Aggregation-induced emission of TTCPy-3: A novel approach for eradicating Nocardia seriolae infections in aquatic fishes.

Aquatic fishes are threatened by the strong pathogenic bacterium Nocardia seriolae, which challenges the current prevention and treatment approaches. This study introduces luminogens with aggregation-induced emission (AIE) as an innovative and non-antibiotic therapy for N. seriolae. Specifically, the AIE photosensitizer, TTCPy-3 is employed against N. seriolae. We evaluated the antibacterial activity of TTCPy-3 and investigated the killing mechanism against N. seriolae, emphasizing its ability to aggregate within the bacterium and produce reactive oxygen species (ROS). TTCPy-3 could effectively aggregate in N. seriolae, generate ROS, and perform real-time imaging of the bacteria. A bactericidal efficiency of 100% was observed while concentrations exceeding 4 µM in the presence of white light irradiation for 10 min. In vivo, evaluation on zebrafish (Danio rerio) confirmed the superior therapeutic efficacy induced by TTCPy-3 to fight against N. seriolae infections. TTCPy-3 offers a promising strategy for treating nocardiosis of fish, paving the way for alternative treatments beyond traditional antibiotics and potentially addressing antibiotic resistance.

Efficient nitrogen removal of a novel Pseudomonas chengduensis strain BF6 mainly through assimilation in the recirculating aquaculture systems.

Biological nitrogen removal has received increasing attention in wastewater treatment. A bacterium with excellent nitrogen removal performance was isolated from biofilters of recirculating aquaculture systems (RAS) and identified as Pseudomonas chengduensis BF6. It was indicated that inorganic nitrogen is transformed into gaseous and biological nitrogen by the metabolic pathways of denitrification, anammox, and assimilation, which is the main nitrogen removal pathway of strain BF6. The strain BF6 could effectively remove nitrogen within 24 h under the conditions of ammonia, nitrate, nitrite, and mixed nitrogen sources with maximum total nitrogen removal efficiencies reaching 97.00 %, 61.40 %, 79.10 %, and 84.98 %, respectively. The strain BF6 exhibited total nitrogen removal efficiency of 91.14 %, altered the microbial diversity and enhanced the relative abundance of Pseudomonas in the RAS biofilter. These findings demonstrate that Pseudomonas sp. BF6 is a highly efficient nitrogen-removing bacterium with great potential for application in aquaculture wastewater remediation.

Characterization of the core gut microbiota of Nile tilapia (Oreochromis niloticus): indication of a putative novel Cetobacterium species and analysis of its potential function on nutrition.

The genus Cetobacterium has been considered a dominant group of gut bacteria in many freshwater fish, and members of this genus contribute to anaerobic metabolism. Because of its significant place in the gut of freshwater fish, many studies on Cetobacterium were performed. Those studies mostly focused on the temporal and spatial changes of its abundance in fish intestine, which were affected by food or other environmental conditions. However, only a few studies isolated strains from genus Cetobacterium and reported their characteristics. In the present study, we performed 16S rRNA sequencing of the intestinal mucosa of Nile tilapia (Oreochromis niloticus) and found that Cetobacterium sp. existed widely in the foregut, midgut and hindgut mucosa, and a strain of Cetobacterium was successfully isolated from the gut of tilapia. We sequenced its whole genome and predicted it to be a novel candidate species of Cetobacterium sp. and named it NK01. The size of its genome was 3,095,946 bp, with a guanine + cytosine content of 28.8%. Among the identified genes, 2855 were predicted to be coding DNA sequences, 84 were tRNA and 34 were rRNA. We found that NK01 produced amino acids, including leucine, isoleucine, valine, glycine, alanine, phenylalanine and proline. Strain NK01 could use starch, sucrose, maltose, glucose, and mannose and synthesize and utilize glycogen. INV, GPI, malQ, malZ, sacA, scrK, glgC, glgA and glk, which were related to carbohydrate metabolism, were detected. yiaY and adhE, which oxidize ethanol to acetaldehyde and participate in a variety of metabolic pathways, were also present in the genome. No coding genes directly involved in acetate or butyrate production were detected. NK01 could also catabolize a variety of vitamins, and all genes involved in folate synthesis were detected, including folP, folC, folA and eutT, which converted vitamin B12s into vitamin B12 coenzyme. Here, we investigated the draft genome and in vitro function of Cetobacterium isolated from the intestinal tract of Nile tilapia. The results provided a preliminary understanding of the core microbiota of fish gut.

