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.

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.

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

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

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.

Sustainable development of factory aquaculture through automation of ultraviolet parasiticide for the prevention and control of cryptocaryoniasis.

BACKGROUND: Cryptocaryon irritans infestations on marine teleosts are a considerable burden on factory mariculture. Ultraviolet (UV) light can kill C. irritans under laboratory conditions. However, a rational method for using UV in factory aquaculture to control cryptocaryoniasis has not been developed. This study focused on evaluating the killing effect of UV on protomonts and tomonts of C. irritans and established an automatic UV parasiticide device for the prevention and control of cryptocaryoniasis in marine teleosts. RESULTS: The survival rate of protomonts and tomonts decreased with an increase in the UV irradiation dose. All the protomonts and tomonts died within 14 and 24 min, respectively. The lowest UV lethal doses of protomonts and tomonts of C. irritans were 2.0 × 106 and 3.5 × 106 µWs cm-2 , respectively. Exposure of protomonts and tomonts to lethal doses of UV radiation led to shrinkage and severe dissolution of the protoplasm, causing abnormal development of cells. The survival rate of artificially infected Larimichthys crocea (treatment group, group A) was 83.33% at the end of the test (day 14) after disinfection using the automatic UV parasiticide device, whereas that of the control group (group C) was 90.00% (p < 0.05). However, all artificially infected L. crocea without disinfection using the automatic UV parasiticide device (untreated group, group B) died on day 8. CONCLUSION: The automation of traditional physical methods conforms to the sustainable development of aquaculture and provides a theoretical reference for the prevention and control of cryptocaryoniasis in mariculture. © 2022 Society of Chemical Industry.

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.

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.

Molecular characterization of a profilin gene from a parasitic ciliate Cryptocaryon irritans.

Profilin, known as one of the core actin-binding proteins, is an integral part of actin-based cytoskeleton involved in cell motility, cytokinesis, neuronal differentiation, and synaptic plasticity. In this study, a putative profilin gene designated as CiProfilin (GenBank accession number: JX987286) was screened out from a cDNA library of Cryptocaryon irritans trophonts. The full-length cDNA of CiProfilin gene is 582 bp, containing an open reading frame (ORF) of 471 bp, which encodes a polypeptide consisting of 156 amino acids with a predicted molecular weight of 17.3 kDa. Quantification of CiProfilin mRNA expression by real-time PCR suggested that CiProfilin was expressed in all stages of C. irritans life cycle with a significantly higher level in trophonts. Five non-universal codons (TAAs) coding glutamines (Gln) were found in the ORF and mutated to CAAs (universal codons for Gln) by site-directed mutagenesis. Then the modified ORF was inserted into the plasmid pGEX-4T-1, the recombinant plasmid was subsequently transformed into Escherichia coli. The bacteria were subsequently induced to express the recombinant CiProfilin protein fused with glutathione S transferase (G-rCiProfilin), which was then purified with glutathione sepharose 4B and thrombin cleavage. The molecular weight and the antigenicity of rCiProfilin were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. The native CiProfilin was found abundant in the peripheral area beneath the cell membrane and around the cytostomes of theronts, suggesting its vital roles in food uptake, stomatogenesis, and parasitic invasion. Co-precipitation assay also revealed the activity of rCiProfilin in actin binding. This study will help further elucidate the specific roles of CiProfilin on the growth of C. irritans and the preliminary mechanism of its invasion to hosts.

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.

First record of Trichodina heterodentata (Ciliophora: Trichodinidae) in banded knifefish Gymnotus carapo (Gymnotidae) cultured in Brazil / Primeiro registro de Trichodina heterodentata (Ciliophora: Trichodinidae) em tuvira Gymnotus carapo (Gymnotidae), cultivado no Brasil

The aim of this study was to report the first record of Trichodina heterodentata in banded knifefish Gymnotus carapo. Banded knifefish cultivated in the municipality of Jataí, state of Goiás, in the central western region of Brazil, experienced fish mortality rates of 3-4% per tank. Macroscopic analysis found that fish had lesions on their skin and gills. Smears of the integument and gills were confectioned and air-dried at room temperature and impregnated with silver nitrate to identify the Trichodinidae species causing the lesions. The trichodinid were identified as T. heterodentata, and their characteristics were compared to those from T. heterodentata recorded from other species of host fish. It was observed that the presence of T. heterodentata was associated with inadequate fish management, low water quality, high rates of stocking density, and inadequate nutritional management.

