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.

Aggregation and proliferation of B cells and T cells in MALTs upon Cryptocaryon irritans infection in large yellow croaker Larimichthys crocea.

Mucosal immunity in mucosa-associated lymphoid tissues (MALTs) plays crucial roles in resisting infection by pathogens, including parasites, bacteria and viruses. However, the mucosal immune response in the MALTs of large yellow croaker (Larimichthys crocea) upon parasitic infection remains largely unknown. In this study, we investigated the role of B cells and T cells in the MALTs of large yellow croaker following Cryptocaryon irritans infection. Upon C. irritans infection, the total IgM and IgT antibody levels were significantly increased in the skin mucus and gill mucus. Notably, parasite-specific IgM antibody level was increased in the serum, skin and gill mucus following parasitic infection, while the level of parasite-specific IgT antibody was exclusively increased in MALTs. Moreover, parasitic infection induced both local and systemic aggregation and proliferation of IgM+ B cells, suggesting that the increased levels of IgM in mucus may be derived from both systemic and mucosal immune tissues. In addition, we observed significant aggregation and proliferation of T cells in the gill, head kidney and spleen, suggesting that T cells may also be involved in the systemic and mucosal immune responses upon parasitic infection. Overall, our findings provided further insights into the role of immunoglobulins against pathogenic infection, and the simultaneous aggregation and proliferation of both B cells and T cells at mucosal surfaces suggested potential interactions between these two major lymphocyte populations during parasitic infection.

Dietary citric acid decreased the theront number of Cryptocaryon irritans (Ciliata) in seawater-adapted tilapia (Oreochromis niloticus).

Cryptocaryoniasis remains a major parasitic disease and economic challenge for marine aquaculture. Cryptocaryoniasis in marine fish is caused by Cryptocaryon irritans (Ciliata). A theront is a motile, free-swimming stage in the life cycle of C. irritans, which is typically the infective stage that actively seeks out a host to initiate infection. Population density and growth rate of theronts were investigated in Nile tilapia, Oreochromis niloticus fed with citric acid-supplemented feed. The experiment involved feeding three diets with graded levels of citric acid (0, control diet, 0.5, 1 and 1.5 g kg-1 diet), to seawater-adapted Nile tilapia (O. niloticus) juveniles for 21 days. The results showed that citric acid in the fish feed had an impact on the theront number of C. irritans in a manner of dose-dependent. In the experimental cohort administered a diet supplemented with 1.5 g kg-1 citric acid, the population density of theronts was observed to be significantly reduced, measured at 29 ± 3.34, as opposed to 473.34 ± 16.48 in the control group at the culmination of the experiment. The observed population growth rate of theronts was significantly higher in the control group than in the group administered the citric acid feed (p < .005). The growth rate (r d-1 ) was 0.12 in control, 0.05 in 0.5 g kg-1 , 0.031 in 1 g kg-1 , and - 0.031 in 1.5 g kg-1 citric acid-supplemented groups. Fish growth and feed conversion ratio were not affected by the citric acid in the feed. In conclusion, the findings of this investigation provide a valuable addition to our understanding of the potential protective effects of citric acid supplementation for fish against the C. irritans parasite. This is evidenced by the observed reduction in theronts present in the water.

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.

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.

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.

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.

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.

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.

Genome-Wide Identification of Trachinotus ovatus Antimicrobial Peptides and Their Immune Response against Two Pathogen Challenges.