Nile tilapia TLR3 recruits MyD88 and TRIF as adaptors and is involved in the NF-κB pathway in the immune response.

TLR3 plays a crucial role in innate immunity. In the present study, OnTLR3 was identified in the Nile tilapia Oreochromis niloticus, with a conserved LRR domain and a C-terminal TIR domain. OnTLR3 was broadly expressed in all tissues tested, with the highest expression levels in the blood and the lowest in the kidney. TLR3 mRNA could be detected from pharyngula (2.5 dpf) to late larva (8.5 dpf) during embryonic and larval development. Moreover, the expression level of OnTLR3 was clearly altered in all five tissues after Streptococcus agalactiae infection in vivo and could be induced by LPS, poly(I:C), S. agalactiae WC1535 and △CPS in Nile tilapia macrophages. When OnTLR3 was overexpressed in 293 T cells, it was distributed in the cytoplasm and could significantly increase NF-κB activation. The pulldown assays showed that OnTLR3 interacted with both OnMyD88 and OnTRIF. The binding assays revealed the specificity of OnTLR3 for pathogen-associated molecular patterns (PAMPs) and bacteria that included S. agalactiae, Aeromonas hydrophila and poly(I:C), LPS and PGN. Taken together, these findings suggest that OnTLR3, as a pattern recognition receptor (PRR), might play an important role in the immune response to pathogen invasion.

Isolation, identification, and pathogenic characteristics of Nocardia seriolae in largemouth bass Micropterus salmoides.

The largemouth bass Micropterus salmoides is an important freshwater aquaculture fish in China. Recently, largemouth bass at a fish farm in Guangdong province experienced an outbreak of a serious ulcer disease. As part of the investigations conducted to identify the aetiology and identify potentially effective control measures, we isolated a pathogenic bacterium (NK-1 strain) from the diseased fish. It was identified as Nocardia seriolae through morphological observation, physiological and biochemical analysis, and molecular identification, and its pathogenicity was verified by experimental infection. Pathological changes in the diseased fish included granulomatous lesions in the liver and spleen, destruction of renal tubules, necrosis of intestinal epithelial cells, infiltration of inflammatory cells in the brain, vacuolation of cells, and swelling and cracking of the mitochondria and endoplasmic reticulum. Bacterial detection using qPCR showed that the spleen and intestine were the main organs targeted by N. seriolae. The mortality of largemouth bass experimentally infected with N. seriolae at 21°C was significantly lower than that in fish infected at higher temperatures between 24 and 33°C; there were no significant differences in the levels of mortality at these higher temperatures. The level of mortality of largemouth bass infected with N. seriolae was lowest at a neutral water pH of 7 but increased significantly at higher and lower pH. Of the tested Chinese herbal medicines, Chinese sumac Galla chinensis and Chinese skullcap Scutellaria baicalensis exhibited the best antibacterial effects. This study lays a foundation for the clinical diagnosis and scientific control of ulcer disease in largemouth bass.

TLR5 recognizes Aeromonas hydrophila flagellin and interacts with MyD88 in Nile tilapia.

