Mucosal immune responses to Ichthyophthirius multifiliis in the ocular mucosa of rainbow trout (Oncorhynchus mykiss, Walbaum), an ancient teleost fish.

The eye, as a specialized visual organ, is directly exposed to the external environment, and, therefore, it faces constant challenges from external pathogenic organisms and toxins. In the ocular mucosa (OM) of mammals, mucosal-associated lymphoid tissues (MALTs) constitute the primary line of defense. However, the immune defense role of the OM remains unknown in aquatic vertebrates. To gain insights into the immune processes within the OM of teleost fish, we developed an infection model of rainbow trout (Oncorhynchus mykiss) OM using a parasite, Ichthyophthirius multifiliis (Ich). Immunofluorescence, qPCR, and H&E staining revealed that Ich successfully infiltrates the OM of rainbow trout, leading to pathological structural changes, as evidenced by A&B staining. Importantly, the qPCR results indicate an up-regulation of immune-related genes following Ich infection in the OM. Moreover, transcriptome analyses were conducted to detect immune responses and impairments in eye function within the OM of rainbow trout with Ich infection. The results of the transcriptome analysis that Ich infection can cause an extensive immune response in the OM, ultimately affecting ocular function. To the best of our knowledge, our findings represent for the first time that the teleost OM could act as an invasion site for parasites and trigger a strong mucosal immune response to parasitic infection. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-023-00199-6.

Investigation of Ichthyophthirius multifiliis infection in fish from natural water bodies in the Lhasa and Nagqu regions of Tibet.

This study aimed to examine the prevalence of Ichthyophthirius multifiliis in fish inhabiting natural water bodies in the Lhasa and Nagqu regions of Tibet in September 2020 and August 2021. The results showed that Schizopygopsis selincuoensis had the highest prevalence of I. multifiliis at 33.73% (56/166), followed by Triplophysa tibetana at 30.00% (6/20), Triplophysa brevicauda at 27.91% (12/43) and Schizopygopsis thermalis at 23.66% (31/131). No infection with I. multifiliis was observed in exotic fish species. In addition, the prevalence of I. multifiliis in Boqu Zangbo (river), Selincuo Lake and Cuona Lake in the Nagqu region was found to be significantly higher than that in Lalu Wetland and Chabalang Wetland in the Lhasa region (P < 0.05). The study revealed a significantly lower prevalence in Lhasa River than in Cuona Lake (P < 0.05). Notably, our findings revealed instances of I. multifiliis infections even in saline water bodies, thereby emphasizing the potential threat that this parasite poses to the preservation of indigenous fish resources in Tibet. Consequently, immediate and effective countermeasures are imperative. This study represents the first systematic investigation of I. multifiliis infection in natural water bodies in Tibet.

The potential mechanism of concentrated mannan-oligosaccharide promoting goldfish's (Carassius auratus Linnaeus) resistance to Ichthyophthirius multifiliis invasion.

Because of the low host specificity, Ichthyophthirius multifiliis (Ich) can widely cause white spot disease in aquatic animals, which is extremely difficult to treat. Prior research has demonstrated a considerable impact of concentrated mannan-oligosaccharide (cMOS) on the prevention of white spot disease in goldfish, but the specific mechanism is still unknown. In this study, transcriptome sequencing, histological analysis, immunofluorescence analysis, phagocytosis activity assay and qRT-PCR assay were used to systematically reveal the potential mechanism of cMOS in supporting the resistance of goldfish (Carrasius auratus) to Ich invasion. According to the transcriptome analysis, the gill tissue of goldfish receiving the cMOS diet showed greater expression of mannose-receptor (MRC) related genes, higher phagocytosis activity, up-regulated expression of phagocytosis-related genes and inflammatory-related genes compared with the control, indicating that cMOS can have an effect on phagocytosis and non-specific immunity of goldfish. After the Ich challenge, transcriptome analysis revealed that cMOS fed goldfish displayed a higher level of phagocytic response, whereas non-cMOS fed goldfish displayed a greater inflammatory reaction. Besides, after Ich infection, cMOS-fed goldfish displayed greater phagocytosis activity, a stronger MRC positive signal, higher expression of genes associated with phagocytosis (ABCB2, C3, MRC), and lower expression of genes associated with inflammation (IL-1ß, IL-17, IL-8, TNF-α, NFKB). In conclusion, our experimental results suggest that cMOS may support phagocytosis by binding to MRC on the macrophage cell membrane and change the non-specific immunity of goldfish by stimulating cytokine expression. The results of this study provide new insights for the mechanism of cMOS on parasitic infection, and also suggest phagocytosis-related pathways may be potential targets for prevention of Ich infection.

