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.

Characterization of miRNAs in Embryonic, Larval, and Adult Lumpfish Provides a Reference miRNAome for Cyclopterus lumpus

MicroRNAs (miRNAs) are endogenous small RNA molecules involved in the post-transcriptional regulation of protein expression by binding to the mRNA of target genes. They are key regulators in teleost development, maintenance of tissue-specific functions, and immune responses. Lumpfish (Cyclopterus lumpus) is becoming an emergent aquaculture species as it has been utilized as a cleaner fish to biocontrol sea lice (e.g., Lepeophtheirus salmonis) infestation in the Atlantic Salmon (Salmo salar) aquaculture. The lumpfish miRNAs repertoire is unknown. This study identified and characterized miRNA encoding genes in lumpfish from three developmental stages (adult, embryos, and larvae). A total of 16 samples from six different adult lumpfish organs (spleen, liver, head kidney, brain, muscle, and gill), embryos, and larvae were individually small RNA sequenced. Altogether, 391 conserved miRNA precursor sequences (discovered in the majority of teleost fish species reported in miRbase), eight novel miRNA precursor sequences (so far only discovered in lumpfish), and 443 unique mature miRNAs were identified. Transcriptomics analysis suggested organ-specific and age-specific expression of miRNAs (e.g., miR-122-1-5p specific of the liver). Most of the miRNAs found in lumpfish are conserved in teleost and higher vertebrates, suggesting an essential and common role across teleost and higher vertebrates. This study is the first miRNA characterization of lumpfish that provides the reference miRNAome for future functional studies.

Development of Recombinant Dihydrolipoamide Dehydrogenase Subunit Vaccine against Vibrio Infection in Large Yellow Croaker

Large yellow croaker (Larimichthys crocea), an economically important marine fish in China, has suffered from serious vibriosis, which has resulted in great economic losses for the large yellow croaker industry. Vaccination has been considered to be a safe and effective method to prevent and control vibriosis. However, due to the complex diversity and serotypes of the Vibrio genus, the progress of Vibrio vaccine development is still slow. In this study, we prepared recombinant Vibrio dihydrolipoamide dehydrogenase (rDLD) protein and investigated its potential as a candidate to be a subunit vaccine against Vibrio. The lysozyme activity and the rDLD-specific antibody level in sera of large yellow croakers immunized with rDLD were significantly higher than those in the control group, and the transcript levels of proinflammatory cytokines (IL-6, IL-8, IL-1β), MHC IIα/β, CD40, CD8α, IL-4/13A, and IL-4/13B were significantly up-regulated in the spleen and head kidney of large yellow croakers immunized with rDLD, suggesting that rDLD could induce both specific and nonspecific immune responses in this species. In addition, rDLD protein increased the survival rate of large yellow croakers against Vibrio alginolyticus and Vibrio parahaemolyticus, with the relative percent of survival (RPS) being 74.5% and 66.9%, respectively. These results will facilitate the development of a potential subunit vaccine against Vibrio in large yellow croaker aquaculture.

Oral Administration of Nanopeptide CMCS-20H Conspicuously Boosts Immunity and Precautionary Effect Against Bacterial Infection in Fish

Massive mortalities caused by bacterial infections in intensive aquaculture result in serious economic losses. In this study, a novel antimicrobial peptide gcIFN-20H was efficiently expressed in Pichia pastoris (GS115) and loaded on carboxylmethyl chitosan (CMCS) to prepare CMCS-20H nanoparticles. Through physical characterization assays (TEM, DLS, BCA, and Raman) and biological activity tests (antimicrobial activity and cytotoxicity), CMCS-20H nanopeptide was verified to be spherical nanoparticles with sustained release, antimicrobial activity, and negligible toxicity. CMCS-20H nanoparticles are more resistant to intestinal degradation than unloaded gcIFN-20H by indirect immunofluorescence assay. Oral administration was then carried out for 42 days. Complement C3 content, lysozyme, and total superoxide dismutase activities are highest in CMCS-20H group by serum biochemistry index assays. After challenge with Aeromonas hydrophila, the survival rate in CMCS-20H group is highest (46%), which is 64% higher than the control group (28%). Meanwhile, the tissue bacterial loads (intestine, spleen, head kidney, trunk kidney, hepatopancreas, muscle, and blood) in the CMCS-20H group are significantly lower than other groups. By PAS staining analysis, the number of intestinal villi goblet cells and the thickness of mucin in the CMCS-20H group obviously increased. CMCS-20H effectively enhances mRNA expressions of some important immune genes (IL-1β, IL-6, TNF-α, IL-2, IFN-γ2, and IgM). The minimal tissue lesions (Intestine, spleen, and trunk kidney) were seen in the CMCS-20H group by histopathological examination. 16S rRNA sequencing showed that oral CMCS-20H maintains the intestinal microbiome homeostasis in bacterial infection. The results indicate that the novel nanopeptide CMCS-20H as the immunopotentiator can remarkably boost fish immunity and precautionary effect by oral administration and address the theoretical mechanisms and insights into the promising application prospect in aquaculture.

