Effects of different curing concentrations and drying times on the microbial community structure and metabolites of dried Spanish mackerel.

Cured Spanish mackerel has a promising market owing to its nutritious nature as well as ease of transportation and preservation. However, the nutritional and flavor formation mechanism of Spanish mackerel after curing and drying is unclear. To overcome this problem, the effects of different processing conditions on the free amino acid, microbial community, and flavor of Spanish mackerel were explored. Staphylococcus and Cobetia are the main microorganisms in cured mackerel and are closely associated with the formation of their quality. Compared with fresh mackerel, cured mackerel contains increased levels of protein, fat, and chloride, contributing to its distinctive flavor. The contents of free amino acids in the BA64 group were substantially higher than those in other groups, particularly the contents of threonine, glycine, and tyrosine. These findings will contribute to the development of high-quality cured Spanish mackerel products and cured aquatic products.

Transcriptome profiling of lumpfish (Cyclopterus lumpus) head kidney to Renibacterium salmoninarum at early and chronic infection stages.

Renibacterium salmoninarum causes Bacterial Kidney Disease (BKD) in several fish species. Atlantic lumpfish, a cleaner fish, is susceptible to R. salmoninarum. To profile the transcriptome response of lumpfish to R. salmoninarum at early and chronic infection stages, fish were intraperitoneally injected with either a high dose of R. salmoninarum (1 × 109 cells dose-1) or PBS (control). Head kidney tissue samples were collected at 28- and 98-days post-infection (dpi) for RNA sequencing. Transcriptomic profiling identified 1971 and 139 differentially expressed genes (DEGs) in infected compared with control samples at 28 and 98 dpi, respectively. At 28 dpi, R. salmoninarum-induced genes (n = 434) mainly involved in innate and adaptive immune response-related pathways, whereas R. salmoninarum-suppressed genes (n = 1537) were largely connected to amino acid metabolism and cellular processes. Cell-mediated immunity-related genes showed dysregulation at 98 dpi. Several immune-signalling pathways were dysregulated in response to R. salmoninarum, including apoptosis, alternative complement, JAK-STAT signalling, and MHC-I dependent pathways. In summary, R. salmoninarum causes immune suppression at early infection, whereas lumpfish induce a cell-mediated immune response at chronic infection. This study provides a complete depiction of diverse immune mechanisms dysregulated by R. salmoninarum in lumpfish and opens new avenues to develop immune prophylactic tools to prevent BKD.

Strain-level diversity of symbiont communities between individuals and populations of a bioluminescent fish.

The bioluminescent symbiosis involving the urchin cardinalfish, Siphamia tubifer, and Photobacterium mandapamensis, a luminous member of the Vibrionaceae, is highly specific compared to other bioluminescent fish-bacteria associations. Despite this high degree of specificity, patterns of genetic diversity have been observed for the symbionts from hosts sampled over relatively small spatial scales. We characterized and compared sub-species, strain-level symbiont diversity within and between S. tubifer hosts sampled from the Philippines and Japan using PCR fingerprinting. We then carried out whole genome sequencing of the unique symbiont genotypes identified to characterize the genetic diversity of the symbiont community and the symbiont pangenome. We determined that an individual light organ contains six symbiont genotypes on average, but varied between 1-13. Additionally, we found that there were few genotypes shared between hosts from the same location. A phylogenetic analysis of the unique symbiont strains indicated location-specific clades, suggesting some genetic differentiation in the symbionts between host populations. We also identified symbiont genes that were variable between strains, including luxF, a member of the lux operon, which is responsible for light production. We quantified the light emission and growth rate of two strains missing luxF along with the other strains isolated from the same light organs and determined that strains lacking luxF were dimmer but grew faster than most of the other strains, suggesting a potential metabolic trade-off. This study highlights the importance of strain-level diversity in microbial associations and provides new insight into the underlying genetic architecture of intraspecific symbiont communities within a host.

Quality changes of Larimichthys crocea pretreated by ultrasonic-assisted immersion under cold storage at 4 °C.