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.

Integrated morphological and transcriptome profiles reveal a highly-developed extrusome system associated to virulence in the notorious fish parasite, Ichthyophthirius multifiliis.

Ichthyophthirius multifiliis is an obligate parasitic ciliate that causes severe economic damage in aquaculture. The parasite contains numerous extrusive organelles (extrusomes) that assist in its pathogenesis and reproduction. However, the structure of these extrusomes and the molecular profiles involved in exocytosis remain unclear. In the present study, through comparative ultrastructural observations across the life cycle of I. multifiliis, we demonstrated that all three of its life stages (theront, trophont, and tomont) exhibited an abundance of extrusomes. In addition, two different types of extrusomes were identified according to their unique structures. Type I extrusomes (mucocysts) are crystalline, oval-shaped, 0.7-1.4 × 0.6-1.1 µm, and distributed as "rosettes" below the trophont membrane. Type II extrusomes, 2.0-3.0 × 0.2-0.3 µm, are rod-shaped with tubular cores and identified as toxicysts, the aggregation of which in the anterior part of the theront and cortex of the trophont revealed their potential roles in I. multifiliis invasion. This was confirmed by our transcriptome investigations of the three stages of I. multifiliis, which revealed that a set of genes involved in proteolysis and DNA/protein biogenesis was highly expressed in the theront and trophont. Furthermore, to map the molecular mechanisms of extrusome release, we characterized 25 Rab family genes in I. multifiliis and determined their expression profiles across the life cycle, reflecting the distribution patterns of the two extrusomes. Collectively, our data revealed that a highly developed extrusome system could play a potential role in the virulence of I. multifiliis, which facilitates a better understanding of the parasite's development.

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.

Predation of Cyclopoid Copepods on the Theronts of Ichthyophthirius multifiliis: Shedding Light on Biocontrol of White Spot Disease.

White spot disease, caused by the parasitic ciliate Ichthyophthirius multifiliis, is a significant threat to the freshwater fish farming industry worldwide, resulting in massive mortality and economic losses. Eliminating the free-swimming theronts from the culture environment is considered crucial for the control of I. multifiliis infection. It is well-documented that planktonic ciliates are valuable food resources for macro-zooplankton in aquatic ecosystems. In this study, we developed a fluorescence labeling method for alive theronts and found that cyclopoid copepods Thermocyclops taihokuensis, Mesocyclops spp., Macrocyclops sp., and Paracyclopina sp. present predation on the theronts in co-culture experiments. Laboratory challenge tests further confirmed that the presence of zooplankton in the culture water body significantly reduced the infection of I. multifiliis in goldfish (p < 0.01). Results from this study revealed that cyclopoid copepods have the potential to be used as biological control agents against white spot disease in aquaculture.

Efficacy of dietary live or heat-killed Bacillus subtilis in goldfish (Carassius auratus) infected with Ichthyophthirius multifiliis.