Effect of Methylmercury Exposure on Bioaccumulation and Nonspecific Immune Respsonses in Hybrid Grouper Epinephelus fuscoguttatus × Epinephelus lanceolatus

Mercury (Hg) is a dangerous heavy metal that can accumulate in fish and is harmful when consumed by humans. This study investigated the bioaccumulation of mercury in the form of methylmercury (MeHg) and evaluated nonspecific immune responses such as phagocytic activity and superoxide anion (O2−) production in hybrid grouper (Epinephelus fuscoguttatus × E. lanceolatus). The hybrid grouper leukocytes were incubated with methylmercury chloride (CH3HgCl) at concentrations of 10–10,000 µg/L to determine cell viability, phagocytic activity, and O2− production in vitro. Subsequently, the grouper were exposed daily to CH3HgCl mixed in the experimental diets at concentrations of 0, 1, 5, and 10 mg/kg for 28 days. The bioaccumulation of MeHg in the liver, head kidney, and muscle tissue was measured, and the phagocytic activity and O2− production were evaluated. In vitro results indicated that cell viability was significantly lower than that of the control group at concentrations > 500 µg/L. The phagocytic rate and O2− production at concentrations ˃ 500 and ˃ 200 µg/L, respectively, were significantly lower than those of the control group. The dietary exposure demonstrated that MeHg accumulated more substantially in the liver and head kidney compared with the muscle tissue in the treatment groups. Moreover, the cumulative concentration significantly increased with higher concentrations and more days of exposure. The phagocytic rate and O2− production in the treatment groups were significantly lower than those in the control group from days 2 and 1, respectively. In conclusion, hybrid grouper accumulated significant MeHg in the liver and head kidney compared with the muscle tissue, and higher concentrations and more exposure days resulted in decreased cell viability, phagocytic activity, and O2− production.

The Early Immune Response of Lymphoid and Myeloid Head-Kidney Cells of Rainbow Trout (Oncorhynchus mykiss) Stimulated with Aeromonas salmonicida

The teleost head kidney is a highly relevant immune organ, and myeloid cells play a major role in this organ’s innate and adaptive immune responses. Because of their complexity, the early phases of the innate immune reaction of fish against bacteria are still poorly understood. In this study, naïve rainbow trout were stimulated with inactivated A. salmonicida and sampled at 12 h, 24 h and 7 d poststimulation. Cells from the head kidney were magnetically sorted with a monoclonal antibody mAB21 to obtain one (MAb21-positive) fraction enriched with myeloid cells and one (MAb21-negative) fraction enriched with lymphocytes and thrombocytes. The gene expression pattern of the resulting cell subpopulations was analysed using a panel of 43 immune-related genes. The results show an overall downregulation of the complement pathway and cytokine production at the considered time points. Some of the selected genes may be considered as parameters for diagnosing bacterial furunculosis of rainbow trout.