The effects of several ultrasonic treatments during cold storage at 4 °C on the quality of large yellow croaker (Pseudosciaena crocea) were suggested. Large yellow croaker fillets were divided into six different groups for treatment. (a) CK (without any treatment), (b) A (single frequency 20 kHz), (c) B (single frequency 40 kHz), (d) C (left and right dual frequency 20 + 40 kHz), (e) D (orthogonal dual frequency: left and right 40 kHz, upper and lower 20 kHz), and (f) E (orthogonal dual frequency: left and right 20 kHz, upper and lower 20 kHz). The samples were divided into six groups, placed in sterile PE bags, and chilled to 4 °C. In order to determine the impact of ultrasonic treatment on the quality of large yellow croaker during cold storage, microbial indicators and physical and chemical indicators were measured every 3 days. The total number of colonies, the percentage of psychrophilic bacteria, the sample's pH, and its TVB-N value were all shown to grow at a much slower pace following ultrasonic treatment. In addition, the antibacterial effect of dual frequency ultrasound was gradually better than that of single frequency ultrasound. In conclusion, Group D has a pretty excellent impact on preserving overall sample quality.

A σE-mediated temperature gauge orchestrates type VI secretion system, biofilm formation and cell invasion in pathogen Pseudomonas plecoglossicida.

Pseudomonas plecoglossicida is a temperature-dependent opportunistic pathogen mediating visceral granulomas in many piscine species including the large yellow croaker (Larimichthys crocea) but the underlying mechanisms are unclear. RpoE is an alternative sigma (σ) factor involved in regulated intramembrane proteolytic (RIP) cascade, enabling bacterial pathogens to coordinate the expression of genetic traits associated with stress adaptation and virulence determinants in response to diverse stimuli in vitro and in vivo of the hosts. In this study, genes associated to RIP cascade in P. plecoglossicida were identified and characterized to show various sequence similarities to their counterparts in Escherichia coli and P. aeruginosa. The expression of P. plecoglossicida RIP locus was induced by higher temperatures. Moreover, RNA sequencing approach revealed that RpoE regulated the expression of ∼297 and ∼261 genes at virulent (18 °C) and non-virulent (28 °C) temperatures, respectively. RpoE regulon genes are involved in various processes associated with bacterial signal transduction, membrane homeostasis, energy metabolism and virulence. In particular, RpoE positively controlled expression of csrA encoding an RNA binding protein essential for central carbon metabolism. In addition, P. plecoglossicida RpoE was validated to regulate type VI secretion system (T6SS) expression, bacteria competition, biofilm formation and reproduction in macrophages. Collectively, RpoE-centered RIP cascade appeared to play important roles in control of the expression of genes involved in adaptation in vivo and in vitro niches by thermal sensing in P. plecoglossicida. These results facilitates to reveal the pathogenic mechanisms of P. plecoglossicida causing fish diseases and provides new perspectives to control bacterial infection.

The galU gene is required for in vivo survival of pseudomonas plecoglossicida in large yellow croaker (Larimichthys crocea).

Pseudomonas plecoglossicida is an important pathogenic bacterium in aquaculture that causes visceral granulomas in large yellow croaker (Larimichthys crocea). Uridine diphosphate glucose phosphorylase encoded by galU plays a key role in biosynthesis of the bacterial envelope, particularly lipopolysaccharide and the capsule. In this study, we inactivated the galU gene in the P. plecoglossicida isolate XSDHY-P. The galU mutant strain showed impaired growth in the early exponential stage and lacked the O polysaccharide side chain in lipopolysaccharide, but almost no defect in biofilm formation was detected. The galU mutant strain also exhibited significantly more sensitivity to the bactericidal action of normal fish serum mediated by the complement system compared to the wild-type strain. In a cell model originating from the head kidney of large yellow croaker, the galU mutant strain showed lower capacities of adhesion, invasion, and intracellular survival compared to the wild-type strain. In addition, the deficiency of the galU mutant drastically decreased bacterial loads in tissues and attenuated P. plecoglossicida virulence in fish. These results suggest that the galU gene of P. plecoglossicida is required for in vivo survival in large yellow croaker.

Effect of Ferredoxin Receptor FusA on the Virulence Mechanism of Pseudomonas plecoglossicida.

