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.

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.

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.

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

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

pH Regulates the Formation and Hatching of Cryptocaryon irritans Tomonts, Which Affects Cryptocaryoniasis Occurrence in Larimichthys crocea Aquaculture.

Cryptocaryon irritans are the main pathogens of white spot disease in marine teleost. However, the occurrence of cryptocaryoniasis is influenced by several abiotic factors including the pH. To explore the effect of pH on the life cycle of C. irritans (encystment, cleavage, and hatchability), protomonts and tomonts of C. irritans were incubated in seawater of 10 different pH levels (2-11). pH 8 was used as the control. The change in morphology and infectivity of theronts that hatched from tomonts against Larimichthys crocea were then recorded. We found that pH 6-9 had no significant effect on the encystment, cleavage, and hatching of the parasites. However, pH beyond this limit decreased the cleavage and hatching of the tomonts. Furthermore, extreme pH decreased the number of theronts hatched by each tomont and the pathogenicity of the theronts, but increased the aspect ratio of the theronts. Infectivity experiments further revealed that extreme pH significantly decreased the infectivity of C. irritans against L. crocea. In conclusion, the C. irritans can survive in pH of 5 to 10, but pH 6-9 is the optimal range for the reproduction and infectivity of C. irritans. However, extreme pH negatively affects these aspects. IMPORTANCECryptocaryon irritans is a ciliate parasite that causes "white spot disease" in marine teleosts. The disease outbreak is influenced by hosts and a range of abiotic factors, such as temperature, salinity, and pH. Studies have shown that change in pH of seawater affects the structure (diversity and abundance of marine organisms) of marine ecosystem. However, how pH affects the life cycle and survival of C. irritans, and how future ocean acidification will affect the occurrence of cryptocaryoniasis, are not well understood. In this study, we explored the effect of pH on the formation and hatching of C. irritans tomonts. The findings of this study provide the foundation of the environmental adaptation of C. irritans, the occurrence of cryptocaryoniasis, and better management of marine fish culture.

Differential immune and metabolic responses underlie differences in the resistance of Siganus oramin and Trachinotus blochii to Cryptocaryon irritans infection.

Numerous studies have demonstrated that Cryptocaryon irritans can efficiently propagate in golden pompano (Trachinotus blochii), especially under intensive high-density culture, which can lead to large-scale infection, bacterial invasion, and major economic losses. By contrast, Siganus oramin is less susceptible to C. irritans infection. Here, we artificially infected S. oramin and T. blochii with C. irritans. We then used RNA-seq to characterize the expression of genes in the gills of S. oramin and T. blochii at different times after infection, conducted bioinformatics analysis of relevant pathways, and compared the differentially expressed genes in the two species. The aim of this study was to enhance our understanding of host-parasite interactions to aid the development of effective prevention and treatment strategies for C. irritans. Infection with C. irritans induced the differential expression of a large number of genes in the gills of S. oramin, indicating that S. oramin may respond to C. irritans infection by modifying the expression of genes at the transcriptional level. Our research showed that the Toll-like receptor signaling pathway, Antigen processing and presentation, Complement and coagulation cascades, and Cytosolic DNA-sensing pathway are involved in the immune response of S. oramin and T. blochii to C. irritans infection. However, T. blochii has a weak ability to mobilize neutrophils to participate in defense against C. irritans infection and differs from S. oramin in its ability to induce specific immune responses. Because of gill tissue damage during infection, dissolved oxygen intake is reduced, which increases physiological and metabolic stress. The metabolic pathways of S. oramin and T. blochii significantly differed; specifically, the main pathways in S. oramin were related to glucose and lipid metabolism, and the main pathways in T. blochii were related to amino acid metabolism. This may reduce the efficiency of ATP biosynthesis in T. blochii and result in dysfunctional energy metabolism. Therefore, differential immune and metabolic responses underlie differences in the resistance of S. oramin and T. blochii to C. irritans.

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.

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.

Scuticociliatosis caused by Philasterides dicentrarchi.

The ciliate Philasterides dicentrarchi has been previously identified as a new agent of scuticociliatosis in marine fish. The parasite can cause high mortalities in fish reared on farms or kept in aquariums. P. dicentrarchi is usually a free-living protozoan but can become an opportunistic histophagous parasite causing rapid lethal systemic infections in cultured fish. This review provides information about the morphology and biology of the scuticociliate P. dicentrarchi, as well as information about the pathological and immunological reactions of the host in response to the infection with the parasite. The epidemiology and the control strategies of the disease are also reviewed.

Outbreak of Cryptocaryon irritans infection in silver pomfret Pampus argenteus cultured in China.