PU.1 Regulates Cathepsin S Expression in Large Yellow Croaker (Larimichthys crocea) Macrophages

Different morphologies have been detected in teleost macrophages. In this study, two macrophage cell lines were sub-cloned from a large yellow croaker head kidney cell line, LYCK. One type of sub-cloned cells was fusiform but the other was round, named LYC-FM and LYC-RM cells respectively, based on their morphologies. Both types showed the characteristics of macrophages, including expression of macrophage-specific marker genes, possession of phagocytic and bactericidal activities, and production of reactive oxygen species (ROS) and nitric oxide (NO). The transcription factor PU.1, crucial for the development of macrophages in mammals, was found to exist in two transcripts, PU.1a and PU.1b, in large yellow croaker, and constitutively expressed in LYC-FM and LYC-RM cells. The expression levels of PU.1a and PU.1b could be upregulated by recombinant large yellow croaker IFN-γ protein (rLcIFN-γ). Further studies showed that both PU.1a and PU.1b increased the expression of cathepsin S (CTSS) by binding to different E26−transformation−specific (Ets) motifs of the CTSS promoter. Additionally, we demonstrated that all three domains of PU.1a and PU.1b were essential for initiating CTSS expression by truncated mutation experiments. Our results therefore provide the first evidence that teleost PU.1 has a role in regulating the expression of CTSS.

Different Fish Meal and Fish Oil Dietary Levels in European Sea Bass: Welfare Implications After Acute Confinement Stress

To provide practical feeding management guidelines preceding a stressful episode during farming practices, European sea bass juveniles (initial weight: 72.3 g) were fed for 60-days different fish meal (FM) and fish oil (FO) dietary levels [high (30% FM, 15% FO, FM30/FO15), intermediate (20% FM, 7% FO, FM20/FO7), and low (10% FM, 3% FO, FM10/FO3)] in triplicate conditions. Fish were then fasted for 36 h and exposed to a 2-h acute crowding (80 kg m–3 biomass). Plasma biochemistry, skin mucus parameters and gene expression of stress and immune-related genes were performed before, at 2 and 24 h after crowding. At the end of the trial, the FM10/FO3 group showed lower final body weight, weight gain, and specific growth rate compared to the other treatments. Most of the plasma parameters were mainly affected by crowding condition rather than diet; however, after stress, lactate was higher in the FM30/FO15 group compared to the other treatments. Similarly, protease, antiprotease, peroxidase and lysozyme in skin mucus were mostly affected by crowding conditions, while fish fed FM10/FO3 displayed higher skin mucosal IgM and bactericidal activity against Vibrio anguillarum and V. harveyi. Most of the stress-related genes considered (hsp70 and gr-1 in the brain; hsp70, gr-1 and gr-2 in the head kidney), showed an overall expression pattern that increased over time after stress, in addition, hsp70 in the head kidney was also up-regulated in fish fed FM30/FO15 after stress. Higher plasmatic lactate together with the up-regulation of some stress-related transcripts suggest a higher reactivity to acute crowding of the stress-response mechanism in fish fed high FM and FO dietary levels. Otherwise, the higher skin mucosal IgM and bactericidal activity observed in fish fed FM10/FO3 dietary levels seems to indicate that acute crowding was able to activate a higher pro-inflammatory response in this treatment. Overall, the results of the present study seem to indicate that 10% FM and 3% FO dietary levels might affect stress and immune responses.

Dietary Glutamine Inclusion Regulates Immune and Antioxidant System, as well as Programmed Cell Death in Fish to Protect against Flavobacterium columnare Infection

The susceptibility of animals to pathogenic infection is significantly affected by nutritional status. The present study took yellow catfish (Pelteobagrus fulvidraco) as a model to test the hypothesis that the protective roles of glutamine during bacterial infection are largely related to its regulation on the immune and antioxidant system, apoptosis and autophagy. Dietary glutamine supplementation significantly improved fish growth performance and feed utilization. After a challenge with Flavobacterium columnare, glutamine supplementation promoted il-8 and il-1β expression via NF-κB signaling in the head kidney and spleen, but inhibited the over-inflammation in the gut and gills. Additionally, dietary glutamine inclusion also enhanced the systematic antioxidant capacity. Histological analysis showed the protective role of glutamine in gill structures. Further study indicated that glutamine alleviated apoptosis during bacterial infection, along with the reduced protein levels of caspase-3 and the reduced expression of apoptosis-related genes. Moreover, glutamine also showed an inhibitory role in autophagy which was due to the increased activation of the mTOR signaling pathway. Thus, our study for the first time illustrated the regulatory roles of glutamine in the fish immune and antioxidant system, and reported its inhibitory effects on fish apoptosis and autophagy during bacterial infection.