Pseudomonas plecoglossicida is an aerobic Gram-negative bacterium, which is the pathogen of "Visceral white spot disease" in large yellow croaker. P. plecoglossicida is a temperature-dependent bacterial pathogen in fish, which not only reduces the yield of large yellow croaker but also causes continuous transmission of the disease, seriously endangering the healthy development of fisheries. In this study, a mutant strain of fusA was constructed using homologous recombination technology. The results showed that knockout of P. plecoglossicida fusA significantly affected the ability of growth, adhesion, and biofilm formation. Temperature, pH, H2O2, heavy metals, and the iron-chelating agent were used to treat the wild type of P. plecoglossicida; the results showed that the expression of fusA was significantly reduced at 4°C, 12°C, and 37°C. The expression of fusA was significantly increased at pH 4 and 5. Cu2+ has a significant inducing effect on the expression of fusA, but Pb2+ has no obvious effect; the expression of fusA was significantly upregulated under different concentrations of H2O2. The expression of the fusA gene was significantly upregulated in the 0.5~4-µmol/l iron-chelating agent. The expression level of the fusA gene was significantly upregulated after the logarithmic phase. It was suggested that fusA included in the TBDR family not only was involved in the transport of ferredoxin but also played important roles in the pathogenicity and environment adaptation of P. plecoglossicida.

Chromosome-Level Genome Assembly of the Bioluminescent Cardinalfish Siphamia tubifer: An Emerging Model for Symbiosis Research.

The bioluminescent symbiosis involving the sea urchin cardinalfish Siphamia tubifer and the luminous bacterium Photobacterium mandapamensis is an emerging vertebrate model for the study of microbial symbiosis. However, little genetic data are available for the host, limiting the scope of research that can be implemented with this association. We present a chromosome-level genome assembly for S. tubifer using a combination of PacBio HiFi sequencing and Hi-C technologies. The final assembly was 1.2 Gb distributed on 23 chromosomes and contained 32,365 protein coding genes with a BUSCO score of 99%. A comparison of the S. tubifer genome to that of another nonluminous species of cardinalfish revealed a high degree of synteny, whereas a comparison to a more distant relative in the sister order Gobiiformes revealed the fusion of two chromosomes in the cardinalfish genomes. The complete mitogenome of S. tubifer was also assembled, and an inversion in the vertebrate WANCY tRNA genes as well as heteroplasmy in the length of the control region were discovered. A phylogenetic analysis based on whole the mitochondrial genome indicated that S. tubifer is divergent from the rest of the cardinalfish family, highlighting the potential role of the bioluminescent symbiosis in the initial divergence of Siphamia. This high-quality reference genome will provide novel opportunities for the bioluminescent S. tubifer-P. mandapamensis association to be used as a model for symbiosis research.

Choreftria shiranui, a new genus and species of cyclopoid copepod (Crustacea: Copepoda) associated with the worm goby from southern Japan, with the proposal of a new family.

A new genus and species of cyclopoid copepod, Choreftria shiranui n. g., n. sp. (Crustacea: Copepoda) is described based on an adult female found from the worm goby, Taenioides snyderi Jordan & Hubbs (Actinopterygii: Perciformes: Gobiidae), from mud flats in the Yatsushiro Sea, southern Japan. The new genus is characterized by bearing the following characters in the female: body distinctly segmented cyclopiform with 4-segmented prosome and 6-segmented urosome; genital somite clearly separated from first abdominal somite; antennule 7-segmented; antenna 4-segmented with one fused serrated claw and long claw on second endopodal segment; mandible reduced with one serrated blade; maxillule rod-like with one element; maxilla 2-segmented with recurved terminal claw; maxilliped 2-segmented; legs 1 to 4 biramous with 3-segmented rami. Since the copepod is not attributable to any of the known cyclopoid families, a new family, Choreftriidae n. fam. is established to accommodate Choreftria n. g. LSID urn:lsid:zoobank.org:pub:C1FB9F60-9871-4D4B-A1BF-3202BA24189F.

Robust host source tracking building on the divergent and non-stochastic assembly of gut microbiomes in wild and farmed large yellow croaker.