BACKGROUND: The beneficial effects of Bacillus subtilis on growth, immune response, and disease resistance against various diseases in different fish species have been proved. However, there are no data concerning this probiotic effect on skin mucosal immunity in fish infected with Ichthyophthirius multifiliis (Ich). Ich has a high mortality rate in both edible and ornamental fish and consequently is concerned with heavy economic losses. OBJECTIVES: Thus, we assessed the efficacy of live and heat-killed B. subtilis on skin immunity and histopathology in goldfish (Carassius auratus) infected with Ich. METHODS: Goldfish (144 fish, 2.38 g average weight) were stocked in nine glass tanks each in three replicates. Fish were fed 109 CFU g-1 live or heat-killed B. subtilis for 80 days. RESULTS: Probiotic administration in both viable and non-viable forms could enhance the growth performance in goldfish. Probiotic therapy also reduced the density of the parasite and histopathological level on skin and gill tissues of the treated fish. Real-time polymerase chain reaction analysis showed a higher expression of lysozyme and tumour necrosis factor-α in the treated groups compared to the control group. CONCLUSIONS: These data demonstrated the beneficial effect of B. subtilis as probiotic and paraprobiotic on growth performance and disease resistance to Ich infestation in goldfish.

Non-specific cytotoxic cell receptor protein-1 (NCCRP-1) is involved in anti-parasite innate CD8+ T cell-mediated cytotoxicity in ginbuna crucian carp.

CD8+ cytotoxic T cells (CTLs) are a main cellular component of adaptive immunity. Our previous research has shown that CD8+ cells demonstrate spontaneous cytotoxic activity against the parasite Ichthyophthirius multifiliis in ginbuna crucian carp, suggesting that CD8+ cells play an important role in innate immunity. Herein, we investigated the molecules and cellular signal pathways involved in the cytotoxic response of ginbuna crucian carp. We considered non-specific cytotoxic receptor protein-1 (NCCRP-1) as candidate molecule for parasite recognition. We detected NCCRP-1 protein in CD8+ cells and the thymus as well as in other cells and tissues. CD8+ cells expressed mRNA for NCCRP-1, Jak2, and T cell-related molecules. In addition, treatment with a peptide containing the presumed antigen recognition site of ginbuna NCCRP-1 significantly inhibited the cytotoxic activity of CD8+ cells against the parasites. The cytotoxic activity of CD8+ cells was significantly inhibited by treatment with the JAK1/2 inhibitor baricitinib. These results suggest that teleost CTLs recognize I. multifiliis through NCCRP-1 and are activated by JAK/STAT signaling.

Parasitic fauna of farmed freshwater ornamental sutchi catfish (Pangasiandon hypophthalmus) and silver dollar (Metynnis hypsauchen) in Alborz province, Iran.

BACKGROUND: Parasitic infestations are one of the most significant problems in global aquaculture. In addition to direct economic losses due to significant mortalities, parasites may have a significant negative impact on the fish's behaviour, energetic demands, trophic position, interspecific competition, growth rate, and reproductive function. OBJECTIVES: We conducted the present study to evaluate the infection status of parasitic pathogens in freshwater sutchi catfish (Pangasianodon hypophthalmus) and silver dollar (Metynnis hypsauchen) farmed in Alborz province of Iran. METHODS: Between January and February 2021, 140 ornamental fish, including 70 sutchi catfish (P. hypophthalmus) and 70 silver dollar (M. hypsauchen), were delivered from various ornamental fish farms for parasitological analysis. Thorough macroscopic and microscopic examinations were performed to detect parasitic infections in delivered freshwater ornamental fish. RESULTS: A total of six parasite species, including five protozoan species (Nyctotherus piscicola, Trichodina heterodentata, Ichthyophthirius multifiliis, Protoopalina sp., and Hexamita sp.) and one monogenean species (Ancyrocephalus sp.), were identified in the examined fish. The overall prevalence of the recovered parasites was 46.43% (65/140) of the fish. CONCLUSIONS: In the current study, several parasites, including Hexamita sp., Ancyrocephalus sp., Protoopalina sp., and N. piscicola, were identified as the first records of the sutchi catfish (P. hypophthalmus) and/or silver dollar (M. hypsauchen), and these two fish species are known as new hosts for the isolated parasites in ornamental fish farms in Iran. Assessing the parasitic fauna of ornamental fish is therefore critical for preventing parasite introduction into adjacent provinces as well as neighbouring countries and promoting fish health.