O objetivo deste estudo foi relatar o primeiro registro de Trichodina heterodentata em tuviras Gymnotus carapo. Um cultivo de tuviras no município de Jataí, estado de Goiás, na região Centro-Oeste do Brasil, apresentou taxas de mortalidade de peixes de 3-4% por tanque. A análise macroscópica constatou que os peixes tinham lesões na pele e brânquias. Raspados do tegumento e brânquias foram confeccionadas e secas ao ar à temperatura ambiente e impregnadas com nitrato de prata para identificar as espécies de Trichodinidae causadoras das lesões. Os tricodinídeos foram identificados como T. heterodentata, e suas características foram comparadas com as de T. heterodentata registradas para outras espécies de peixes hospedeiros. Observou-se que a presença de T. heterodentata estava associada ao manejo inadequado dos peixes, baixa qualidade da água, altas taxas de densidade de estocagem e manejo nutricional inadequado.

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.

Molecular characterization and phylogenetic analyses of Tripartiella macrosoma Basson and Van As, 1987 and Tripartiella obtusa Ergens and Lom, 1970 based on 18S rRNA gene sequence data.

In the present study, we provided the first 18S rRNA gene sequence data of two Tripartiella species, Tripartiella macrosoma Basson and Van As, 1987 and Tripartiella obtusa Ergens and Lom, 1970, which were isolated from Tachysurus fulvidraco (Richardson, 1846) and Hemibarbus maculatus Bleeker, 1871 in Chongqing, China, respectively. Morphologically, both species fall within the morphometry range of the original descriptions and are very similar to the original populations in the overall appearance of the adhesive disc. Tripartiella macrosoma can be easily distinguished from the other Tripartiella species by possessing the denticle with a long strip and conspicuously inclined backward blade and a robust and short ray. Tripartiella obtusa is mainly characterized by a broad blade and a relatively long ray. Phylogenetically, T. macrosoma clustered with Trichodinella myakkae (Mueller, 1937) Raabe, 1950 and further with Trichodinella sp., which was sister to a group that includes four populations of Trichodinella epizootica (Raabe, 1950) Srámek-Husek, 1953; finally, they formed a small clade with T. obtusa. This result suggested that T. macrosoma had a closer relationship with Trichodinella spp. than with T. obtusa and T. obtusa diverged earlier than T. macrosoma and Trichodinella spp. By combining morphological and molecular data, the polyphyletic characteristics of Tripartiella and Trichodinella were further analyzed, and the results revealed that the validity of the genus Tripartiella is doubtful.

Parasitos em trutas-arco-íris (Oncorhynchus mykiss) criadas em sistema intensivo na região serrana de Santa Catarina, Brasil / [Parasites in rainbow trout (Oncorhynchus mykiss) raised in an intensive system in the mountain region of Santa Catarina, Brazil]

With the aim to determine the prevalence and mean parasite intensity in Oncorhynchus mykiss, 120 specimens were analyzed between April and September 2018. The cavity analysis was done by visual inspection, scraping of mucus, and extraction of the branchial arches. In the evisceration process, the intestine, the liver, and the kidney were separated, while the musculature was analyzed using the "candling table" method. All the collected material was preserved in 10% buffered formaldehyde and sent to the Laboratory of Parasitology and Parasitic Diseases, of the Center for Agroveterinary Sciences (CAV) of the University of the State of Santa Catarina (UDESC) for making the slides and identifying the parasites. Parasites were detected only in the analysis of the scraping of body mucus. Only specimens of the genus Trichodina were identified, in 34 of the 120 fish analyzed, in two collections, resulting in a prevalence of 28.33%. In total, 144 specimens of Trichodina were observed. The overall mean intensity was of 4.24 parasites in each fish analyzed. Characteristic lesions of infection by protozoa were not identified. This is the first report of the occurrence of Trichodina spp. in O. mykiss bred in an intensive system in Brazil, with low rates of parasitic infection in the mountain region of Santa Catarina.(AU)

Role of major histocompatibility complex II antigen-presentation pathway genes in orange-spotted grouper infected with Cryptocaryon irritans.

