Immunological and molecular diagnostic techniques in fish health: present and future prospectus.

Fish health management is critical to aquaculture and fisheries as it directly affects sustainability and productivity. Fish disease diagnosis has taken a massive stride because of advances in immunological and molecular diagnostic tools which provide a sensitive, quick, and accurate means of identifying diseases. This review presents an overview of the main molecular and immunological diagnostic methods for determining the health of fish. The immunological techniques help to diagnose different fish diseases by detecting specific antigens and antibodies. The application of immunological techniques to vaccine development is also examined in this review. The genetic identification of pathogens is made possible by molecular diagnostic techniques that enable the precise identification of bacterial, viral, and parasitic organisms in addition to evaluating host reactions and genetic variation associated with resistance to disease. The combination of molecular and immunological methods has resulted in the creation of novel techniques for thorough evaluation of fish health. These developments improve treatment measures, pathogen identification and provide new information about the variables affecting fish health, such as genetic predispositions and environmental stresses. In the framework of sustainable fish farming and fisheries management, this paper focuses on the importance of these diagnostic techniques that play a crucial role in protecting fish populations and the aquatic habitats. This review also examines the present and potential future directions in immunological and molecular diagnostic techniques in fish health.

Identification of the grass carp interleukin-23 receptor and its proinflammatory role in intestinal inflammation.

The interleukin 23 receptor (IL-23R) is associated with a variety of inflammatory diseases in humans and other mammals. However, whether IL-23R is involved in inflammatory diseases in teleost fish is less understood. Thus, to investigate the potential involvement of IL-23R in fish inflammatory diseases, the full-length cDNA of IL-23R from grass carp Ctenopharyngodon idella was cloned and used to generate a recombinant protein (rgcIL-23R) containing the extracellular domain of IL-23R, against which a polyclonal antibody (rgcIL-23R pAb) was then developed. qPCR analysis revealed that IL-23R mRNA was significantly upregulated in most grass carp tissues in response to infection with Gram-negative Aeromonas hydrophila. Treatment with rgcIL-23R significantly induced IL-17A/F1 expression in C. idella kidney (CIK) cells. By contrast, knockdown of IL-23R caused significant decreases in IL-23R, STAT3, and IL-17N expression in CIK cells after lipopolysaccharide (LPS) stimulation. Similarly, rgcIL-23R pAb treatment effectively inhibited the LPS-induced increase in the expression of IL-23 subunit genes and those of the IL-23/IL-17 pathway in CIK cells. Furthermore, intestinal symptoms identical to those caused by A. hydrophila were induced by anal intubation with rgcIL-23R, but suppressed by rgcIL-23R pAb. Therefore, these results suggest that IL-23R has a crucial role in the regulation of intestinal inflammation and, thus, is a promising target for controlling inflammatory diseases in farmed fish.

DNA Methylation Analysis Reveals Potential Mechanism in Takifugu rubripes Against Cryptocaryon irritans Infection.

Takifugu rubripes (T. rubripes) is a valuable commercial fish, and Cryptocaryon irritans (C. irritans) has a significant impact on its aquaculture productivity. DNA methylation is one of the earliest discovered ways of gene epigenetic modification and also an important form of modification, as well as an essential type of alteration that regulates gene expression, including immune response. To further explore the anti-infection mechanism of T. rubripes in inhibiting this disease, we determined genome-wide DNA methylation profiles in the gill of T. rubripes using whole-genome bisulfite sequencing (WGBS) and combined with RNA sequence (RNA-seq). A total of 4659 differentially methylated genes (DMGs) in the gene body and 1546 DMGs in the promoter between the infection and control group were identified. And we identified 2501 differentially expressed genes (DEGs), including 1100 upregulated and 1401 downregulated genes. After enrichment analysis, we identified DMGs and DEGs of immune-related pathways including MAPK, Wnt, ErbB, and VEGF signaling pathways, as well as node genes prkcb, myca, tp53, and map2k2a. Based on the RNA-Seq results, we plotted a network graph to demonstrate the relationship between immune pathways and functional related genes, in addition to gene methylation and expression levels. At the same time, we predicted the CpG island and transcription factor of four immune-related key genes prkcb and mapped the gene structure. These unique discoveries could be helpful in the understanding of C. irritans pathogenesis, and the candidate genes screened may serve as optimum methylation-based biomarkers that can be utilized for the correct diagnosis and therapy T. rubripes in the development of the ability to resist C. irritans infection.