Toll-like receptor 5 (TLR5) is responsible for bacterial flagellin recognition in vertebrates. In the present study, TLR5M was identified in the Nile tilapia Oreochromis niloticus (OnTLR5), containing a conserved LRR domain, a transmembrane region and a C-terminal TIR domain, similar to that of other fishes and mammals. OnTLR5 was broadly expressed in all the tissues examined, presenting the highest expression levels in the blood and the lowest in the kidney. OnTLR5 was detected from 2 d postfertilization (dpf) to 8 dpf during embryonic development. Moreover, expression levels of OnTLR5 were clearly altered in all five tissues examined in response to Streptococcus agalactiae infection in vivo. Overexpression of OnTLR5 in HEK293T cells revealed that OnTLR5 was distributed in the cytoplasm and significantly increased NF-κB activation. In response to cotransfection with OnMyd88, OnTLR5 significantly upregulated OnMyd88-induced NF-κB activation. Pulldown assays showed that OnTLR5 interacts with OnMyd88 and revealed an interaction between TLR5 and Aeromonas hydrophila flagellin. Taken together, these findings suggest that OnTLR5 plays important roles in TLR/IL-1R signalling pathways and the immune response to pathogen invasion.

DDX43 recruits TRIF or IPS-1 as an adaptor and activates the IFN-ß pathway in Nile tilapia (Oreochromis niloticus).

DDX43 is one of the members of the DExD/H-box protein family, and emerging data suggest that it may play an important role in antiviral immunity across mammals. However, little is known about DDX43 in the fish immune response. In this study, we isolated the cDNA sequence of ddx43 in Nile tilapia (Oreochromis niloticus). The ddx43 gene was 2338 bp in length, contained an open reading frame (ORF) of 2064 bp and encoded a polypeptide of 687 amino acids. The predicted protein of OnDDX43 has three conserved domains, including the RNA binding domain KH, DEAD-like helicase superfamily DEXDc and C-terminal HELICc domain. In healthy Nile tilapia, the Onddx43 transcript was broadly expressed in all examined tissues, with the highest expression levels in the muscle and brain and the lowest in the liver. After challenge with Streptococcus agalactiae, lipopolysaccharides (LPS) and polyinosinic polycytidylic acid (Poly I:C), the expression level of Onddx43 mRNA was upregulated or downregulated in all of the tissues tested. Overexpression of OnDDX43 in 293 T cells showed that it has a positive regulatory effect on IFN-ß. The subcellular localization showed that OnDDX43 was expressed in the cytoplasm. We performed further pull-down assays and found that OnDDX43 interacted with both interferon-ß promoter stimulator1 (IPS-1) and TIR domain-containing adaptor inducing interferon-ß (TRIF).

Genome survey of Misgurnus anguillicaudatus to identify genomic information, simple sequence repeat (SSR) markers, and mitochondrial genome.

BACKGROUND: The dojo loach Misgurnus anguillicaudatus is an important economic species in Asia because of its nutritional value and broad environmental adaptability. Despite its economic importance, genomic data for M. anguillicaudatus is currently unavailable. METHODS AND RESULTS: In the present study, we conducted a genome survey of M. anguillicaudatus using next-generation sequencing technology. Its genome size was estimated to be 1105.97 Mb by using K-mer analysis, and its heterozygosity ratio, repeat sequence content, GC content were 1.45%, 58.98%, and 38.03%, respectively. A total of 376,357 microsatellite motifs were identified, and mononucleotides, with a frequency of 42.57%, were the most frequently repeated motifs, followed by 40.83% dinucleotide, 7.49% trinucleotide, 8.09% tetranucleotide, and 0.91% pentanucleotide motifs. The AC/GT, AAT/ATT, and ACAG/CTGT repeats were the most abundant motifs among dinucleotide, trinucleotide, and tetranucleotide motifs, respectively. Besides, the complete mitochondrial genome was sequenced. Based on the Maximum Likelihood and Bayesian inference analyses, M. anguillicaudatus yingde in this study was the "introgressed" mitochondrial type. Seventy microsatellite loci were randomly selected from detected SSR loci to test polymorphic, of which, 20 microsatellite loci were assessed in 30 individuals from a wild population. The number of alleles (Na), observed heterozygosity (Ho), and expected heterozygosity (He) per locus ranged from 7 to 19, 0.400 to 0.933, and 0.752 to 0.938, respectively. All 20 loci were highly informative (PIC > 0.700). Eight loci deviated from Hardy-Weinberg equilibrium after Bonferroni correction (P < 0.05). CONCLUSIONS: This is the first report of genome survey sequencing in M. anguillicaudatus, genome information, mitochondrial genome, and microsatellite markers will be valuable for further studies on population genetic analysis, natural resource conservation, and molecular marker-assisted selective breeding.

