach to controlling Cryptocaryon irritans infection in fish.

Marine cultured fish often suffer from Cryptocaryon irritans infection, which causes enormous mortality. C. irritans is resistant to oxidative damage induced by zinc. To develop an effective drug to control the parasite, a putative thioredoxin glutathione reductase (CiTGR) from C. irritans was cloned and characterized. CiTGR was designed as a target to screen for inhibitors by molecular docking. The selected inhibitors were tested both in vitro and in vivo. The results showed that CiTGR is located in the nucleus of the parasite, possesses a common pyridine-oxidoreductases redox active center, and lacks a glutaredoxin active site. Recombinant CiTGR exhibited high TrxR activity but low glutathione reductase activity. Shogaol was found to significantly suppress TrxR activity and enhance toxicity of zinc on C. irritans (P < 0.05). The abundance of C. irritans on the fish body decreased significantly after oral administration of shogaol (P < 0.05). These results implied that CiTGR could be used to screen for drugs that weaken the resistance of C. irritans to oxidative stress, which is critical for controlling the parasite in fish. This paper deepens the understanding of the interaction between ciliated parasites and oxidative stress.

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.

Proteomics and Phosphoproteomic Analysis to Identify Spleen of Takifugu rubripes Infected Cryptocaryon irritans.

Takifugu rubripes is important commercially fish species in China and it is under serious threat from white spot disease (cyptocaryoniasis), which leads to heavy economic losses. In this study, we used proteomics and phosphoproteomic analysis to identify differentially abundant proteins in the spleen of T. rubripes infected with the Cryptocaryon irritans. We identified 5,307 proteins and 6,644 phosphorylated sites on 2,815 phosphoproteins using high-throughput proteomics analysis of the spleen of T. rubripes based on 26,421 unique peptides and 5,013 modified peptides, respectively. The 5,307 quantified host proteins, 40 were upregulated and 43 were downregulated in the infection group compared to the control group. Among the 2815 phosphoproteins, 44/120 were upregulated/downregulated, and 62/151 were upregulated/downregulated in the 6644 quantified phosphosites. Using the combination of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses, screening for significantly different phosphoproteins, motif analysis and protein-protein interaction analysis, we ultimately identified three phosphorylated proteins (G-protein-signaling modulator 1-like, zinc finger protein 850-like, and histone H1-like) and three phosphorylated protein kinases (serine/threonine-protein kinase homolog isoform X2, mitogen-activated protein kinase 5-like, and protein kinase C theta type) as potential biomarkers for T. rubripes immune responses. We then screened the phosphorylation sites of these biomarker proteins for further verification. Based on our results, we speculate that phosphorylation modification of the phosphorylation sites is involved in the immunity of T. rubripes against C. irritans.

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.

Antibacterial activity study of a novel piscidin 5-like type 4 from Larimichthys crocea.

As one of the key components of innate immune system, piscidins are likely to play pivotal role in the first defense line in fish. Piscidins own multiple resistance activity. A novel piscidin 5-like type 4 was excavated from Larimichthys crocea (termed Lc-P5L4) liver transcriptome immuned by Cryptocaryon irritans, and upregulated at 7 days post infection when secondary bacterial infection occurred. In the study, we characterized the antibacterial activity of Lc-P5L4. The liquid growth inhibition assay detected the recombinant Lc-P5L4 (rLc-P5L) had potent antibacterial activity to Photobacterium damselae. Scanning electron microscope (SEM) observed the cell surface of P. damselae collapsed to form pit, and membrane of some bacteria ruptured after co-incubation with rLc-P5L. Further, transmission electron microscope (TEM) was also employed to observe the intracellular microstructural damage, rLc-P5L4 caused cytoplasm contraction, pores formation and contents leakage. After knowing about its antibacterial effects, the preliminary antibacterial mechanism was also explored, western blot analysis showed rLc-P5L4 could bind to P. damselae through targeting to LPS. Agarose gel eletrophoresis analysis further showed rLc-P5L4 could also penetrate into cells and brought about genome DNA degradation. Therefore, rLc-P5L4 was of potential being a candidate to explore new antimicrobial drug or additive agent, especially to P. damselae.

Integrated mRNA and miRNA expression analyses for Cryptocaryon irritans resistance in large yellow croaker (Larimichthys crocea).