Silver pomfret Pampus argenteus is a major cultivated marine fish species with a high market value. In summer 2021, Cryptocaryon irritans, a ciliate parasite, infected the cultured silver pomfret in aquaculture ponds in Ningbo, Zhejiang Province, China. The symptoms of infected fish include white spots on the skin and fins, increased body surface mucus, loss of appetite, irritability, and shedding of scales. After collecting white spots from moribund fish, the 18S ribosomal RNA sequence of the pathogen on the fish skin was amplified by PCR; phylogenetic analysis showed that it was closely related to C. irritans strains from Ningde, Fujian, China. Four groups of silver pomfret were tested in an artificial infection experiment over the course of 72 h, consisting of 3 infected groups (1600, 4000, and 8000 theronts fish-1) and 1 healthy group. White spots were observed on the skin and fins of the infected fish, but not on their gills. Samples were taken from the gills, liver, kidney, and spleen of both infected and healthy fish and were compared to evaluate any significant histopathological differences. As the dose of infection increased, symptoms became more pronounced. At 72 h, mortality rates were 8.3, 50, and 66.7% for the 3 different concentrations, respectively. The median lethal concentration was calculated to be 366 theronts g-1 at 72 h, 298 theronts g-1 at 84 h, and 219 theronts g-1 at 96 h. This study emphasizes the importance of developing early diagnosis methods and appropriate prevention strategies to decrease the impact of C. irritans infection in the silver pomfret aquaculture industry.

Genetic diversity of Ichthyophthirius multifiliis (Fouquet, 1876) infecting farmed rainbow trout (Oncorhynchus mykiss Walbaum, 1792) in Turkey.

We assessed genetic diversities among Ichthyophthirius multifiliis (Ich) field isolates collected from farmed rainbow trout (Oncorhynchus mykiss) in Turkey. The overall prevalence of Ich was 35.3% (634/1798). Five novel Ich genotypes (ImulTR1 and ImulTR3-ImulTR6) were described based on mitochondrial cox-1 and nad1_b genes. The remaining genotype ImulTR2 was identical to the previously reported NY3 (or Ark9 and TW7) genotype from the United States and South Asia. Phylogenetic analysis indicated Turkish Ich isolates separated genetically into at least four distinct groups. Our study presents the first data on the genotypes of Ich in Turkey. We also provide evidence for the wide distribution of the NY3 genotype (or Ark9 and TW7) from the United States and South Asia to Turkey. Genetic diversities within the mitochondrial genes provided adequate resolution for describing novel genotypes and identifying the known genotype within Turkish Ich isolates. Description of the Ich genotypes allows for tracking of pathogen genotypes worldwide. Thus, we can better understand the connections between Ich outbreaks in the fisheries aquaculture.

Quantitative detection of parasitic ciliate Cryptocaryon irritans in seawater with an optimized sample processing method.

The protozoan Cryptocaryon irritans is one of the most important ectoparasites of marine fish, causing 'white spot disease' and mass mortality in aquaculture. To accurately predict disease outbreaks and develop prevention strategies, improved detection methods are required that are sensitive, convenient and rapid. In this study, a pair of specific primers based on the C. irritans 18S rRNA gene was developed and used in a real-time PCR (qPCR) assay. This assay was able to detect five theronts in 1 L of natural seawater. Furthermore, a linear model was established to analyse the log of Ct value and parasite abundance in seawater (y = -2.9623x + 24.2930), and the coefficient of determination (R2 ) value was 0.979. A lysis buffer was optimized for theront DNA extraction and used for storage sample. This method was superior to the commercial water DNA kit, and there was no significant degradation of DNA at room temperature for 24-96 hr. A dilution method was developed to manage qPCR inhibitors and used to investigate natural seawater samples in a net cage farm with diseased fish, and the findings were consistent with the actual situation. This study provides a valuable tool for assisting in the early monitoring and control of cryptocaryoniasis in aquaculture.

Severe Natural Outbreak of Cryptocaryon irritans in Gilthead Seabream Produces Leukocyte Mobilization and Innate Immunity at the Gill Tissue.