Comparison of droplet digital PCR and real-time quantitative PCR for quantitative detection of the parasitic ciliate Ichthyophthirius multifiliis in the water environment.

Ichthyophthiriasis, caused by the parasitic ciliate Ichthyophthirius multifiliis (Ich), is considered one of the most harmful diseases affecting freshwater fish globally. It can cause mass mortalities of fish in intensive farming systems. In such systems, it is thus necessary to detect and quantify the number of Ich in the water so that control measures can be implemented before Ichthyophthiriasis breaks out. In recent years, molecular diagnostic methods have become increasingly important in aquaculture. Real-time quantitative polymerase chain reaction (qPCR) and droplet digital polymerase chain reaction (ddPCR) have become robust assays for detecting pathogens. In this study, a set of specific primers and a TaqMan-minor groove binder probe targeting the small-subunit rDNA (SSU rDNA) of Ich were developed. They were used in qPCR and ddPCR assays to compare the performance of these two different methods in quantitatively detecting Ich. After optimizing the reaction conditions, both qPCR and ddPCR assays were found to have high linearity and quantitative correlations for standard plasmid DNA. When used for the detection of Ich eDNA in water samples, the qPCR assay had a wider detection range, making it a suitable method to screen for the prevalence of Ichthyophthiriasis. However, the ddPCR approach had higher sensitivity, which would help provide advance notice of the disease in complex water environmental samples.

In Vitro Assessment of Berberine against Ichthyophthirius multifiliis in Goldfish.

Ichthyophthirius multifiliis is a pathogenic ciliate parasite, which infects almost all freshwater teleost fish and leads to significant economic losses. The present study aimed to evaluate the acute toxicity of berberine to the free-living stages of I. multifiliis, that is, theronts and tomonts. Our results indicated that 99.30% of I. multifiliis theronts were killed by a concentration of 15 mg/L berberine during the 4 h exposure time, while berberine had no effect on protomonts. Nevertheless, berberine at a concentration of 5 mg/L could effectively reduce the release of theronts from tomonts treated for 4 h. Additionally, according to the transmission electron microscopy results, berberine at 15 mg/L could strongly change the shape of protomonts, destroy their organelles, and significantly decrease the number of ribosomes. The median lethal concentration (LC50) of berberine for goldfish at 96 h was 528.44 mg/L, which was almost 67 times the median effective concentration (EC50) of berberine for killing theronts (7.86 mg/L). The results demonstrated that berberine could be an effective and safe potential parasiticide for killing I. multifiliis.

Antiparasitical efficacy of sophoraflavanone G isolated from Sophora flavescens against parasitic protozoa Ichthyophthirius multifiliis.

Ichthyophthirius multifiliis, a global distributed protozoan parasite, causes "White spot disease" and leads to serious mortality of freshwater fish in aquaculture. The present study was conducted to assess the anti-I. multifiliis efficacy of active compound isolated from Sophora flavescens. The isolated active compound was identified as sophoraflavanone G (SG) with ESI-MS and NMR. In vitro tests, SG at concentrations of 0.5 mg/L and 2 mg/L resulted in death of all theronts and tomonts, respectively; SG at concentrations of 0.125 mg/L and 0.25 mg/L notably decreased theronts infectivity (p < 0.05). Additionally, the in vivo test results showed that a cumulative delivery of SG at concentration of 2 mg/L for 7 days protected fish from I. multifiliis infection. The 96-h LC50 (median lethal concentration) and safety concentration of SG to grass carp were 46.6 mg/L and 11.3 mg/L, respectively. The present work indicated that SG was a potential safe and effectively therapeutic agent in treating I. multifiliis.

cMOS enhanced the mucosal immune function of skin and gill of goldfish (Carassius auratus Linnaeus) to improve the resistance to Ichthyophthirius multifiliis infection.