TLR1 in Nile tilapia: The conserved receptor cannot interact with MyD88 and TIRAP but can activate NF-κB in vitro.

Toll-like receptors (TLRs) play a critical role in the innate immune response of fish. In this study, we isolated the cDNA sequence of Nile tilapia TLR1 (OnTLR1). The deduced OnTLR1 protein contains a signal peptide, 7 leucine-rich repeats (LRRs), a C-terminal LRR (LRR-CT), a transmembrane region and a highly conserved TIR domain. In healthy Nile tilapia, the OnTLR1 transcript was broadly expressed in all examined tissues, with the highest expression levels in the spleen. After infection with Streptococcus agalactiae, the OnTLR1 transcripts were upregulated in the gill and kidney. After stimulation with polyinosinic-polycytidylic acid (poly(I:C)), the expression levels of OnTLR1 were significantly downregulated in the intestine, whereas OnTLR1 transcripts were significantly upregulated in the kidney. After challenge with lipopolysaccharide (LPS), the expression levels of OnTLR1 were significantly upregulated in the spleen and kidney. The subcellular localization showed that OnTLR1 was expressed in the cytoplasm. TLR1 significantly increased MyD88-dependent NF-κB activity. However, the results of a pull-down assay showed that OnTLR1 did not interact with MyD88 or TIRAP. Binding assays revealed the specificity of OnTLR1 for pathogen-associated molecular patterns (PAMPs) and bacteria that included S. agalactiae, Aeromonas hydrophila and poly(I:C) and LPS. Taken together, these findings suggest that OnTLR1, as a pattern recognition receptor (PRR), might play an important role in the immune response to pathogen invasion.

A Chromosome-Level Genome Assembly of Mozambique Tilapia (Oreochromis mossambicus) Reveals the Structure of Sex Determining Regions.

The Mozambique tilapia (Oreochromis mossambicus) is a fascinating taxon for evolutionary and ecological research. It is an important food fish and one of the most widely distributed tilapias. Because males grow faster than females, genetically male tilapia are preferred in aquaculture. However, studies of sex determination and sex control in O. mossambicus have been hindered by the limited characterization of the genome. To address this gap, we assembled a high-quality genome of O. mossambicus, using a combination of high coverage of Illumina and Nanopore reads, coupled with Hi-C and RNA-Seq data. Our genome assembly spans 1,007 Mb with a scaffold N50 of 11.38 Mb. We successfully anchored and oriented 98.6% of the genome on 22 linkage groups (LGs). Based on re-sequencing data for male and female fishes from three families, O. mossambicus segregates both an XY system on LG14 and a ZW system on LG3. The sex-patterned SNPs shared by two XY families narrowed the sex determining regions to ∼3 Mb on LG14. The shared sex-patterned SNPs included two deleterious missense mutations in ahnak and rhbdd1, indicating the possible roles of these two genes in sex determination. This annotated chromosome-level genome assembly and identification of sex determining regions represents a valuable resource to help understand the evolution of genetic sex determination in tilapias.

Efficient Inhibition of Streptococcus agalactiae by AIEgen-Based Fluorescent Nanomaterials.