Large yellow croaker (Larimichthys crocea) is one of the most important mariculture fish in China. However, cryptocaryonosis caused by Cryptocryon irritans infection has brought huge economic losses and threatened the healthy and sustainable development of L. crocea industry. Recently, a new C. irritans resistance strain of L. crocea (RS) has been bred using genomic selection technology in our laboratory work. However, the molecular mechanisms for C. irritans resistance of RS have not been fully understood. MicroRNAs (miRNAs) are endogenous small non-coding RNAs that are post-transcriptional regulators, and they play vital roles in immune process of bony fish. Identification of anti-C.irritans relevant miRNA signatures could, therefore, be of tremendous translational value. In the present study, integrated mRNA and miRNA expression analysis was used to explore C. irritans resistance mechanisms of the L. crocea. RS as well as a control strain (CS) of L. crocea, were artificially infected with C. irritans for 100 h, and their gill was collected at 0 h (pre-infection), 24 h (initial infection), and 72 h (peak infection) time points. The total RNA from gill tissues was extracted and used for transcriptome sequencing and small RNA sequencing. After sequencing, 23,172 known mRNAs and 289 known miRNAs were identified. The differential expression was analyzed in these mRNAs and mRNAs and the interactions of miRNA-mRNA pairs were constructed. KEGG pathway enrichment analyses showed that these putative target mRNAs of differentially expressed miRNAs (DEMs) were enriched in different immune-related pathways after C. irritans infection in RS and CS. Among them, necroptosis was the immune-related pathway that was only significantly enriched at two infection stages of RS group (RS-24 h/RS-0h and RS-72 h/RS-0h). Further investigation indicates that necroptosis may be activated by DEMs such as miR-133a-3p, miR-142a-3p and miR-135c, this promotes inflammation responses and pathogen elimination. These DEMs were selected as miRNAs that could potentially regulate the C. irritans resistance of L. crocea. Though these inferences need to be further verified, these findings will be helpful for the research of the molecular mechanism of C. irritans resistance of L. crocea and miRNA-assisted molecular breeding of aquatic animals.

Characterization of the MMP9 Gene and Its Association with Cryptocaryon irritans Resistance Traits in Trachinotus ovatus (Linnaeus, 1758).

The MMPs are endogenous proteolytic enzymes that require zinc and calcium as cofactors. MMP9 is one of the most complex matrix metalloproteinases in the gelatinase family and has many biological functions. In mammals, mmp9 is thought to be closely associated with cancer. However, studies in fish have rarely been reported. In this study, to understand the expression pattern of the ToMMP9 gene and its association with the resistance of Trachinotus ovatus to Cryptocaryon irritans, the sequence of the MMP9 gene was obtained from the genome database. The expression profiles were measured by qRT-PCR, the SNPs were screened by direct sequencing, and genotyping was performed. The ToMMP9 gene contained a 2058 bp ORF encoding a putative amino acid sequence of 685 residues. The homology of the ToMMP9 in teleosts was more than 85%, and the genome structure of ToMMP9 was conserved in chordates. The ToMMP9 gene was expressed in different tissues of healthy individuals and was highly expressed in the fin, the gill, the liver and the skin tissues. The ToMMP9 expression in the skin of the infected site and its adjacent sites increased significantly after C. irritans infection. Two SNPs were identified in the ToMMP9 gene, and the SNP (+400A/G) located in the first intron was found to be significantly associated with the susceptibility/resistance to C. irritans. These findings suggest that ToMMP9 may play an important role in the immune response of T. ovatus against C. irritans.

Effects of Cryptocaryon irritans infection on the histopathology, oxidative stress, immune response, and intestinal microbiota in the orange-spotted grouper Epinephelus coioides.

Cryptocaryon irritans is a parasitic ciliate of marine fish, causing serious mortality and economic loss of grouper. In this study, the orange-spotted grouper (Epinephelus coioides) were separately exposed to C. irritans infection for 72 h at a dose of 5000 or 10000 active theronts per fish, and we evaluated the changes in histopathology, oxidative stress, immune response, and intestinal microbiota composition. The results showed that C. irritans infection caused pathological alteration on the skin, gills, and liver of E. coioides. Oxidative stress responses occurred in the liver and gills, reflected in the corresponding antioxidant enzyme and gene indexes. The mRNA expression levels of inflammation-related genes (IL-1ß, IL-6, and IL-8) and the mediators of apoptosis (casp3, casp9, and cytc) were increased in the liver and gills of the fish. C. irritans infection also affected the diversity and composition of intestinal microbiota. Specifically, the relative abundance of Firmicutes was increased, whereas that of Proteobacteria was decreased. Several potentially beneficial bacteria (Pandoraea, Clostridium sensu stricto 1, Christensenellaceae R-7 group, and Weissella) were decreased, whereas pathogenic bacteria (Streptococcus and Acinetobacter) were increased. In conclusion, this study reveals that C. irritans infection caused histopathology, immune disorders, and intestinal microbial community variation in E. coioides.

Host responses against the fish parasitizing ciliate Cryptocaryon irritans.

The parasitic ciliate Cryptocaryon irritans, which infects almost all marine fish species occurring in both tropical and subtropical regions throughout the world. The disease, cryptocaryonosis, accounts for significant economic losses to the aquaculture industry. This review attempts to provide a comprehensive overview of the biology of the parasite, host-parasite interactions and both specific and non-specific host defense mechanisms are responsible for the protection of fish against challenge infections with this ciliate. Also, this article reflects the current interest in this subject area and the quest to develop an available vaccine against the disease. Due to the high frequency of clinical fish cryptocaryonosis, the study of fish immune responses to C. irritans provides an optimal experimental model for understanding immunity against extracellular protozoa.