Golden pompano, Trachinotus ovatus, as a highly nutritious commercially valuable marine fish, has become one of the preferred species for many fish farmers due to its rapid growth, wide adaptability, and ease of feeding and management. However, with the expansion of aquaculture scale, bacterial and parasitic diseases have also become major threats to the golden pompano industry. This study, based on comparative genomics, shows the possibility of preferential evolution of freshwater fish over marine fish by analyzing the phylogenetic relationships and divergence times of 14 marine fish and freshwater fish. Furthermore, we identified antimicrobial peptide genes from 14 species at the genomic level and found that the number of putative antimicrobial peptides may be related to species evolution. Subsequently, we classified the 341 identified AMPs from golden pompano into 38 categories based on the classification provided by the APD3. Among them, TCP represented the highest proportion, accounting for 23.2% of the total, followed by scolopendin, lectin, chemokine, BPTI, and histone-derived peptides. At the same time, the distribution of AMPs in chromosomes varied with type, and covariance analysis showed the frequency of its repeat events. Enrichment analysis and PPI indicated that AMP was mainly concentrated in pathways associated with disease immunity. In addition, our transcriptomic data measured the expression of putative AMPs of golden pompano in 12 normal tissues, as well as in the liver, spleen, and kidney infected with Streptococcus agalactiae and skin infected with Cryptocaryon irritans. As the infection with S. agalactiae and C. irritans progressed, we observed tissue specificity in the number and types of responsive AMPs. Positive selection of AMP genes may participate in the immune response through the MAPK signaling pathway. The genome-wide identification of antimicrobial peptides in the golden pompano provided a complete database of potential AMPs that can contribute to further understanding the immune mechanisms in pathogens. AMPs were expected to replace traditional antibiotics and be developed into targeted drugs against specific bacterial and parasitic pathogens for more precise and effective treatment to improve aquaculture production.

Effect of Biokos, a natural lipopeptide surfactant extracted from a bacterium of the Pseudomonas genus, on infection of Cryptocaryon irritans.

Cryptocaryoniasis (marine white spot disease), caused by Cryptocaryon irritans, is a major threat to marine fish cultures in tropical and subtropical waters, and a serious nuisance to hobbyists with saltwater fish tanks. With only classical treatment schedules such as copper salts or hyposaline baths being available, control of the disease remains a challenge. In this study, we investigated the effect of Biokos, a viscosin-like lipopeptide surfactant extracted from a bacterium of the Pseudomonas genus, on the external life stages of C. irritans, including theronts, protomonts and tomonts. The present study demonstrated that the compound has an antiparasitic effect on all tested external stages of the parasite. In particular, when Biokos was used at 48 mg/L, it was able to kill almost all theronts and protomonts within 1 h in in vitro experiments, and using the same concentration in an in vivo challenge experiment, the parasitic load was reduced by more than 95% compared to the control group with no Biokos. Additionally, cultured fish cells were able to proliferate, and fish showed no adverse signs at Biokos concentrations that were effective in killing the parasite. Thus, Biokos may be a promising way for preventing or reducing the burden of this parasitic disease in the future.

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.

Treatments of orange-spotted grouper (Epinephelus coioides) against Cryptocaryon irritans with •OH, ClO2 or HCHO: Survival, physiological and histological response.

Cryptocaryon irritans causes one of the most serious diseases in various wild and cultured marine fish, leading to mass mortality and economic loss. In this study, hydroxyl radical (•OH) solution produced by strong ionization discharge combined with water jet cavitation effect was injected into orange-spotted grouper (Epinephelus coioides) aquaculture tanks for C. irritans control. The results showed that all C. irritans theronts were inactivated by •OH solution at concentrations of 0.5 mg/L within 2 min. •OH could induce alteration of shape, the absence of motility and macronucleus dispersion in theronts. A possible explanation was that the macronucleus of C. irritans might be damaged by •OH; as a result, its metabolism and life activities were disturbed. The •OH treatment increased the survival rate of E. coioides challenged with C. irritans from 64.7 ± 8.0% (mean ± SD) to 100% and reduced their infection intensity significantly. Stress response biomarkers such as malonaldehyde, glutathione, glutathione peroxidase, superoxide dismutase (SOD) and catalase levels in the gills of E. coioides at different time points were analysed. The SOD activity in the •OH group first decreased and then recovered to the initial level at the end of the experiment. The other stress response biomarkers had no significant difference from that in the uninfected control group after •OH treatment. Additionally, the gill of E. coioides in the •OH group exhibited slight and reversible transformation compared with the uninfected control group. Compared with •OH treatment, chlorine dioxide and formalin treatment reduced the survival rate, induced oxidative damage and changed the histological gill structure in E. coioides. In conclusion, •OH could be applied effectively to control C. irritans infection without affecting the normal physiological condition of E. coioides.