Streptococcus agalactiae, referred to as group B streptococcus (GBS), is a prominent co-pathogenic bacterium causing the onset and death of human, animal, and aquatic products. Although antibiotics are efficient against GBS, antibiotic resistance through antibiotic overuse is an equally serious problem. Therefore, the treatment of GBS infection appears strongly dependent on nonantibiotic therapy, such as photodynamic therapy. Different from other photosensitizers (PSs), luminogens with aggregation-induced emission (AIEgen) can efficiently generate fluorescence and reactive oxygen species (ROS). Herein, TBP-1, an efficient AIE PSs, is chosen to resist GBS, and its antibacterial activity and the killing mechanism toward GBS are investigated. The ROS generation performance and the images of GBS treated with TBP-1 in the dark or under white light irradiation were investigated. TBP-1 with its high ROS generation ability can efficiently kill GBS and serve as a novel treatment strategy against GBS infection.

Distant hybridization and gynogenesis between Nile tilapia Oreochromis niloticus and Jaguar cichlid Parachromis managuensis.

To investigate the distant hybridization and gynogenesis between Nile tilapia Oreochromis niloticus and Jaguar cichlid Parachromis managuensis, reciprocal crossing was first performed between the two species. No offspring, however, were viable when there were these hybridizations. Gynogenesis was induced in O. niloticus and P. managuensis using ultraviolet (UV)-irradiated spermatozoa from P. managuensis and O. niloticus, respectively. The morphology during embryonic development indicated gynogenetic offspring of both O. niloticus and the P. managuensis were normal and deformed, and the results from flow cytometric analysis indicated normal fry were diploid and deformed fry were haploid. Gynogenetic O. niloticus and P. managuensis had the same DNA content and chromosome number as their species of origin, indicating that gynogenetic individuals were produced in both species. The presence of only females for both gynogenetic P. managuensis and O. niloticus was indicative of an XX genotype in the female P. managuensis and O. niloticus. Results from studies on genetic diversity indicated the average heterozygosity of the gynogenetic diploid population of O. niloticus were less than that of the cultured population, but the genetic homozygosity of the gynogenetic diploid population of O. niloticus was greater than that of the cultured population after one generation of gynogenesis, which achieved the goal of rapidly establishing genetic homozygosity.

Nile tilapia Toll-like receptor 7 subfamily: Intracellular TLRs that recruit MyD88 as an adaptor and activate the NF-κB pathway in the immune response.

Toll-like receptor 7 (TLR7) subfamily members are important pattern recognition receptors that participate in the recognition of pathogen-associated molecular patterns. In the present study, three TLR family members, OnTLR7, OnTLR8 and OnTLR9, were identified in the Nile tilapia Oreochromis niloticus. TLR7-, TLR8-and TLR9-deduced proteins have typical structural characteristics of TLRs, including Toll/interleukin-1 receptor (TIR), leucine-rich repeat (LRR) and transmembrane region (TM). OnTLR7, OnTLR8 and OnTLR9 were broadly expressed in all of the tissues tested, with the highest expression levels in the brain (TLR7) and spleen (TLR8 and TLR9). Moreover, the expression levels of OnTLR7, OnTLR8 and OnTLR9 were significantly increased in most tested tissues after Streptococcus agalactiae infection in vivo. After LPS stimulation, OnTLR7 and OnTLR9 mRNA expression levels were downregulated in the intestine and upregulated in the liver, spleen and kidney; however, OnTLR8 mRNA expression levels were upregulated in the kidney only after LPS stimulation for 5 d. After Poly I:C stimulation, OnTLR7 and OnTLR9 mRNA expression levels were upregulated in the intestine, liver, spleen and kidney, and the highest expression was found in the liver, while OnTLR8 mRNA expression levels were upregulated in the intestine, liver and kidney and downregulated in the spleen. Subcellular localization of OnTLR7, OnTLR8, and OnTLR9 in 293T cells showed that OnTLR9 was distributed in both the cytoplasm and nucleus while OnTLR8 and OnTLR7 were distributed mainly in the cytoplasm. Overexpression of OnTLR7, OnTLR8 and OnTLR9 in 293T cells had no significant effect on the activity of NF-κB, but they could significantly enhance MyD88-mediated NF-κB activity after cotransfection with MyD88. Pulldown assays showed that OnTLR7, OnTLR8, and OnTLR9 could interact with OnMyD88. Taken together, these results indicate that TLR7 subfamily genes play a role in the immune response to pathogen invasion of Nile tilapia.