Immune and endocrine alterations at the early stage of inflammatory assemblage in toads after stimulation with heat-killed bacteria (Aeromonas hydrophila).

The red-leg syndrome in amphibians is a condition commonly associated with the bacteria Aeromonas hydrophila and has led to population declines. However, there is little information concerning the inflammatory assemblage in infected anurans. We evaluated immune and endocrine alterations induced by stimulation with heat-killed A. hydrophila injected in Rhinella diptycha toads. Control animals were not manipulated, while the others were separated into groups that received intraperitoneal injection of 300 µl of saline or heat-killed bacteria: groups A1 (3 × 107 cells), A2 (3 × 108 cells), and A3 (3 × 109 cells). Animals were bled and euthanized six hours post-injection. We evaluated neutrophil: lymphocyte ratio (NLR), plasma bacterial killing ability (BKA), testosterone (T), melatonin (MEL), and corticosterone (CORT) plasma levels. Heat-killed A. hydrophila increased CORT and NLR, and decreased MEL, especially at higher concentrations. There was no effect of treatment on T and BKA. We then selected the saline and A3 groups to conduct mRNA expression of several genes including glucocorticoid receptor (GR), toll-like receptor-4 (TLR-4), interferon-γ (IFN-γ), interleukin (IL)-1ß, IL-6, and IL-10. We found higher expression of IL-6, IL-1ß, IL-10, and IFN-γ in group A3 compared to the saline group. These results indicate the beginning of an inflammatory assemblage, notably at the two highest concentrations of bacteria, and give a better understanding of how anurans respond to an infection within an integrated perspective, evaluating different physiological aspects. Future studies should investigate later phases of the immune response to elucidate more about the inflammation in amphibians challenged with A. hydrophila.

Participation of Hepcidins in the Inflammatory Response Triggered by λ-Carrageenin in Gilthead Seabream (Sparus aurata).

The role of hepcidins, antimicrobial peptides involved in iron metabolism, immunity, and inflammation, is studied. First, gilthead seabream (Sparus aurata L.) head-kidney leucocytes (HKLs) were incubated with λ-carrageenin to study the expression of hepcidin and iron metabolism-related genes. While the expression of most of the genes studied was upregulated, the expression of ferroportin gene (slc40a) was downregulated. In the second part of the study, seabream specimens were injected intramuscularly with λ-carrageenin or buffer (control). The expression of the same genes was evaluated in the head kidney, liver, and skin at different time points after injection. The expression of Hamp1m, ferritin b, and ferroportin genes (hamp1, fthb, and slc40a) was upregulated in the head kidney of fish from the λ-carrageenin-injected group, while the expression of Hamp2C and Hamp2E genes (hamp2.3 and hamp2.7) was downregulated. In the liver, the expression of hamp1, ferritin a (ftha), slc40a, Hamp2J, and Hamp2D (hamp2.5/6) genes was downregulated in the λ-carrageenin-injected group. In the skin, the expression of hamp1 and (Hamp2A Hamp2C) hamp2.1/3/4 genes was upregulated in the λ-carrageenin-injected group. A bioinformatic analysis was performed to predict the presence of transcription factor binding sites in the promoter region of hepcidins. The primary sequence of hepcidin was conserved among the different mature peptides, although changes in specific amino acid residues were identified. These changes affected the charge, hydrophobicity, and probability of hepcidins being antimicrobial peptides. This study sheds light on the poorly understood roles of hepcidins in fish. The results provide insight into the regulatory mechanisms of inflammation in fish and could contribute to the development of new strategies for treat inflammation in farm animals.

RNAi-directed knockdown in the cnidarian fish blood parasite Sphaerospora molnari.

