Novel identification of mixed infection of Lactococcus garvieae and Cryptocaryon irritans isolated from cultured Trachinotus ovatus in China.

Lactococcus garvieae has recently been identified and listed as one of the causative agents of hyperacute hemorrhagic sepsis in fish. In intensive recirculating aquaculture systems where there are high fish densities and minimal water changes, not only will it be conducive to the growth of bacteria, but Cryptocaryon irritans as a marine protozoan fish parasite is also prone to appear. This study reports the disease status of Trachinotus ovatus in an aquaculture area in Yangjiang City, Guangdong Province. Through the diagnosis of clinical symptoms of the diseased fish, identification of specific primers, 16s rRNA sequences phylogenetic tree analysis, physiological and biochemical identification, and observation of histopathological sections, the result of the experiment is that the mass death of T. ovatus is caused by a mixture of L. garvieae and C. irritants infections. Subsequently, regression infection experiments were performed to verify Koch's law. It was confirmed that the pathogen had strong virulence to T. ovatus. This is the first time that the co-infection of L. garvieae and C. irritans to T. ovatus was found in South China. The research results of this experiment have certain enlightenment significance for the epidemic trend of fish diseases in relevant sea areas.

Metabolomic changes in Cryptocaryon irritans from Larimichthys crocea after exposure to copper plate.

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.

Effects of Gene Alternative Splicing Events on Resistance to Cryptocaryonosis of Large Yellow Croaker (Larimichthys crocea).

Large yellow croaker (L. crocea) is a productive species in marine aquaculture with great economic value in China. However, the sustainable development of large yellow croaker is hampered by various diseases including cryptocaryonosis caused by Cryptocaryon irritans. The genetic regulation processes for cryptocaryonosis in large yellow croaker are still unclear. In this present study, we analyzed differential alternative splicing events between a C. irritans resistance strain (RS) and a commercial strain (CS). We identified 678 differential alternative splicing (DAS) events from 453 genes in RS and 719 DAS events from 500 genes in CS. A set of genes that are specifically alternatively spliced in RS was identified including mfap5, emp1, and trim33. Further pathway analysis revealed that the specifically alternative spliced genes in RS were involved in innate immune responses through the PRR pathway and the Toll and Imd pathway, suggesting their important roles in the genetic regulation processes for cryptocaryonosis in large yellow croaker. This study would be helpful for the studies of the pathogenesis of cryptocaryonosis and dissection of C. irritans resistance for L. crocea.

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.

Immunological effects of DNA vaccination and interleukin utilization as an adjuvant in Astyanax lacustris immunized against Ichthyophthirius multifiliis.

The increasing significance of the aquaculture sector and commercially valuable species underscores the need to develop alternatives for controlling diseases such as Ichthyophthirius multifiliis-induced ichthyophthiriasis. This ciliated protozoan parasite threatens nearly all freshwater fish species, causing substantial losses in the fishery industry. Despite this, effective large-scale treatments are lacking, emphasizing the necessity of adopting preventive strategies. While the pathogenesis of ichthyophthiriasis and its immune stimulation allows for vaccination strategies, precise adjustments are crucial to ensure the production of an effective vaccine compound. Therefore, this study aimed to evaluate the impact of immunizing Astyanax lacustris with a genetic vaccine containing IAG52A from I. multifiliis and the molecular adjuvant IL-8 from A. lacustris. Transcript analysis in immunized A. lacustris indicated mRNA production in fish muscles, demonstrating an expression of this mRNA. Fish were divided into five groups, receiving different vaccine formulations, and all groups received a booster dose 14 days after the initial immunization. Samples from vaccinated fish showed increased IL-1ß mRNA expression in the spleen within 6 h post the second dose and after 14 days. In the head kidney, IL-1ß mRNA expression showed no significant difference at 6 and 24 h but an increase was noted in fish injected with IAG and IAG + IL-8 after 14 days. IL-8 mRNA expression in the spleen and kidney did not significantly differ from the control group. Histological analysis revealed no variation in leukocyte concentration at 6 and 24 h post-vaccination; however, after 14 days, the groups injected with IAG and IAG + IL-8 exhibited a higher leukocyte density at the application sites than the control. The obtained data suggest that the used vaccine is transcribed, indicating its potential to stimulate innate immune response parameters through mRNA cytokine expression and leukocyte migration.

Functional role of TrIL-1ß in Takifugu rubripes defense against Cryptocaryon irritans infection.

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-1ß (IL-1ß), 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-1ß (TrIL-1ß) in fish infected with C. irritans. Phylogenetic analysis indicated that the TrIL-1ß protein sequence was most closely related to that of Atlantic salmon (Salmo salar) (67.2 %). The incubation experiments revealed that TrIL-1ß may reduce trophont activity by destroying membranes. Immunofluorescence experiments showed that recombinant TrIL-1ß promoted the expression of endogenous IL-1ß, which penetrated and disrupted the cell membranes of trophonts. Transmission electron microscopy showed that the IL-1ß group had less tissue damage compared with control groups of fish. IL-1ß-small interfering RNA and IL-1ß overexpression experiments were performed in head kidney primary cells, and challenge experiments were performed in vitro. Quantitative RT-PCR results showed that TrIL-1ß regulated and activated MyD88/NF-κB and MyD88/MAPK/p38 signaling pathways during C. irritans infection. TrIL-1ß 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-1ß could protect fish against C. irritans infection. These results enrich current knowledge about the molecular structure of TrIL-1ß. They also suggested that recombinant TrIL-1ß 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.