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.

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.

Comparative transcriptome analysis reveals immunoregulation mechanism of lncRNA-mRNA in gill and skin of large yellow croaker (Larimichthys crocea) in response to Cryptocaryon irritans infection.

BACKGROUND: Cryptocaryonosis caused by Cryptocaryon irritans is one of the major diseases of large yellow croaker (Larimichthys crocea), which lead to massive economic losses annually to the aquaculture industry of L. crocea. Although there have been some studies on the pathogenesis for cryptocaryonosis, little is known about the innate defense mechanism of different immune organs of large yellow croaker. RESULTS: In order to analyze the roles of long non-coding RNAs and genes specifically expressed between immune organs during the infection of C. irritans, in this study, by comparing transcriptome data from different tissues of L. crocea, we identified tissue-specific transcripts in the gills and skin, including 507 DE lncRNAs and 1592 DEGs identified in the gills, and 110 DE lncRNAs and 1160 DEGs identified in the skin. Furthermore, we constructed transcriptome co-expression profiles of L. crocea gill and skin, including 7,503 long noncoding RNAs (lncRNAs) and 23,172 protein-coding genes. Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that the DEGs and the target genes of the DE lncRNAs in the gill were specifically enriched in several pathways related to immune such as HIF-1 signaling pathway. The target genes of DE lncRNAs and DEGs in the skin are specifically enriched in the complement and coagulation cascade pathways. Protein-protein interaction (PPI) network analysis identified 3 hub genes including NFKBIA, TNFAIP3 and CEBPB, and 5 important DE lncRNAs including MSTRG.24134.4, MSTRG.3038.5, MSTRG.27019.3, MSTRG.26559.1, and MSTRG.10983.1. The expression patterns of 6 randomly selected differentially expressed immune-related genes were validated using the quantitative real-time PCR method. CONCLUSIONS: In short, our study is helpful to explore the potential interplay between lncRNAs and protein coding genes in different tissues of L. crocea post C. irritans and the molecular mechanism of pathogenesis for cryptocaryonosis. HIGHLIGHTS: Skin and gills are important sources of pro-inflammatory molecules, and their gene expression patterns are tissue-specific after C. irritans infection. 15 DEGs and 5 DE lncRNAs were identified as hub regulatory elements after C. irritans infection The HIF-1 signaling pathway and the complement and coagulation cascade pathway may be key tissue-specific regulatory pathways in gills and skin, respectively.

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.

Mucosal immunoglobulin response in Epinephelus coioides after Cryptocaryon irritans infection.

The teleost mucosal immune system consists mainly of the skin, gills and gut, which play crucial roles in local immune responses against invading organisms. Immunoglobulins are essential molecules in adaptive immunity that perform crucial biological functions. In our study, a mucosal immunity model was constructed in Epinephelus coioides groupers after Cryptocaryon irritans infection, according to previous experience. Total IgM and IgT in the groupers increased in the serum and mucus in the immune group, whereas only pathogen-specific IgM were detected existence. More critically, pathogen-specific IgM was detected in the head kidney, gill and skin supernatants, thus suggesting that the systematic immune and mucosal immune system secreted immunoglobulins. Furthermore, an early response in the skin was observed, on the basis of the detection of pathogen-specific IgM in the skin supernatant. In conclusion, this research characterized the grouper IgM and IgT in mucosal immune responses to pathogens in the gills and skin, thus providing a theoretical basis for future studies on vaccines against C. irritans.

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.