RNA interference (RNAi) is an effective approach to suppress gene expression and monitor gene regulation. Despite its wide application, its use is limited in certain taxonomic groups, including cnidarians. Myxozoans are a unique group of cnidarian parasites that diverged from their free-living ancestors about 600 million years ago, with several species causing acute disease in farmed and wild fish populations. In this pioneering study we successfully applied RNAi in blood stages of the myxozoan Sphaerospora molnari, combining a dsRNA soaking approach, real-time PCR, confocal microscopy, and Western blotting. For proof of concept, we knocked down two unusual actins, one of which is known to play a critical role in S. molnari cell motility. We observed intracellular uptake of dsRNA after 30 min and accumulation in all cells of the typical myxozoan cell-in-cell structure. We successfully knocked down actin in S. molnari in vitro, with transient inhibition for 48 h. We observed the disruption of the cytoskeletal network within the primary cell and loss of the characteristic rotational cell motility. This RNAi workflow could significantly advance functional research within the Myxozoa, offering new prospects for investigating therapeutic targets and facilitating drug discovery against economically important fish parasites.

Nervous necrosis virus induced vacuolization is a Rab5- and actin-dependent process.

Cytoplasmic vacuolization is commonly induced by bacteria and viruses, reflecting the complex interactions between pathogens and the host. However, their characteristics and formation remain unclear. Nervous necrosis virus (NNV) infects more than 100 global fish species, causing enormous economic losses. Vacuolization is a hallmark of NNV infection in host cells, but remains a mystery. In this study, we developed a simple aptamer labelling technique to identify red-spotted grouper NNV (RGNNV) particles in fixed and live cells to explore RGNNV-induced vacuolization. We observed that RGNNV-induced vacuolization was positively associated with the infection time and virus uptake. During infection, most RGNNV particles, as well as viral genes, colocalized with vacuoles, but not giant vacuoles > 3 µm in diameter. Although the capsid protein (CP) is the only structural protein of RGNNV, its overexpression did not induce vacuolization, suggesting that vacuole formation probably requires virus entry and replication. Given that small Rab proteins and the cytoskeleton are key factors in regulating cellular vesicles, we further investigated their roles in RGNNV-induced vacuolization. Using live cell imaging, Rab5, a marker of early endosomes, was continuously located in vacuoles bearing RGNNV during giant vacuole formation. Rab5 is required for vacuole formation and interacts with CP according to siRNA interference and Co-IP analysis. Furthermore, actin formed distinct rings around small vacuoles, while vacuoles were located near microtubules. Actin, but not microtubules, plays an important role in vacuole formation using chemical inhibitors. These results provide valuable insights into the pathogenesis and control of RGNNV infections.

Characterization of caspase gene family in Sebastes schlegelii and their expression profiles under Aeromonas salmonicida and Vibrio anguillarum infection.

The caspase, functioning as a proteinase, plays a crucial role in eukaryotic cell apoptosis, regulation of apoptosis, cellular growth, differentiation, and immunity. The identification of caspase gene family in Sebastes schlegelii is of great help to understand its antimicrobial research. In S. schlegelii, we totally identified nine caspase genes, including four apoptosis initiator caspases (caspase 2, caspase 8, caspase 9 and caspase 10), four apoptosis executioners (caspase 3a, caspase 3b, caspase 6, and caspase 7) and one inflammatory executioner (caspase 1). The duplication of caspase 3 genes on chr3 and chr8 may have been facilitated by whole genome duplication (WGD) events or other complex evolutionary processes. In general, the number of caspase genes relatively conserved in high vertebrates, while exhibiting variation in teleosts. Furthermore, syntenic analysis and phylogenetic relationships analysis supported the correct classification of these caspase gene family in S. schlegelii, especially for genes with duplicated copies. Additionally, the expression patterns of these caspase genes in different tissues of S. schlegelii under healthy conditions were assessed. The results revealed that the expression levels of most caspase genes were significantly elevated in the intestine, spleen, and liver. To further investigate the potential immune functions of these caspase genes in S. schlegelii, we challenged individuals with A. salmonicida and V. anguillarum, respectively. After infection with A. salmonicida, the expression levels of caspase 1 in the liver and spleen of S. schlegelii remained consistently elevated throughout the infection time points. The expression levels of most caspase family members in the intestine exhibited significant divergence following V. anguillarum infection. This study provides a comprehensive understanding of the caspase gene families in S. schlegelii, thereby establishing a solid foundation for further investigations into the functional roles of these caspase genes.

Grass carp superoxide dismutases exert antioxidant function and inhibit autophagy to promote grass carp reovirus (GCRV) replication.