BACKGROUND: Given the lack of genetic background, the source tracking unknown individuals of fish species with both farmed and wild populations often cannot be robustly achieved. The gut microbiome, which is shaped by both deterministic and stochastic processes, can serve as a molecular marker of fish host source tracking, particularly as an alternative to the yet-to-be-established host genetic marker. A candidate for testing the feasibility is the large yellow croaker, Larimichthys crocea, which is carnivorous and ranks the top mariculture fish in China. Wild resource of this fish was depleted decades ago and might have potential problematic estimation because of escaping of farmed individuals. RESULTS: The rectums of wild (n = 212) and farmed (n = 79) croakers from multiple batches were collected for the profiling of their gut bacterial communities. The farmed individuals had a higher alpha diversity and lower bacterial load than the wild individuals. The gut microbiota of the two sources exhibited divergence and high inter-batch variation, as featured by the dominance of Psychrobacter spp. in the wild group. Predicted functional capacity of the gut microbiome and representative isolates showed differences in terms of host source. This difference can be linked to the potential diet divergence between farmed and wild fishes. The non-stochastic distribution pattern of the core gut microbiota of the wild and farmed individuals supports the feasibility of microbiota-based host source tracking via the machine learning algorithm. A random forest classifier based on the divergence and non-stochastic assembly of the gut microbiome was robust in terms of host source tracking the individuals from all batches of croaker, including a newly introduced batch. CONCLUSIONS: Our study revealed the divergence of gut microbiota and related functional profiles between wild and farmed croakers. For the first time, with representative datasets and non-stochastic patterns, we have verified that gut microbiota can be robustly applied to the tracking of host source even in carnivorous fish. Video abstract.

Dispersed Crude Oil Induces Dysbiosis in the Red Snapper Lutjanus campechanus External Microbiota.

The fish external microbiota competitively excludes primary pathogens and prevents the proliferation of opportunists. A shift from healthy microbiota composition, known as dysbiosis, may be triggered by environmental stressors and increases host susceptibility to disease. The Deepwater Horizon (DWH) oil spill was a significant stressor event in the Gulf of Mexico. Despite anecdotal reports of skin lesions on fishes following the oil spill, little information is available on the impact of dispersed oil on the fish external microbiota. In this study, juvenile red snapper (Lutjanus campechanus) were exposed to a chemically enhanced water-accommodated fraction (CEWAF) of Corexit 9500/DWH oil (CEWAF) and/or the bacterial pathogen Vibrio anguillarum in treatments designed to detect changes in and recovery of the external microbiota. In fish chronically exposed to CEWAF, immunoglobulin M (IgM) expression significantly decreased between 2 and 4 weeks of exposure, coinciding with elevated liver total polycyclic aromatic hydrocarbons (PAHs). Dysbiosis was detected on fish chronically exposed to CEWAF compared to seawater controls, and addition of a pathogen challenge altered the final microbiota composition. Dysbiosis was prevented by returning fish to clean seawater for 21 days after 1 week of CEWAF exposure. Four fish exhibited lesions during the trial, all of which were exposed to CEWAF but not all of which were exposed to V. anguillarum. This study indicates that month-long exposure to dispersed oil leads to dysbiosis in the external microbiota. As the microbiota is vital to host health, these effects should be considered when determining the total impacts of pollutants in aquatic ecosystems. IMPORTANCE Fish skin is an immunologically active tissue. It harbors a complex community of microorganisms vital to host homeostasis as, in healthy fish, they competitively exclude pathogens found in the surrounding aquatic environment. Crude oil exposure results in immunosuppression in marine animals, altering the relationship between the host and its microbial community. An alteration of the healthy microbiota, a condition known as dysbiosis, increases host susceptibility to pathogens. Despite reports of external lesions on fishes following the DWH oil spill and the importance of the external microbiota to fish health, there is little information on the effect of dispersed oil on the external microbiota of fishes. This research provides insight into the impact of a stressor event such as an oil spill on dysbiosis and enhances understanding of long-term sublethal effects of exposure to aid in regulatory decisions for protecting fish populations during recovery.

Polyactin A and CpG enhance inactivated Pseudomonas plecoglossicida vaccine potency in large yellow croaker (Larimichthys crocea).

Pseudomonas plecoglossicida is the causative agent of visceral granulomas disease (VGD) in large yellow croaker (LYC, Larimichthys crocea) farming. However, multi-antibiotic resistant of P. plecoglossicida creates an urgent need of an efficient vaccine to combat this pathogen. In this study, an inactivated vaccine added polyactin (PA), CpG-riched plasmid (pCpG) and aluminum adjuvant (Al) was developed. As a result, its relative percentage survival (RPS) against P. plecoglossicida were up to 64%. Comparatively, RPS of groups that vaccinated with vaccines adjuvanted with PA and Al or CpG and Al were 49% and 39%. However, an interesting result that the vaccine combined with PA, CpG and Al did not show the strongest activation of total serum protein and antibody levels in serum among three vaccinated groups. According to expressions of some cellular immune related genes, we found that the inactivated vaccine combined with PA, CpG and Al was more likely to induce a cellular immune response rather than humoral immune response. Totally, our study demonstrated that the mixture of PA, CpG and aluminum adjuvant is a potential adjuvant system for LYC vaccine development.