Cryptocaryon irritans, a pathogen model for fish mucosal immunity, causes skin mucosal and systematic humoral immune response. Where and how MHC II antigen presentation occurs in fish infected with C. irritans remain unknown. In this study, the full-length cDNA of the grouper cysteine protease CTSS was cloned. The expression distributions of six genes (CTSB, CTSL, CTSS, GILT, MHC IIA and MHC IIB) involved in MHC II antigen presentation pathway were tested. These genes were highly expressed in systematic immune tissues and skin and gill mucosal-associated immune tissues. All six genes were upregulated in skin at most time points. Five genes expected CTSS was upregulated in spleen at most time points. CTSB, CTSL and MHC IIA were upregulated in the gill and head kidney at some time points. These results indicate that the presentation of MHC II antigen intensively occurred in local infected skin and gill. Spleen, not head kidney, had the most extensive systematic antigen presentation. In skin, six genes most likely peaked at day 2, earlier than in spleen (5-7 days), marking an earlier skin antibody peak than any recorded in serum previously. This significant and earlier mucosal antigen presentation indicates that specific immune response occurs in local mucosal tissues.

Characteristics delineation of piscidin 5 like from Larimichthys crocea with evidence for the potent antiparasitic activity.

Several researches reported that piscidin members of teleosts owned strong antiparasitic activity. Cryptocaryon irritans, a type of ectoparasite, could infect most of the marine teleosts. Larimichthys crocea could severely suffer from marine white spot disease caused by C. irritans, and their mortality rate was significantly high. Concentrating on this problem, we have done many related works. Piscidin 5 like (termed Lc-P5L) was another piscidin member isolated from a comparative transcriptome of C. irritans-immuned L. crocea. In the paper, quantitative Real-time PCR (qRT-PCR) showed Lc-P5L was upregulated in examined tissues, including gill, head kidney, muscle, liver, spleen and intestine after challenged by C. irritans, the significant upregulation time was in accordance to key developmental stages of C. irritans, which implied different infection stages could result in host immune response. Furthermore, using microscope techniques, we observed theronts or trophonts became weakly motile, cilia became detached, cells were out of shape, membranes eventually lysed in different cell positions and cytoplasmic contents leaked. Laser confocal scanning microscope (LCSM) observed theronts macronucleus grew swell and depolymerized after treated by recombinant Lc-P5L (rLc-P5L). Data suggested rLc-P5L was significantly lethal to C. irritans, and the death state of the parasite incubated with rLc-P5L was remarkably similar to other piscidin members or other antiparasitic peptides (APPs). Thus, these data provided new insights into L. crocea immunity against C. irritans and potential of rLc-P5L as a therapeutic agent against pathogen invasion.

Distribution of Mpeg1+ cells in healthy grouper (Epinephelus coioides) and after Cryptocaryon irritans infection.

Cryptocaryon irritans is an extremely harmful ciliated obligate parasite that is responsible for large economic losses in aquaculture. C. irritans infection can cause an insect-resistant immune response in fish, and many immune cells can be observed in the local infection site. However, it is unclear whether macrophages are involved in the host defense against C. irritans infection. The Mpeg1 protein can form pores and destroy the cell membrane of invading pathogens, and is also used as a macrophage-specific marker in mammals. Therefore, a polyclonal antibody against grouper recombinant Mpeg1a was produced to mark macrophages in this study, which could recognize both isoforms of Mpeg1 (Mpeg1a/b). Immunofluorescence revealed that EcMpeg1 positive cells were mostly distributed in the head kidney and spleen in healthy grouper. Immunofluorescence and immunohistochemistry showed that the number of EcMpeg1 positive cells increased in the gills after infection with C. irritans, implying that EcMpeg1 positive cells may be involved in the process of grouper resistance against C. irritans infection.