Superoxide dismutases (SODs) are potent antioxidants crucial for neutralizing reactive oxygen species (ROS) and protecting organisms from oxidative damage. In this study, we successfully cloned and analyzed two SOD genes, CiSOD1 and CiSOD2, from grass carp (Ctenopharyngodon idellus). CiSOD1 consists of two CuZn signature motifs and two conserved cysteine residues, while CiSOD2 contains a single Mn signature motif. The expression of CiSODs was found to be ubiquitous across all examined tissues, with their expression levels significantly altered after stimulation by grass carp reovirus (GCRV) or pathogen-associated molecular patterns (PAMPs). CiSOD1 was observed to be uniformly distributed in the cytoplasm, whereas CiSOD2 localized in the mitochondria. Escherichia coli transformed with both CiSODs demonstrated enhanced host resistance to H2O2 and heavy metals. Additionally, purified recombinant CiSOD proteins effectively protected DNA against oxidative damage. Furthermore, overexpression of CiSODs in fish cells reduced intracellular ROS, inhibited autophagy, and then resulted in the promotion of GCRV replication. Knockdown of CiSODs showed opposite trends. Notably, these roles of CiSODs in autophagy and GCRV replication were reversed upon treatment with an autophagy inducer. In summary, our findings suggest that grass carp SODs play an important role in decreasing intracellular ROS levels, inhibiting autophagy, and subsequently promoting GCRV replication.

Lufenuron treatment temporarily represses gene expression and affects the SUMO pathway in liver of Atlantic salmon.

Lufenuron is a benzoylurea insecticide currently in use to combat sea lice infestation in salmon aquaculture in Chile. With pending approval in Norway, the aim of this work was to study the uptake and toxicity of lufenuron in liver tissue of Atlantic salmon. Juvenile salmon weighing 40 g were given a standard 7-day oral dose, and bioaccumulation and transcriptional responses in the liver were examined 1 day after the end-of-treatment (day 8) and after 1 week of elimination (day 14). Bioaccumulation levels of lufenuron were 29 ± 3 mg/kg at day 8 and 14 ± 1 mg/kg at day 14, indicating relatively rapid clearance. However, residues of lufenuron were still present in the liver after 513 days of depuration. The exposure gave a transient inhibition of transcription in the liver at day 8 (2437 significant DEGs, p-adj < .05), followed by a weaker compensatory response at day 14 (169 significant DEGs). Pathways associated with RNA metabolism such as the sumoylation pathway were most strongly affected at day 8, while the apelin pathway was most profoundly affected at day 14. In conclusion, this study shows that lufenuron easily bioaccumulates and that a standard 7-day oral dose induces a transient inhibition of transcription in liver of salmon.

Phenotypic and genomic insights into the pathogenicity and antimicrobial resistance of an Enterobacter roggenkampii strain isolated from diseased silver arowana (Osteoglossum bicirrhosum).

Enterobacter roggenkampii is an opportunistic pathogen that causes infections in a wide range of hosts. A bacterial strain named EOBSR_19 was isolated from diseased silver arowana, Osteoglossum bicirrhosum. This bacterium was identified as E. roggenkampii based on the phenotypic characteristics and sequence analysis of the16S rDNA and gyrB genes. Average nucleotide identity and phylogenetic analysis based on the whole genome sequence further confirmed the bacterial taxonomy of EOBSR_19. Artificial experimental infection indicated that EOBSR_19 was pathogenic to fish. Antimicrobial susceptibility test showed it was multi-drug resistant. The EOBSR_19 was found to be resistant to 18 antibiotics belonging to quinolones, macrolides, sulfonamides, aminoglycosides, and ß-lactams classes. The whole genome sequencing analysis showed that EOBSR_19 carried 730 virulence genes that were annotated for different functional modules, such as adhesion and invasion, secretion system, siderophore transport system and bacterial toxin. Among them, the virulence genes related to adhesion and invasion were the most abundant. In addition, drug resistance genes involved in multiple mechanisms of antimicrobial resistance were identified in its genomics, including multidrug resistance efflux pumps, antibiotic inactivating enzymes, and antibiotic binding site mutations. Its genomic analysis via whole-genome sequencing provided insights into the pathogenicity and antimicrobial resistance.

Characterization and in vitro antibacterial activity of grass carp (Ctenopharyngodon idella) serum amyloid A.