Brain immunity response of fish Eleginops maclovinus to infection with Francisella noatunensis.

The brain's immune system is selective and hermetic in most species, including fish, favoring immune responses mediated by soluble immunomodulatory factors such as serotonin and the availability of nutrients against infectious processes. Francisella noatunensis coexist with fish such as Eleginops maclovinus, which raises questions about the susceptibility and immune response of the brain of E. maclovinus against Francisella. In this study, we inoculated fish with different doses of Francisella and took samples for 28 days. We detected bacteria in the brain of fish injected with a high concentration of Francisella at all time points. qPCR analysis of immune genes indicated a response mainly in the medium-dose and early expression of genes involved in iron metabolism. Finally, brain serotonin levels were higher than in uninfected fish in all conditions, suggesting possible immunomodulatory participation in an infectious process.

Effect of High Hydrostatic Pressure Treatment on Urease Activity and Inhibition of Fishy Smell in Mackerel (Scomber japonicus) during Storage.

In this study, the physicochemical changes related to fishy smell were determined by storing high hydrostatic pressure (HHP)-treated mackerel (Scomber japonicus) meat in a refrigerator for 20 days. The inhibition of crude urease activity from Vibrio parahaemolyticus using HHP treatment was also investigated. The mackerel meat storage experiment demonstrated that production of trimethylamine (TMA) and volatile basic nitrogen (VBN), the main components of fishy smell, was significantly reduced on the 20th day of storage after the HHP treatment compared to the untreated mackerels. The results demonstrated that the increased ammonia nitrogen rates in the 2000, 3000, and 4000 bar, HHP-treated groups decreased by 23.8%, 23.8%, and 31.0%, respectively, compared to the untreated groups. The enzyme activity of crude urease was significantly reduced in the HHP-treated group compared to that in the untreated group. Measurement of the volatile organic compounds (VOCs) in mackerel meat during storage indicated that the content of ethanol, 2-butanone, 3-methylbutanal, and trans-2-pentenal, which are known to cause off-flavor due to spoilage, were significantly reduced by HHP treatment. Collectively, our results suggested that HHP treatment would be useful for inhibiting the activity of urease, thereby reducing the fishy smells from fish and shellfish.

Lumpfish (Cyclopterus lumpus) Is Susceptible to Renibacterium salmoninarum Infection and Induces Cell-Mediated Immunity in the Chronic Stage.

Renibacterium salmoninarum is a Gram-positive, intracellular pathogen that causes Bacterial Kidney Disease (BKD) in several fish species in freshwater and seawater. Lumpfish (Cyclopterus lumpus) is utilized as a cleaner fish to biocontrol sea lice infestation in Atlantic salmon (Salmo salar) farms. Atlantic salmon is susceptible to R. salmoninarum, and it can transfer the infection to other fish species. Although BKD outbreaks have not been reported in lumpfish, its susceptibility and immune response to R. salmoninarum is unknown. In this study, we evaluated the susceptibility and immune response of lumpfish to R. salmoninarum infection. Groups of lumpfish were intraperitoneally (i.p.) injected with either R. salmoninarum (1×107, 1×108, or 1×109 cells dose-1) or PBS (control). R. salmoninarum infection kinetics and mortality were followed for 98 days post-infection (dpi). Transcript expression levels of 33 immune-relevant genes were measured in head kidney (n = 6) of fish infected with 1×109 cells/dose and compared to the control at 28 and 98 dpi. Infected lumpfish displayed characteristic clinical signs of BKD. Lumpfish infected with high, medium, and low doses had a survival rate of 65%, 93%, and 95%, respectively. Mortality in the high-dose infected group stabilized after 50 dpi, but R. salmoninarum persisted in the fish tissues until 98 dpi. Cytokines (il1ß, il8a, il8b), pattern recognition receptors (tlr5a), interferon-induced effectors (rsad2, mxa, mxb, mxc), and iron regulation (hamp) and acute phase reactant (saa5) related genes were up-regulated at 28 dpi. In contrast, cell-mediated adaptive immunity-related genes (cd4a, cd4b, ly6g6f, cd8a, cd74) were down-regulated at 28 dpi, revealing the immune suppressive nature of R. salmoninarum. However, significant upregulation of cd74 at 98 dpi suggests induction of cell-mediated immune response. This study showed that R. salmoninarum infected lumpfish in a similar fashion to salmonid fish species and caused a chronic infection, enhancing cell-mediated adaptive immune response.