Expression analysis of taste receptor genes (T1R1, T1R3, and T2R4) in response to bacterial, viral and parasitic infection in rainbow trout, Oncorhynchus mykiss.

Emerging evidence suggests that bitter and sweet Taste receptors (TRs) in the airway are important sentinels of innate immunity. TRs are G protein-coupled receptors that trigger downstream signaling cascades in response to activation of specific ligands. Among them, the T1R family consists of three genes: T1R1, T1R2, and T1R3, which function as heterodimers for sweet tastants and umami tastants. While the other TRs family components T2Rs function as bitter tastants. To understand the relationship between TRs and mucosal immunity in teleost, here, we firstly identified and analyzed the molecular characteristics of three TRs (T1R1, T1R3, and T2R4) in rainbow trout (Oncorhynchus mykiss). Secondly, by quantitative real-time PCR (qPCR), we detected the mRNA expression levels of T1R1, T1R3 and T2R4 and found that the three genes could be tested in all detected tissues (pharynx, buccal cavity, tongue, nose, gill, eye, gut, fin, skin) and the expression levels of T1R3 and T2R4 were higher in buccal mucosa (BM) and pharyngeal mucosa (PM) compare to other tissues. It may suggest that T1R3 and T2R4 play important roles in BM and PM. Then, to analyses the changes of expression levels of the three genes in rainbow trout infected with pathogens, we established three infection models Flavobacterium columnare (F. cloumnare), infectious hematopoietic necrosis virus (IHNV) and Ichthyophthirius multifiliis (Ich). Subsequently, by qPCR, we detected the expression profiles of TRs in the gustatory tissues (BM, PM and skin) of rainbow trout after infection with F. cloumnare, IHNV, and Ich, respectively. We found that under three different infection models, the expression of the T1R1, T1R3 and T2R4 showed their own changes in mRNA levels. And the expression levels of the T1R1, T1R3 and T2R4 changed significantly at different time points in response to three infection models, respectively, suggesting that TRs may be associated with mucosal immunity.

Larval Geoduck (Panopea generosa) Proteomic Response to Ciliates.

The innate immune response is active in invertebrate larvae from early development. Induction of immune response pathways may occur as part of the natural progression of larval development, but an up-regulation of pathways can also occur in response to a pathogen. Here, we took advantage of a protozoan ciliate infestation of a larval geoduck clam culture in a commercial hatchery to investigate the molecular underpinnings of the innate immune response of the larvae to the pathogen. Larval proteomes were analyzed on days 4-10 post-fertilization; ciliates were present on days 8 and 10 post-fertilization. Through comparisons with larval cultures that did not encounter ciliates, proteins implicated in the response to ciliate presence were identified using mass spectrometry-based proteomics. Ciliate response proteins included many associated with ribosomal synthesis and protein translation, suggesting the importance of protein synthesis during the larval immune response. There was also an increased abundance of proteins typically associated with the stress and immune responses during ciliate exposure, such as heat shock proteins, glutathione metabolism, and the reactive oxygen species response. These findings provide a basic understanding of the bivalve molecular response to a mortality-inducing ciliate and improved characterization of the ontogenetic development of the innate immune response.

Scuticociliatosis caused by Uronema sp. in ten different ornamental aquarium reef fish in Brazil.