Serum amyloid A (SAA) predominantly synthesized by hepatocytes is a classical acute phase protein and has been extensively studied in mammals. However, the studies on the structure and properties of fish SAA are limited although SAA genes have been cloned and identified from various fishes. In the present study, a cDNA of grass carp (Ctenopharyngodon idella) SAA (gcSAA) was cloned and characterized, displaying a high homology with its counterparts in vertebrates. gcSAA mRNA was expressed with highest abundance in the liver and its levels were increased by a 24-hour infection of Aeromonas hydrophila (A. hydrophila) for more than 5 folds in the intestine, 15 folds in the spleen, 75 folds in the head kidney and 100 folds in the liver, implying that it is an acute phase protein in grass carp. Subsequently, recombinant gcSAA protein (rgcSAA) was prepared from a prokaryotic expression system after codon optimization of its coding sequence. The direct antibacterial activity assay and the plate count assay disclosed that gcSAA inhibited the growth and survival of A. hydrophila but not Edwardsiella piscicida (E. piscicida) which both are common bacterial pathogens in aquaculture. The propidium iodide (PI) uptake assay confirmed the bactericidal property of gcSAA, showing that it is able to enhance the uptake of PI in A. hydrophila but not E. piscicida. These findings revealed the molecular features of gcSAA and its roles in host defense against bacterial infection.

Characteristics and Complete Genome Analysis of a Pathogenic Aeromonas Veronii SJ4 from Diseased Siniperca Chuatsi.

As an opportunistic pathogen, Aeromonas veronii can cause hemorrhagic septicemia of various aquatic animals. In our present study, a dominant strain SJ4, isolated from naturally infected mandarin fish (Siniperca chuatsi), was identified as A. veronii according to the morphological, physiological, and biochemical features, as well as molecular identification. Intraperitoneal injection of A. veronii SJ4 into S. chuatsi revealed clinical signs similar to the natural infection, and the median lethal dosage (LD50) of the SJ4 to S. chuatsi in a week was 3.8 × 105 CFU/mL. Histopathological analysis revealed that the isolate SJ4 could cause cell enlargement, obvious hemorrhage, and inflammatory responses in S. chuatsi. Detection of virulence genes showed the isolate SJ4 carried act, fim, flgM, ompA, lip, hly, aer, and eprCAL, and the isolate SJ4 also produce caseinase, dnase, gelatinase, and hemolysin. In addition, the complete genome of A. veronii SJ4 was sequenced, and the size of the genome of A. veronii SJ4 was 4,562,694 bp, within a G + C content of 58.95%, containing 4079 coding genes. Nine hundred ten genes encoding for several virulence factors, such as type III and VI secretion systems, flagella, motility, etc., were determined based on the VFDB database. Besides, 148 antibiotic resistance-related genes in 27 categories related to tetracyclines, fluoroquinolones, aminoglycosides, macrolides, chloramphenicol, and cephalosporins were also annotated. The present results suggested that A. veronii was etiological agent causing the bacterial septicemia of S. chuatsi in this time, as well as provided a valuable base for revealing pathogenesis and resistance mechanism of A. veronii.

Genomic characterization and transcription analysis of European sea bass (Dicentrarchus labrax) rtp3 genes.

Fish RTP3, belonging to the receptor-transporting protein family, display several functions, including a putative antiviral role as virus-responsive gene. In this work, we have identified and characterized two different European sea bass rtp3 genes. In addition, an in vivo transcription analysis in response to LPS, poly I:C and betanodavirus infection (RGNNV genotype) has been performed. The sequence analysis showed that European sea bass displays two rtp3 genes, X1 and X2, composed of two exons and a single intron (1007-bp and 888-bp long, respectively), located within the ORF sequence. The full-length cDNA is 1969 bp for rtp3 X1, and 1491 bp for rtp3 X2. Several ATTTA motifs have been found in the intron sequence of both genes, whereas rtp3 X1 also contains this motif in both untranslated regions. The transcription analyses revealed significant level of rtp3 X2 mRNA in brain and head kidney after LPS and poly I:C inoculation; however, the induction elicited by RGNNV infection was much higher, suggesting an essential role for this protein in controlling NNV infections.

A circRNA therapy based on Rnf103 to inhibit Vibrio anguillarum infection.