Multilocus sequence analysis reveals different lineages of Pseudomonas anguilliseptica associated with disease in farmed lumpfish (Cyclopterus lumpus L.).

The bacterium Pseudomonas anguilliseptica has in recent years emerged as a serious threat to production of lumpfish in Norway. Little is known about the population structure of this bacterium despite its association with disease in a wide range of different fish species throughout the world. The phylogenetic relationships between 53 isolates, primarily derived from diseased lumpfish, but including a number of reference strains from diverse geographical origins and fish species, were reconstructed by Multi-Locus Sequence Analysis (MLSA) using nine housekeeping genes (rpoB, atpD, gyrB, rpoD, ileS, aroE, carA, glnS and recA). MLSA revealed a high degree of relatedness between the studied isolates, altough the seven genotypes identified formed three main phylogenetic lineages. While four genotypes were identified amongst Norwegian lumpfish isolates, a single genotype dominated, irrespective of geographic origin. This suggests the existence of a dominant genotype associated with disease in production of lumpfish in Norwegian aquaculture. Elucidation of the population structure of the bacterium has provided valuable information for potential future vaccine development.

Impact of dietary porcine blood by-products in meagre (Argyrosomus regius) physiology, evaluated by welfare biomarkers and the antibacterial properties of the skin mucus.

Tools are required for quick and easy preliminary evaluation of functional feeds efficiency on fisheries. The analysis of skin mucus biomarkers is a recent alternative approach providing a faster feed-back from the laboratory which is characterized by being less invasive, more rapid and with reduced costs. The effect of replacing fishmeal and fish protein hydrolysates by means of two porcine by-products, the porcine spray-dried plasma (SDPP) and pig protein hydrolysate (PPH), in compound diets (50.4% crude protein, 16.2% crude protein, 22.1 MJ/kg feed) was evaluated in juvenile meagre (Argyrosomus regius) during a two-months period. To determine the impact of these dietary replacements, growth and food performance were measured together with digestive enzymes activities and filet proximal composition. Additionally, skin mucus was collected and characterized by determining main mucus biomarkers (protein, glucose, lactate, cortisol, and antioxidant capacity) and its antibacterial properties, measured by the quick in vitro co-culture challenges. In comparison to the control group, the inclusion of PPH and SDPP, in meagre diets reduced growth (7.4-8.8% in body weight), increased feed conversion ratios (9.0-10.0%), results that were attributed to a reduction in feed intake values (24.2-33.0%) (P < 0.05). Porcine blood by-products did not modify the activity of gastric and pancreatic digestive enzymes as well as those involved in nutrient absorption (alkaline phosphatase) nor liver oxidative stress condition (P > 0.05). In contrast, a reduction in fillet lipid content associated to an increase in fillet protein levels were found in fish fed SDPP and PPH diets (P < 0.05). As compared to the control diet, the dietary replacement did not alter the levels of the skin mucus biomarkers related to stress (cortisol and antioxidant capacity) or nutritional status (soluble protein, glucose and lactate) (P > 0.05). Interestingly, regardless of the worst performance in somatic growth, meagre fed diets containing both tested porcine by-products showed a significantly improved antibacterial capacity of their skin mucus. This enhancement was more prominent for fish fed with the PPH diet, which may be attributed to a higher content of immunomodulatory bioactive compounds in PPH. Further research will be necessary to provide insights on how the inclusion of SDPP and PPH, at the expense of dietary fishmeal and fish protein hydrolysates, affects feed intake and growth performance in meagre. However, the use of skin mucus biomarkers has been demonstrated to be an excellent methodology for a preliminary characterization of the functional feeds, in particular for their prophylactic properties by the study of mucus antibacterial activity.