Scuticociliatosis, caused by an opportunistic ciliate protozoan, is responsible for significant economic losses in marine ornamental fish. This study reports the occurrence of Uronema spp., parasitizing ten species of marine reef fish at an ornamental fish wholesaler: Blue green damselfish (Chromis viridis), Vanderbilt's Chromis (Chromis vanderbilti), Pennant coralfish (Heniochus acuminatus), Threespot angelfish (Apolemichthys trimaculatus), Goldspotted angelfish (Apolemichthys xanthopunctatus), Sea goldie (Pseudanthias squamipinnis), Orchid dottyback (Pseudochromis fridmani), Threadfin butterflyfish (Chaetodon auriga), Vagabond butterflyfish (Chaetodon vagabundus), and Bluecheek butterflyfish (Chaetodon semilarvatus). Diseased fish showed disorders such as hemorrhages and ulcerative lesions on the body surface. Histopathological analysis of the muscle, liver, gut, kidney, spleen, gills, and stomach revealed hemorrhages and degeneration of muscle fiber, vacuolar degeneration of hepatocyte, inflammatory process and granuloma in the liver, atrophy of intestinal villi, inflammatory process and granuloma in the kidney, melanomacrophage centers, as well as inflammatory process in the spleen, epithelial cells hyperplasia and granuloma formation in the gills, and vacuolar degeneration and eosinophils in the stomach. Due to the severity of the disease, it is necessary to implement biosecurity measures with rapid and accurate diagnosis to minimize the risk of economic losses caused by Uronema spp.

Transcriptomic analysis of Nibea albiflora skin in response to infection by Cryptocaryon irritans.

Massive infection caused by Cryptocaryon irritans is detrimental to the development of marine aquaculture. Recently, our lab found that Nibea albiflora has low sensitivity and low mortality to C. irritans infection. The present study was designed to investigate the mechanisms of the N. albiflora response to C. irritans infection by analyzing transcriptome changes in the skin. Skin samples of control and experimental groups with C. irritans infection were collected at 24 and 72 h (24 h control, 24 h post-infection, 72 h control, and 72 h post-infection). Three parallels were set for each group and sample time, and a total of 12 skin samples were collected for sequencing. Overall, 297,489,843 valid paired-end reads and 48,817 unigenes were obtained with an overall length of 59,010,494 nt. In pairwise comparisons, changes in expression occurred in 1621 (764 upregulated and 857 downregulated), 285 (180 upregulated and 105 downregulated), 993 (489 upregulated and 504 downregulated), and 37 (8 upregulated and 29 downregulated) genes at 24 h control vs 24 h post-infection, 72 h control vs 72 h post-infection, 24 h post-infection vs 72 h post-infection, and 24 h control vs 72 h control, respectively. Gene Ontology (GO) analysis of differentially expressed genes (DEGs) indicated that the number of genes enriched in GO sub-categories were ordered 24 h control vs 24 h post-infection > 24 h post-infection vs 72 h post-infection >72 h control vs 72 h post-infection > 24 h control vs 72 h control. Further analysis showed that immune-related GO terms (including immune system process, complement activation, and humoral immunity) were significantly enriched at both 72 h control vs 72 h post-infection and 24 h post-infection vs 72 h post-infection, but no immune-related GO terms were significantly enriched in the 24 h control vs 72 h control and at 24 h control vs 24 h post-infection, indicating that C. irritans infection mainly affected the physiological metabolism of N. albiflora at an early stage (24 h), and immune-related genes play an important role at a later stage (72 h) of infection. In KEGG pathway analysis, the complement and coagulation cascade pathway are involved in early infection. Hematopoietic cell lineage, natural killer (NK) cell-mediated cytotoxicity, and the intestinal immune network for IgA production are involved in later infection. Further analysis showed that the alternative pathway of complement and coagulation cascades plays an important role in the resistance of N. albiflora to early C. irritans infection. During late infection, CD34, IgM, and IgD were significantly upregulated in the hematopoietic cell lineage pathway. CCR9 was significantly downregulated, and IGH and PIGR were significantly upregulated in the intestinal immune network for IgA production. GZMB and IGH were significantly downregulated in NK cell-mediated cytotoxicity. These findings indicate that acquired immunity at the mRNA level was initiated during later infection. In addition, the IL-17 signaling pathway was enriched by downregulated DEGs at 24 h post-infection vs 72 h post-infection, suggesting the inflammatory response at 24 h was stronger than at 72 h and the invasion of the parasite has a greater impact on the host.