White spots amidst the gold: ultrastructural and histological aspects of the chronic inflammatory response of goldfish with ichthyophthiriasis.

Ichthyophthirius multifiliis, the causative agent of white spot disease, is a ciliated protozoan parasite that infects freshwater fish and induces high mortality. Outbreaks occur both in natural and production sites. The aim of the present study was to describe the lesions caused by chronic infection by I. multifiliis in goldfish (Carassius auratus) from an ornamental fish farm, highlighting important ultrastructural aspects of this protozoan. Damaged skin and gills, collected from fish with white or ulcerative skin lesions, were routinely processed for histological analysis and transmission electron microscopy. The parasitic forms present in the skin were associated with an inflammatory infiltrate consisting of macrophages, lymphocytes and other polymorphonuclear cells. The lesions associated with the presence of the parasite were organized in the form of granulomas, with macrophages in the layers closest to the parasites. A trophont-thickened membrane and induction of granulomatous inflammation were identified in this study as mechanisms for evasion of the immune response. We concluded that the presence of I. multifiliis trophonts resulted in the formation of granulomatous inflammation, whether associated or not with pathogen lysis, suggesting that the parasite can use an inflammatory response to evade the immune response.

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.

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.

Antiparasitic efficacy of flavonoids identified from Psoralea corylifolia against Tetrahymena piriformis in guppy (Poecilia reticulate).

Tetrahymena piriformis belongs to the ciliated protists (ciliates), causing severe economic losses in aquaculture. Chemical drugs currently used usually have toxic side effects, and there is no specific drug against Tetrahymena. Therefore, it is an urgent need to identify new antiparasitic lead compounds. In the present study, the in vitro parasiticidal activity of ethyl acetate (EtOAc) extracts and water extracts from 22 selected traditional Chinese medicines (TCMs) were evaluated against T. piriformis. The EtOAc extract of P. corylifolia turned out to be the most active with the minimum parasiticidal concentration of 100 mg/L within 3 h. Thus, it was separated into 12 fractions by the first-dimensional (D1) normal phase liquid chromatography (NPLC), meanwhile combining with in vitro antiparasitic tests for activity tracking. Subsequently, 8 flavonoids were identified in the active fractions by the second-dimensional (D2) reverse phase liquid chromatography (RPLC) tandem high-resolution mass spectrometry. According to the results, 5 flavonoids were selected for in vitro antiparasitic test, of which isobavachalcone showed the minimum parasiticidal concentration of 3.125 mg/L in 2 h. Bathing treatment of infected guppies with isobavachalcone could significantly reduce the burden of T. piriformis, obtaining a 24-h median effective concentration (24-h EC50) value of 1.916 mg/L. And the concentration of isobavachalcone causing guppies to die within 24 h is 39 times than that of 24-h EC50. The results demonstrated that isobavachalcone has the potential to be developed into a novel commercial fish drug against T. piriformis.

Moniezia benedeni infection increases IgE+ cells in sheep (Ovis aries) small intestine.

The concentration of immunoglobulin (Ig) E is the lowest among serum Igs, but it can induces type I hypersensitivity and plays an important role in anti-parasitic infection. The present study aimed to explore the residence characteristics of IgE+ cells in the sheep small intestine and the impact of Moniezia benedeni infection on them. The recombinant plasmids pET-28a-IgE were constructed and induced and expressed in Escherichia coli. BL21 (DE3). The rabbit anti-sheep IgE polyclonal antibody was prepared using the obtained recombinant protein as antigen. Finally, the levels of IgE+ cells in the small intestine of healthy (Control group) and naturally M. benedeni-infected (Infected group) sheep were detected analyzed. The results showed that the rabbit anti-sheep IgE polyclonal antibody with good immunogenicity (titer = 1: 128000) could specifically bind to the heavy chain of natural sheep IgE. In the Control group, the IgE+ cells were mainly distributed in lamina propria of the small intestine, and the densities were significantly decreased from duodenum to ileum (P<0.05), with respective values of (4.28 cells / 104 µm2, 1.80 cells / 104 µm2, and 1.44 cells / 104 µm2 in duodenum, jejunum, and ileum. In the Infected group, IgE+ cells density were 6.26 cells / 104 µm2, 3.01 cells / 104 µm2, and 2.09 cells / 104 µm2 in duodenum, jejunum and ileum respectively, which were significantly higher in all segments compared to the Control group (P<0.05), increasing by 46.26%, 67.22% and 45.14%, respectively. In addition, compared with the Control group, the IgE protein levels were significantly increased in all intestinal segments of the Infected group (P<0.01), however, there was no significant differences among the different intestinal segments within the same group (P>0.05). The results demonstrated that M. benedeni infection could significantly increase the content of IgE and the distribution density of its secreting cells in sheep small intestine. The intestinal mucosal immune system of sheep presented obvious specificity against M. benedeni infection. This lays a good foundation for further exploring molecular mechanisms of the intestinal mucosal immune system monitoring and responding to M. benedeni infection.