The protozoan parasite Cryptocaryon irritans causes marine white spot disease in a wide range of fish hosts, including gilthead seabream, a very sensitive species with great economic importance in the Mediterranean area. Thus, we aimed to evaluate the immunity of gilthead seabream after a severe natural outbreak of C. irritans. Morphological alterations and immune cell appearance in the gills were studied by light microscopy and immunohistochemical staining. The expression of several immune-related genes in the gills and head kidney were studied by qPCR, including inflammatory and immune cell markers, antimicrobial peptides (AMP), and cell-mediated cytotoxicity (CMC) molecules. Serum humoral innate immune activities were also assayed. Fish mortality reached 100% 8 days after the appearance of the C. irritans episode. Gill filaments were engrossed and packed without any space between filaments and included parasites and large numbers of undifferentiated and immune cells, namely acidophilic granulocytes. Our data suggest leukocyte mobilization from the head kidney, while the gills show the up-regulated transcription of inflammatory, AMPs, and CMC-related molecules. Meanwhile, only serum bactericidal activity was increased upon infection. A potent local innate immune response in the gills, probably orchestrated by AMPs and CMC, is triggered by a severe natural outbreak of C. irritans.

Barcode sequence could be a good target for developing a species-specific anti-parasite agent based on CRISPR-Cas9.

Parasitic infections are a severe issue in many regions of the world. We assume that if a chemical can destroy a DNA barcode sequence, then this chemical could be developed as a species-specific parasiticidal agent. To test this hypothesis, we designed sgRNAs that target the sequences of both a DNA barcode (ITS-2) and a control (5.8S rDNA) in Cryptocaryon irritans. In in vivo tests, we found that exposure to Cas9 mRNA mixed with sgRNAs was able to significantly reduce the hatching rate of tomont and the survival rate of theront. Quantitative Real-time PCR demonstrated that the DNAs of tomont and theront exposed to sgRNAs and Cas9 mRNA were significantly disrupted, no matter whether they were exposed to a single sgRNA or a mixture of two sgRNAs. DNA sequencing also suggested the test group that was exposed to a single sgRNA mixed with Cas9-induced mutation at sgRNA targeted fragments and the test group exposed to two sgRNAs combined with Cas9-induced deletion of large pieces. The findings and principles provided by this study contribute to the development of novel nucleic acid therapeutic drugs for cryptocaryoniasis and other parasitic diseases and provide insight into the development of species-specific parasiticidal agents.

Local immune responses to two stages of Ichthyophthirius multifiliis in ginbuna crucian carp.

Ichthyophthirius multifiliis is a ciliated protozoan parasite and is known to infect many freshwater teleosts. Characterizing the immune system in epithelial tissues, where the parasites penetrate and settle, is key to understanding host-parasite interactions. This study examined local immune responses in vivo to the infective stage (theront and trophont) of the parasites using intra-fin administration, which has been developed to analyze in vivo immune responses using fish fin. CD8α+ and CD4+ T-cell compositions were increased significantly in the fin cavity injected with theront or trophont antigens. The expression of GATA-3 and T-bet mRNA, which regulate differentiation of helper T-cells, was upregulated significantly in leukocytes from the trophont antigen-injected site. In contrast, the percentages of macrophages and neutrophils, which are innate immunity components, were decreased significantly in the injection sites. These results suggest that I. multifiliis antigens inhibit the migration of macrophages and neutrophils, and T-cells are the first responders to I. multifiliis. Thus, to better understand the interaction of host immunity and I. multifiliis, further studies should focus on exploring the inhibitory factors from I. multifiliis or examining innate functions of teleost T-cells.

Transcriptomic analysis reveals innate immune mechanisms of an underlying parasite-resistant grouper hybrid (Epinephelus fuscogutatus × Epinephelus lanceolatus).

Hybridization is an artificial breeding strategy for generating potentially desirable offspring. Recently, a novel Hulong grouper hybrid (Epinephelus fuscogutatus × Epinephelus lanceolatus) yielded significant growth superiority over its parent. Improved innate immunity is considered as another desirable feature during hybridization. However, whether this Hulong grouper achieved disease resistance has not yet been revealed. In this study, we first examine the infection intensity of C. irritans in the Hulong grouper, and found that the Hulong grouper is less susceptible to C. irritans primary infection. A higher immobilization titer was found in the infected Hulong grouper at Day 2 when compared with the control grouper. Furthermore, severe hyperplasia was observed in the orange-spotted grouper, but not in the Hulong grouper's skin epidermis. To further understand the innate immune mechanism against C. irritans, we conducted a comparative transcriptome analysis of the Hulong grouper during the infection. There are 6464 differentially expressed genes (DEGs) identified in the skin between the control and infected Hulong grouper. This indicates that the innate immune components, such as the complement system, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, Interleukin 17 (IL-17) signaling pathway, and Toll-like receptor (TLR) signaling pathway were up-regulated during the infection. These results show that the C. irritans infection can induce a remarkable inflammatory response in the Hulong grouper. Moreover, a total of 75 pairs of orthologs with the ratio of nonsynonymous (Ka) to synonymous (Ks) substitutions ＞1, considered rapidly evolving genes (REGs), was identified between the Hulong and orange-spotted grouper. More critically, most REGs were enriched in the immune system, suggesting that rapid evolution of the immune system might occur in the Hulong grouper. These results provide a more comprehensive understanding of the innate immunity mechanism of the hybrid Hulong grouper.