The circular RNA circBCL2L1 regulates innate immune responses via microRNA-mediated downregulation of TRAF6 in teleost fish.

Growing numbers of studies have shown that circular RNAs (circRNAs) can function as regulatory factors to regulate the innate immune response, cell proliferation, cell migration, and other important processes in mammals. However, the function and regulatory mechanism of circRNAs in lower vertebrates are still unclear. Here, we discovered a novel circRNA derived from the gene encoding Bcl-2-like protein 1 (BCL2L1) gene, named circBCL2L1, which was related to the innate immune responses in teleost fish. Results indicated that circBCL2L1 played essential roles in host antiviral immunity and antibacterial immunity. Our study also identified a microRNA, miR-30c-3-3p, which could inhibit the innate immune response by targeting inflammatory mediator TRAF6. And TRAF6 is a key signal transduction factor in innate immune response mediated by TLRs. Moreover, we also found that the antiviral and antibacterial effects inhibited by miR-30c-3-3p could be reversed with the expression of circBCL2L1. Our data revealed that circBCL2L1 functioned as a competing endogenous RNA (ceRNA) of TRAF6 by competing for binding with miR-30c-3-3p, leading to activation of the NF-κB/IRF3 inflammatory pathway and then enhancing the innate immune responses. Our results suggest that circRNAs can play an important role in the innate immune response of teleost fish.

Dietary supplementation of fermented lemon peel enhances lysozyme activity and susceptibility to Photobacterium damselae for orange-spotted grouper, Epinephelus coioides.

The waste recycling of lemon peel, as a functional feed additive in aquafeed was evaluated by estimating the effects of fermented lemon peel (FLP) supplementation in diet on growth performance, innate immune responses, and susceptibility to Photobacterium damselae of grouper, Epinephelus coioides. A basal diet was added FLP at 0%, 1%, 3%, and 5%. Four tested diets were each fed to juvenile grouper (initial weight: 15.89 ± 0.10 g, triplicate groups) in a recirculation rearing system for eight weeks. Fish fed diets with 0%-3% FLP exhibited higher (p < 0.05) final weight, weight gain, and feed efficiency than fish fed the 5% FLP-diet. After challenge test, fish fed the 3% FLP-diet appeared the lowest mortality, followed by fish fed the 1% FLP-diet, and lowest in fish fed 0% and 5% FLP-diets. Plasma lysozyme activities were higher in fish fed diets with FLP than in fish fed the FLP-free control diet before challenge test. After challenge, fish fed diets with 1% and 3% FLP showed highest lysozyme activities, followed by fish fed the diet with 5% FLP, and lowest in fish fed the control diet. Hepatic malondialdehyde content was higher in fish fed the control diet than in fish fed diets with 1%-3% FLP. Results found that diets supplemented with 1%-3% fermented lemon peel can enhance lysozyme activity and resistance to pathogen P. damselae of grouper.

Preliminary studies on the different roles of T6SSs in pathogenicity of Pseudomonas plecoglossicida NB2011.

Pseudomonas plecoglossicida, the causative agent of visceral granulomas in the large yellow croaker (Larimichthys crocea) in China, encodes three sets of type Ⅵ secretion systems (T6SS1-3). The purpose of this study was to characterize the different roles of T6SSs involved in infection. In-frame deletion of T6SSs was constructed, which resulted in 8 mutants. Competition against E. coli DH5α, virulence against the croaker and in vivo survival ability of the mutants were tested. The expression and secretion of Hcp by P. plecoglossicida NB2011 were investigated. The results showed T6SS2 mutant failed to inhibit the growth of E. coli, which is an indication of T6SS2 acting against environmental bacteria. The LD50 value of T6SS1 mutant strongly increased; T6SS2 and T6SS3 mutants were similar to that of the wild type; and the virulence of double deletion or triple deletion mutant was drastically alleviated, indicating that T6SS1 being one of the major virulence factors, and T6SS2 and T6SS3 directly or indirectly being involved in the pathogenicity. T6SS1 mutant disappeared in the fish spleen in 3 days, while other strains kept increasing, indicating the T6SS1 stimulation bacteria replication in vivo. Hcp1 secreted at 12-28°C and Hcp2 secreted at 12-35°C, while Hcp3 secretion not detected in vitro. This study has thrown some insights on the understanding of pathogenicity mechanisms of this pathogen.