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 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.

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.

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.

Integration of transcriptomic and metabolomic profiling of encystation in Cryptocaryon irritans regulated by rapamycin.

Encystation in Cryptocaryon irritans is a fundamental process for environmental resistance and development. Autophagy participates in the encystation of ciliates, and rapamycin can induce autophagy in the cells. A set of genes and metabolites related to autophagy and encystation are highly elaborative. The existence of these genes and metabolites and their role are well characterized. However, little is known about their role in protozoans such as ciliates. The newly produced C. irritans protomonts were exposed to an optimal concentration of rapamycin (1400 nM), and the survival, encystation, microstructure/ultrastructure, transcriptomic and metabolomic profile in treated and control protomonts were investigated. The results showed that exposure of protomonts to rapamycin at 4 h significantly lowered the survival and encystation rates to 91.62 % and 98.44 % compared to the control group (100 %, p ≤ 0.05). Morphological alterations observed in light microscopy and transmission electron microscopy (TEM) demonstrated that the drug significantly changed cell symmetry by causing the formation of various autophagic vacuoles/vesicles. The transcriptome sequencing of rapamycin-treated protomont revealed that 2249 (1837 up-regulated and 977 down-regulated) differentially expressed genes (DEGs) were identified. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that 226 DEGs were successfully annotated in 21 pathways (p˂0.05), including most enriched pathways apoptosis and phagosome with 25 and 24 DEGs, respectively. Most unigenes were assigned to autophagy-related pathways; 24 DEGs were classified into phagosomes, and 15 DEGs were assigned to lysosome pathways. Cytoskeleton and cell progression-associated genes were down-regulated. Besides, cell death-inducing proteins were up-regulated. The metabolomic analysis revealed exposure to rapamycin treatment enhanced protomont metabolites, including L-Cysteine, which is related to autophagy. Rapamycin had influenced the gene and metabolites of protomont; activating autophagy with inhibition of mechanistic target of rapamycin, (mTOR). The process negatively influences protomont morphology, encystation, and survival. Further autophagy-related gene silencing can be investigated via genome sequencing of C. irritans to study encystation.

Outbreak of Ichthyophthirius multifiliis associated with Aeromonas hydrophila in Pangasianodon hypophthalmus: The role of turmeric oil in enhancing immunity and inducing resistance against co-infection.

Ichthyophthirius multifiliis, a ciliated parasite causing ichthyophthiriasis (white spot disease) in freshwater fishes, results in significant economic loss to the aquaculture sector. One of the important predisposing factors for ichthyophthiriasis is low water temperature (i.e., below 20°C), which affects the health and makes freshwater fishes more susceptible to parasitic infections. During ichthyophthiriasis, fishes are stressed and acute immune reactions are compromised, which enables the aquatic bacterial pathogens to simultaneously infect the host and increase the severity of disease. In the present work, we aimed to understand the parasite-bacteria co-infection mechanism in fish. Later, Curcuma longa (turmeric) essential oil was used as a promising management strategy to improve immunity and control co-infections in fish. A natural outbreak of I. multifiliis was reported (validated by 16S rRNA PCR and sequencing method) in Pangasianodon hypophthalmus from a culture facility of ICAR-CIFRI, India. The fish showed clinical signs including hemorrhage, ulcer, discoloration, and redness in the body surface. Further microbiological analysis revealed that Aeromonas hydrophila was associated (validated by 16S rRNA PCR and sequencing method) with the infection and mortality of P. hypophthalmus, confirmed by hemolysin and survival assay. This created a scenario of co-infections, where both infectious agents are active together, causing ichthyophthiriasis and motile Aeromonas septicemia (MAS) in P. hypophthalmus. Interestingly, turmeric oil supplementation induced protective immunity in P. hypophthalmus against the co-infection condition. The study showed that P. hypophthalmus fingerlings supplemented with turmeric oil, at an optimum concentration (10 ppm), exhibited significantly increased survival against co-infection. The optimum concentration induced anti-stress and antioxidative response in fingerlings, marked by a significant decrease in cortisol and elevated levels of superoxide dismutase (SOD) and catalase (CAT) in treated animals as compared with the controls. Furthermore, the study indicated that supplementation of turmeric oil increases both non-specific and specific immune response, and significantly higher values of immune genes (interleukin-1ß, transferrin, and C3), HSP70, HSP90, and IgM were observed in P. hypophthalmus treatment groups. Our findings suggest that C. longa (turmeric) oil modulates stress, antioxidant, and immunological responses, probably contributing to enhanced protection in P. hypophthalmus. Hence, the application of turmeric oil treatment in aquaculture might become a management strategy to control co-infections in fishes. However, this hypothesis needs further validation.

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.