Updating specific PCR primer for detection of Cryptocaryon irritans from reared Larimichthys polyactis.

Artificial breeding of small yellow croaker (Larimichthys polyactis) was recently achieved, providing a bright future for its commercial farming. In May 2019, a disease outbreak occurred among small yellow croakers in an aquaculture farm near Xiangshan Bay, charactering by white spots spotted on the surface of fish skin, gills and fins. The parasite was preliminarily identified as Cryptocaryon irritans based on morphological feature of the parasite and the symptoms on fish. However, the previously published specific primer pairs failed to confirm the existence of C. iriitans. Six nucleotides mismatches were discovered after mapping specific forward primer back to targeted gene. Therefore, an updated PCR specific primer was developed within the 9th highly variable region of 18S rRNA gene and conserved in all C. irritans sequences available in GenBank database. The specificity was verified in silico by Primer-BLAST against GenBank nucleotide. Laboratory cultured ciliates (Mesanophrys, Pseudokeronopsis and Uronema) as well as natural microbial community samples collected from sea water and river water was used as negative control to verify the specificity of the primer in situ. Besides, tank transfer method was used to evaluate the treatment of the parasite infection. By tank transfer method, 2.00 ± 0.61 out of 10 fish that already sever infected were successfully survived after 8 days treatment, meanwhile the control group died out at d 6. More loss to the treatment group during first five days was observed and may attribute to the combined effect from infection and stress the recent domesticated fish suffered during rotation. Therefore, tank transfer method was also effective to prevent small yellow croaker from further infection, however the loss of the small yellow croaker suffered from stress during rotation also needs to be carefully concerned. In conclusion, this study reported the first diagnose of C. irritans infection on small yellow croaker, provided updated specific primer to detect C. irritans infection on fish body and reported the effect of tank transfer on small yellow croaker treatment.

Scuticociliate (Philasterides dicentrarchi) infection cluster in a multispecies marine aquarium system.

Scuticociliatosis, caused by ciliated protozoa of the subclass Scuticociliatia, has been associated with high mortalities in marine fish. Environmental factors such as an increase in water temperature can enhance this disease. The aim of the present report is to describe the occurrence of a cluster of cases of scuticociliatosis in a multispecies marine cold-water system in a public aquarium. Philasterides dicentrarchi was identified by PCR in formalin-fixed tissues of some of the fish showing meningitis or meningoencephalitis, dermatitis and myositis with intralesional protozoa. An increase in water temperature of approximately 2°C was identified as a potential contributing factor for this cluster of infections. Higher temperature may have enhanced the propagation or pathogenicity of scuticociliates or increased host susceptibility of some species of fish, especially wolf-eel Anarrhichthys ocellatus and spotted ratfish Hydrolagus colliei. This report also highlights the complexity of dealing with mixed species systems housing fish from different natural ecozones.

Transcriptome analysis in Takifugu rubripes and Dicentrarchus labrax gills during Cryptocaryon irritans infection.

Takifugu rubripes and Dicentrarchus labrax are important commercial fish in China that are under serious threat from Cryptocaryon irritans. C. irritans is a ciliated obligate parasite that causes marine white spot disease and leads to heavy economic losses. We analysed the transcriptome in the gills of T. rubripes and D. labrax to compare differentially expressed genes (DEGs) and pathways during infection with C. irritans. In total, we identified 6,901 and 35,736 DEGs from T. rubripes and D. labrax, respectively. All DEGs were annotated into GO terms; 6,901 DEGs from T. rubripes were assigned into 991 sub-categories, and 35,736 DEGs from D. labrax were assigned into 8,517 sub-categories. We mapped DEGs to the KEGG database and obtained 153 and 350 KEGG signalling pathways from T. rubripes and D. labrax, respectively. Immune-related categories included Toll-like receptors, MAPK, lysosome, C-type lectin receptor and NOD-like receptor signalling pathways were significantly enriched pathways. In immune-related signalling pathways, we found that AP-1, P38, IL-1ß, HSP90 and PLA were significantly up-regulated DEGs in T. rubripes, but P38 and PLA were significantly down-regulated in D. labrax. In this study, transcriptome was used to analyse the difference between scaly and non-scaly fish infection by C. irritans, which not only provided a theoretical basis for the infection mechanism of C. irritans, but also laid a foundation for effectively inhibiting the occurrence of this disease. Our work provides further insight into the immune response of host resistance to C. irritans.