METHODS: of supporting mucosal immune barrier integrity and prevention of some pathogenic infections in aquatic species, are key areas of active study, often focusing on feed additives. The objectives of this study were to explore the effects of feeding cMOS (concentrated mannan oligosaccharide) on the gill and skin mucosal barriers of goldfish (Carassius auratus Linnaeus) and evaluate health status during Ichthyophthirius multifiliis infection. After feeding the cMOS-containing diet for 60 days, Hematoxylin and eosin (H&E) staining showed greater length of gill lamella and thicker dermal dense layer, while Alcian Blue and Periodic acid-Schiff (AB-PAS) staining showed higher numbers of mucin cells in cMOS fed fish. Chemical analysis showed that fish fed cMOS had greater enzyme activity of lysozyme (LZM) and alkaline phosphatase (AKP) in gill and skin tissues, while qRT-PCR revealed higher expression of Muc-2 and IL-1ß, as well as lower expression of IL-10. After Ichthyophthirius multifiliis challenge, goldfish fed the cMOS diet had lower mortality and infection rates, as well as fewer visible white spots on the body surfaces. Histologically, the gill and skin of these fish presented less tissue damage and fewer parasites, and had a greater number of mucus cells. In addition, the expression of Muc-2 and IL-10 were notably higher while the expression of IL-1ß was significantly lower in cMOS fed goldfish than control fed fish. In this study, cMOS fed goldfish had stronger immune barrier function of skin and gill mucous, and better survival following Ichthyophthirius multifiliis infection.

Transcriptome analysis of grass carp (Ctenopharyngodon idella) and Holland's spinibarbel (Spinibarbus hollandi) infected with Ichthyophthirius multifiliis.

Ichthyophthirius multifiliis is a protozoan ciliate that causes white spot disease (also known as ichthyophthiriasis) in freshwater fish. Holland's spinibarbel (Spinibarbus hollandi) was less susceptible to white spot disease than grass carp (Ctenopharyngodon Idella). In this study, grass carp and Holland's spinibarbel are infected by I. multifiliis and the amount of infection is 10,000 theronts per fish. All grass carp died within 12 days after infection, and the survival rate of Holland's spinibarbel was more than 80%. In order to study the difference in sensitivity of these two fish species to I. multifiliis, transcriptome analysis was conducted using gill, skin, liver, spleen and head kidney of Holland's spinibarbel and grass carp at 48 h post-infection with I. multifiliis. A total of 489,296,696 clean reads were obtained by sequencing. A total of 105 significantly up-regulated immune-related genes were obtained by Gene Ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis in grass carp. Cluster of differentiation 40 (CD40), cluster of differentiation 80 (CD 80), tumor necrosis factor-alpha (TNF-α), toll-like receptor 4 (TLR-4), interleukin 1 beta (IL-1ß) and other inflammatory-related genes in grass carp were enriched in the cytokine-cytokine receptor interaction pathway and toll-like receptor pathway. In Holland's spinibarbel, a total of 46 significantly up-regulated immune-related genes were obtained by GO classification and KEGG pathway enrichment analysis. Immune-related genes, such as Immunoglobin heavy chain (IgH), cathepsin S (CTSS), complement C1q A chain (C1qA), complement component 3 (C3) and complement component (C9) were enriched in phagosome pathway, lysosome pathway and complement and coagulation concatenation pathway. C3 was significantly up-regulated in gill and head kidney. Fluorescence in situ hybridization (FISH) showed that the C3 gene was highly expressed in gill tissue of Holland's spinibarbel infected with I. multifiliis. A small amount of C3 gene was expressed in the gill arch of grass carp after infected with I. multifiliis. In conclusion, the severe inflammatory response in vivo after infecting grass carp with I. multifiliis might be the main cause of the death of grass carp. The extrahepatic expression of the gene of Holland's spinibarbel might play an important role in the immune defense against I. multifiliis.