The losses caused by Vibrio infections in the aquaculture industry are challenging to quantify. In the face of antibiotic resistance, a natural and environmentally friendly alternative is urgently needed. In this study, we identify E3 ubiquitin-protein ligase RNF103 (rnf103) as a crucial target involved in immune evasion by Vibrio anguillarum. Our research demonstrates that Rnf103 promotes immune escape by inhibiting Traf6. Interestingly, we discover a circular RNA (circRNA), circRnf103, formed by reverse splicing of the Rnf103 gene. Predictive analysis and experimentation reveal that circRnf103 encodes Rnf103-177aa, a protein that competes with Rnf103 and binds to Traf6, preventing its degradation. Notably, circRnf103 therapy induces Rnf103-177aa protein production in zebrafish. In zebrafish models, circRnf103 exhibits significant effectiveness in treating V. anguillarum infections, reducing organ burden. These findings highlight the potential of circRNA therapy as a natural and innovative approach to combat infectious diseases sustainably, particularly in aquaculture and environmental management.

The transcriptome sequencing analysis reveals immune mechanisms of soybean fermented powder on the loach (Misgurnus anguillicaudatus) in response to Lipopolysaccharide (LPS) infection.

The loach (Misgurnus anguillicaudatus), a small commercial fish that is widely cultivated for its high-quality protein, vitamins, minerals, and essential amino acid, is a member of the genus Misgurnus and the family Cyprinidae. In this study, we gave the LPS-injected loach fermented soybean meal and used transcriptome sequencing to investigate the impact of the fermented soybean powder on the loach's immune system. 3384 up-regulated genes and 12116 down-regulated genes were found among the 15500 differentially expressed genes, according to the results. The differentially expressed genes were shown to be involved in cellular processes, metabolic processes, cellular anatomical entities, and binding, according to the Go functional annotation. Meanwhile, the KEGG enrichment analysis indicated that the soybean fermented powder treated groups showed significant differences in DNA replication, Nucleotide excision repair, Fanconi anemia pathway, and Base excision repair pathways, suggesting that these pathways are closely related to the enhancement of the immune function of loach by soybean fermented powder. The particular conclusions not exclusively can provide a new conception for the rational utilization of soybean fermented powder but also can provide theoretical guidance for the subsequent healthy breeding of loach.

16S rRNA and transcriptome analysis of the FOS-mediated alleviation of Aeromonas hydrophila-induced intestinal damage in Megalobrama amblycephala.

This study was conducted to elucidate the effects of FOS that alleviate Aeromonas hydrophila-induced intestinal damage. The results showed that A. hydrophila disrupted the intestinal structure and increased intestinal permeability, causing abnormalities in mucosal pathology. Additionally, A. hydrophila induced an imbalance in the intestinal flora and disturbed its stability. Dietary FOS ameliorated the injury to the intestinal structure of fish, but also in part improved the condition of the intestinal tight junction complex. Transcriptomic analysis showed that 120 genes were up-regulated and 320 genes were down-regulated. The intestinal immune network for the IgA production signalling pathway was enriched following A. hydrophila infection, and the change in the FOS group was mainly in the Tight junction signalling pathway. Similarly, dietary FOS reduced the disruption of the intestinal microbiota induced by A. hydrophila and improved the intestinal microbiota's stability; FOS was also partially implicated in the upregulation of Tight junction and Adhesion junction pathways by transcriptomic analysis. After further analysis, it was found that fish fed FOS had upregulated expression of genes related to apoptosis, antigen presentation, and the T-cell-mediated immune response in the intestine compared with those in the A. hydrophila group, which may be related to changes in the intestinal microbiome.

Transcriptomic landscape of Atlantic salmon (Salmo salar L.) skin.

