Cryptocaryon irritans is a highly detrimental parasite in mariculture, causing significant economic losses to the aquaculture industry of Larimichthys crocea. In recent years, copper and copper alloy materials have been used to kill parasites. In this study, the effect of copper plates on the tomont period of C. irritans was explored. The findings indicated that copper plates effectively eradicated tomonts, resulting in a hatching rate of 0. The metabolomic analysis revealed that a total of 2,663 differentially expressed metabolites (1,032 up-regulated and 1,631 down-regulated) were screened in the positive ion mode, and 2,199 differentially expressed metabolites (840 up-regulated and 1,359 down-regulated) were screened in the negative ion mode. L-arginine and L-aspartic acid could be used as potential biomarkers. Copper plate treatment affected 25 metabolic pathways in the tomont, most notably influencing histidine metabolism, retinol metabolism, the biosynthesis of phenylalanine, tyrosine, and tryptophan, as well as arginine and proline metabolism. It was shown that high concentrations of copper ions caused a certain degree of disruption to the metabolome of tomonts in C. irritans, thereby impacting their metabolic processes. Consequently, this disturbance ultimately leads to the rapid demise of tomonts upon exposure to copper plates. The metabolomic changes observed in this study elucidate the lethal impact of copper on C. irritans tomonts, providing valuable reference data for the prevention and control of C. irritans in aquaculture.

The Japanese puffer, Takifugu rubripes, is a commercially important fish species in China that is under serious threat from white spot disease (cyptocaryoniasis), which leads to heavy economic losses. We previously found that interleukin-1β (IL-1β), an important cytokine with a potential role in resistance against pathogens, was one of the most significantly differentially up-regulated proteins in the gills and spleen of T. rubripes infected by the protozoan parasite Cryptocaryon irritans. In this study, we assessed the potential function of T. rubripes IL-1β (TrIL-1β) in fish infected with C. irritans. Phylogenetic analysis indicated that the TrIL-1β protein sequence was most closely related to that of Atlantic salmon (Salmo salar) (67.2 %). The incubation experiments revealed that TrIL-1β may reduce trophont activity by destroying membranes. Immunofluorescence experiments showed that recombinant TrIL-1β promoted the expression of endogenous IL-1β, which penetrated and disrupted the cell membranes of trophonts. Transmission electron microscopy showed that the IL-1β group had less tissue damage compared with control groups of fish. IL-1β-small interfering RNA and IL-1β overexpression experiments were performed in head kidney primary cells, and challenge experiments were performed in vitro. Quantitative RT-PCR results showed that TrIL-1β regulated and activated MyD88/NF-κB and MyD88/MAPK/p38 signaling pathways during C. irritans infection. TrIL-1β also promoted the differential expression of IgM, showing that it was involved in humoral immunity of T. rubripes. The cumulative mortality experiment show that TrIL-1β could protect fish against C. irritans infection. These results enrich current knowledge about the molecular structure of TrIL-1β. They also suggested that recombinant TrIL-1β could be used as an adjuvant in a subunit vaccine against C. irritans infection, which is of profound importance for the prevention and control of parasitic diseases in T. rubripes.

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.

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.

This study presents a genome-wide identification of NOD-like receptors (NLRs) in the golden pompano, key to its innate immunity. We identified 30 ToNLRs, analyzing their chromosomal positions, characteristics, evolutionary relationships, evidence of positive selection, and synteny with the yellowtail kingfish. Our findings categorize these NLRs into three main subgroups: NLRA, NLRC, and the distinct ToNLRX1. Post-exposure to Streptococcus agalactiae, most ToNLRs increased expression in the spleen, whereas NLRC3like13, NLRC3like16, and NLRC3like19 so in the kidneys. Upon Cryptocaryon irritans exposure, we categorized our groups based on the site of infection into the control group (BFS), the trophont-attached skin (TAS), and the nearby region skin (NRS). ToAPAF1 and ToNOD1 expressions rose in the NRS, in contrast to decreased expressions of ToNLRC5, ToNWD1 and ToCIITA. Other ToNLRs showed variable expressions in the TAS. Overall, this research lays the groundwork for further exploration of innate immunity in the golden pompano.

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.

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.

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.

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.

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.