Molecular cloning and expression analysis of CD79a and CD79b in rainbow trout (Oncorhynchus mykiss) after bacterial, parasitic, and viral infection.

CD79a and CD79b heterodimers are important components that consist of B cell receptor compound, which play a crucial role in transduction activation signal of the antigen binding BCR, and B cell development and antibody production. In order to investigate the characters and potential functions of CD79a and CD79b in rainbow trout (Oncorhynchus mykiss), we firstly cloned and analyzed the expression of CD79a and CD79b and found that the cDNA sequences of CD79a and CD79b both contained open reading frame of 711 and 645 bp in length for encoding the protein of 237 and 215 amino acid residues, respectively. The predicted amino acid sequences from trout were highly conserved with those of other teleost fishes in structure. Phylogenetic tree was constructed to analyze the evolutionary relationship between the trout and other known species, the result indicated that CD79a and CD79b of trout clustered at high bootstrap values with Salmo salar. Moreover, three trout infection models with F. columnare G4, I. multifiliis and infectious hematopoietic necrosis virus (IHNV) were constructed, which resulted in morphological changes and serious lesions in skin and gills. Importantly, the high expression of CD79a and CD79b occurred in skin, gills, and followed by head kidney in response to bacterial, parasitic, and viral infection, as its expression was closely related to that of Igs. Our findings indicated that CD79a and CD79b play vital roles in both systemic and mucosal immune responses of rainbow trout during bacterial, parasitic, and viral infection, which will contribute to explore the roles of CD79 subunits in B cell signaling during ontogeny and disease.

Effects of Extract of Betel Leaf (Piperaceae) against Ectoparasite "Ich" on Pangasius Catfish.

<b>Background and Objective:</b> Freshwater fish aquaculture in Indonesia has grown rapidly, especially the aquaculture of catfish (<i>Pangasianodon hypophthalmus</i>). This species is very good because it is fast-growing and very popular in the market and is important for national food security in many Asian countries. One of the problems faced by freshwater fish aquaculture is ectoparasite <i>Ichthyophthirius multifiliis</i> infection, which often results in significant economic losses to freshwater fish aquaculture. This study aimed to check the effect extract of betel leaf against the ectoparasite, <i>Ichthyophthirius multifiliis</i> in pangasius catfish in an eco-friendly manner. <b>Materials and Methods:</b> A total of 120 fishes with a mean weight of 4.17±0.96 g and a length of 8.5±0.67 cm were examined. Preliminary research was carried out to detect ectoparasites in fish. All fish was infected with ectoparasitic Ich (100%) and were identified as a salt-like granule white spot and a large C-shaped macronucleus. Infected fishes were transferred and equally distributed to the tank (20 L water) which had previously been treated with betel leaf extract for 24 hrs, 3 days, at doses 2.5, 5 and 7.5 g L¹ and control. <b>Results:</b> The results showed that the betel leaf extract solution effect decreased significantly to the number of ectoparasites <i>Ichthyophthirius multifiliis</i>, both in mucus and pangasius catfish and a dose of 7.5 g L¹ was the optimum dose. <b>Conclusion:</b> Betel leaf extract has the potential to control the decrease in the number of ectoparasites, though further phytochemical studies will need to be performed.

Molecular Characterization and Expression Analysis of Intercellular Adhesion Molecule-1 (ICAM-1) Genes in Rainbow Trout (Oncorhynchus mykiss) in Response to Viral, Bacterial and Parasitic Challenge.