In this study, we present the first spatial transcriptomic atlas of Atlantic salmon skin using the Visium Spatial Gene Expression protocol. We utilized frozen skin tissue from 4 distinct sites, namely the operculum, pectoral and caudal fins, and scaly skin at the flank of the fish close to the lateral line, obtained from 2 Atlantic salmon (150 g). High-quality frozen tissue sections were obtained by embedding tissue in optimal cutting temperature media prior to freezing and sectioning. Further, we generated libraries and spatial transcriptomic maps, achieving a minimum of 80 million reads per sample with mapping efficiencies ranging from 79.3 to 89.4%. Our analysis revealed the detection of over 80,000 transcripts and nearly 30,000 genes in each sample. Among the tissue types observed in the skin, the epithelial tissues exhibited the highest number of transcripts (unique molecular identifier counts), followed by muscle tissue, loose and fibrous connective tissue, and bone. Notably, the widest nodes in the transcriptome network were shared among the epithelial clusters, while dermal tissues showed less consistency, which is likely attributable to the presence of multiple cell types at different body locations. Additionally, we identified collagen type 1 as the most prominent gene family in the skin, while keratins were found to be abundant in the epithelial tissue. Furthermore, we successfully identified gene markers specific to epithelial tissue, bone, and mesenchyme. To validate their expression patterns, we conducted a meta-analysis of the microarray database, which confirmed high expression levels of these markers in mucosal organs, skin, gills, and the olfactory rosette.

Transcriptomic Variation of Amphiprion Percula (Lacepède, 1802) in Response to Infection with Cryptocaryon Irritans Brown, 1951.

Cryptocaryon irritans (Brown 1951) frequently infect the Pomacentridae fishes causing severe economic losses. However, the anti-C. irritans' molecular mechanism in these fishes remains largely unknown. To address this issue, we conducted RNA-Seq for C. irrtians-infected gills of the clownfish Amphiprion percula (Lacepède 1802) at the early (day 1) and late (day 3) stages of infection. A total of 1655 differentially expressed genes (DEGs) were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of DEGs showed a vast genetic variation related to the following aspects: ECM-receptor interaction, P13K-Akt signalling, cytokine-cytokine receptor interaction, and endocytosis. During the early phase of infection, key genes involved in ATP production, energy homeostasis, and stress control were abruptly increased. In the late phase, however, acute response molecules of the peripheral nervous system (synaptic transmission and local immunity), metabolic system triggering glycogen synthesis, energy maintenance, and osmoregulation were found to be critical. The highest number of upregulated genes (URGs) recovered during the early phase was included under the 'biological process' category, which primarily functions as response to stimuli, signalling, and biological regulation. In the late phase, most of the URGs were related to gene regulation and immune system processes under 'molecular function' category. The immune-related URGs of early infection include major histocompatibility complex (MHC) class-II molecules apparently triggering CD4+ T-cell-activated Th responses, and that of late infection include MHC class-1 molecules for the possible culmination of CD8+ T-cell triggered cytotoxicity. The high level of genic single nucleotide polymorphisms (SNPs) identified during the late phase of infection is likely to influence their susceptibility to secondary infection. In summary, the identified DEGs and their related metabolic and immune-related pathways and the SNPs may provide new insights into coordinating the immunological events and improving resistance in Pomacentridae fishes against C. irritans.

Silencing of the Vasa gene by RNA Interference Affects Embryonic Development and Reproductive Output in the Sea Louse Caligus rogercresseyi.

The sea louse Caligus rogercresseyi is a major ectoparasitic copepod that causes significant economic losses in the salmon farming industry. Despite recent advancements, the mechanisms underlying germline and embryo development in this species remain poorly understood. The Vasa gene encodes a highly conserved DEAD box helicase that is required for germ cell formation and function in many species. In this study, the Vasa gene was characterized in C. rogercresseyi, and its expression and function were analyzed. Phylogenetic analysis showed that the Cr-Vasa gene product formed clusters in clades with Vasa proteins from closely related species of crustaceans. Cr-Vasa gene expression patterns were assessed by qPCR, and the results showed a significantly higher relative expression level in adult females compared to copepodid, chalimus, and adult male stages. Tissue-specific localization of Cr-Vasa mRNA in C. rogercresseyi was determined using chromogenic in situ hybridization, and strong positive signal was observed in male testes, but also in the intestine and cuticle, while in females, it was observed in the ovaries, oocytes, cuticle, intestine, and egg strings. RNAi-mediated gene silencing of Cr-Vasa impacted embryonic development and reproductive output in adult female lice. Females from the dsVasa-treated group displayed unusual phenotypes, including shorter egg strings with numerous extra-embryonic inclusions, irregularly shaped abnormal embryos, and aborted egg strings. This study provides insights into the role of the Vasa gene in C. rogercresseyi embryonic development and reproductive output, which may have implications for the control of this parasitic copepod in the salmon farming industry.