The intercellular adhesion molecule-1 (ICAM-1), known as CD54, is a transmembrane cell surface glycoprotein that interacts with two integrins (i.e., LFA-1 and Mac-l) important for trans-endothelial migration of leukocytes. The level of ICAM-1 expression is upregulated in response to some inflammatory stimulations, including pathogen infection and proinflammatory cytokines. Yet, to date, our knowledge regarding the functional role of ICAM-1 in teleost fish remains largely unknown. In this study, we cloned and characterized the sequence of ICAM-1 in rainbow trout (Oncorhynchus mykiss) for the first time, which exhibited that the molecular features of ICAM-1 in fishes were relatively conserved compared with human ICAM-1. The transcriptional level of ICAM-1 was detected in 12 different tissues, and we found high expression of this gene in the head kidney, spleen, gills, skin, nose, and pharynx. Moreover, upon stimulation with infectious hematopoietic necrosis virus (IHNV), Flavobacterium columnare G4 (F. columnare), and Ichthyophthirius multifiliis (Ich) in rainbow trout, the morphological changes were observed in the skin and gills, and enhanced expression of ICAM-1 mRNA was detected both in the systemic and mucosal tissues. These results indicate that ICAM-1 may be implicated in the mucosal immune responses to viral, bacterial, and parasitic infections in teleost fish, meaning that ICAM-1 emerges as a master regulator of mucosal immune responses against pathogen infections in teleost fish.

Dual-Organ Transcriptomic Analysis of Rainbow Trout Infected With Ichthyophthirius multifiliis Through Co-Expression and Machine Learning.

Ichthyophthirius multifiliis is a major pathogen that causes a high mortality rate in trout farms. However, systemic responses to the pathogen and its interactions with multiple organs during the course of infection have not been well described. In this study, dual-organ transcriptomic responses in the liver and head kidney and hemato-serological indexes were profiled under I. multifiliis infection and recovery to investigate systemic immuno-physiological characteristics. Several strategies for massive transcriptomic interpretation, such as differentially expressed genes (DEGs), Poisson linear discriminant (PLDA), and weighted gene co-expression network analysis (WGCNA) models were used to investigate the featured genes/pathways while minimizing the disadvantages of individual methods. During the course of infection, 6,097 and 2,931 DEGs were identified in the head kidney and liver, respectively. Markers of protein processing in the endoplasmic reticulum, oxidative phosphorylation, and the proteasome were highly expressed. Likewise, simultaneous ferroptosis and cellular reconstruction was observed, which is strongly linked to multiple organ dysfunction. In contrast, pathways relevant to cellular replication were up-regulated in only the head kidney, while endocytosis- and phagosome-related pathways were notably expressed in the liver. Moreover, interestingly, most immune-relevant pathways (e.g., leukocyte trans-endothelial migration, Fc gamma R-mediated phagocytosis) were highly activated in the liver, but the same pathways in the head kidney were down-regulated. These conflicting results from different organs suggest that interpretation of co-expression among organs is crucial for profiling of systemic responses during infection. The dual-organ transcriptomics approaches presented in this study will greatly contribute to our understanding of multi-organ interactions under I. multifiliis infection from a broader perspective.

Parasiticidal effects of Tanacetum vulgare extract against Ichthyophthirius multifiliis.

Ichthyophthiriasis, which is caused by Ichthyophthirius multifiliis (Ich) infections, has a severe impact on productivity in freshwater aquaculture. These infections were previously treated effectively with malachite green, a compound that is now banned on fish farms due to its carcinogenicity. To find efficacious drugs to control Ich, flowers of tansy Tanacetum vulgare were evaluated for their antiprotozoal activity. Tanacetum vulgare extract significantly reduced the survival of Ich trophonts and theronts. In vitro, the extract killed all trophonts at 3200 mg l-1, terminated tomont reproduction at 50 mg l-1, and caused mortality of all theronts at 100 mg l-1. T. vulgare extract may be a new and efficacious drug for the control of Ich.