Structurally diverse genes encode TLR13 in Nile tilapia: The two receptors can recognize Streptococcus 23S RNA and conduct signal transduction through MyD88.

Toll-like receptors (TLRs) play a crucial role in the innate immune system, which is the first line of defence against pathogens and pathogenic products in fish. In the present study, we cloned the full-length cDNA and genome sequences of two TLR13 s (OnTLR13a, OnTLR13b) from Nile tilapia (Oreochromis niloticus). TLR family motifs, i.e., the leucine-rich repeat (LRR) domains and Toll/interleukin (IL)-1 receptor (TIR) domains, were conserved in the putative proteins OnTLR13a and OnTLR13b, with fifteen LRR domains and one TIR domain. Four exons and three introns were identified in the OnTLR13a genome sequence, and three exons and two introns were identified in the OnTLR13b genome sequence. In healthy Nile tilapia tissues, OnTLR13a and OnTLR13b were ubiquitously expressed in all 11 tested tissues/organs. The highest expression levels were observed in the spleen (OnTLR13a) and blood (OnTLR13b), and the lowest expression levels were observed in the liver (OnTLR13a) and stomach (OnTLR13b). The expression level of OnTLR13b at 5.5 days postfertilization (dpf) was significantly higher than that at the other 8 time points (2.5, 3.5, 4.5, 5, 6, 6.5, 7.5 and 8.5 dpf). Upon stimulation with an intraperitoneal injection of 200 µL (107 CFU/mL) Streptococcus agalactiae, the expression levels of OnTLR13a and OnTLR13b were significantly upregulated in the intestine and gill. After cotransfection with MyD88, OnTLR13a significantly increased MyD88-dependent NF-κB activation in 293 T cells. However, OnTLR13b significantly impaired MyD88-dependent NF-κB activation. In addition, TLR13a slightly increased MyD88-dependent AP-1 activation, and TLR13b significantly increased MyD88-dependent AP-1 activation. TLR13a significantly increased MyD88-dependent interferon-ß (IFN-ß) activation, and TLR13b had no effect on MyD88-dependent IFN-ß activation. These findings suggest that although the deduced protein structure of OnTLR13 is evolutionarily conserved between OnTLR13 and other TLR members, its signal transduction function is markedly different. Co-immunoprecipitation (Co-IP) assays showed that both OnTLR13a and OnTLR13b could interact with OnMyD88. RNA pulldown assays showed that TLR13a and TLR13b could combine with the 23S rRNA of S. agalactiae. These results indicate that TLR13a and TLR13b play important roles in the innate immune response against bacterial infection in Nile tilapia.

R-spondin1 in loach (Misgurnus anguillicaudatus): Identification, characterization, and analysis of its expression patterns and DNA methylation in response to high-temperature stress.

With a well-understood function in mammals, R-spondin1 (Rspo1) is an important regulator of ovarian development via the Wnt/ß-catenin pathway. Rspo1 deficiency causes retardation of ovarian development in XX fish, and increases Rspo1 function induces femininity and sex reversal in XY fish. In this study, Rspo1 was successfully cloned from loach (Misgurnus anguillicaudatus), and its expression profile was analyzed. The full-length cDNA of Misgurnus anguillicaudatus Rspo1 (MaRspo1) comprised 1322 bp and included an open reading frame (ORF) of 795 bp, which encoded a predicted polypeptide measuring 264 amino acids in length. Phylogenetic and gene structure analyses showed a highly conserved sequence of MaRspo1 (identical to the Rspo1 genes of other species), consisting of an N-terminal signal peptide (SP), two furin-like cysteine-rich domains (FU1 and FU2), a thrombospondin type 1 repeat (TSP1) and a C-terminal region. Real-time PCR revealed the female-biased expression profile of MaRspo1, with the highest expression level among tested tissues detected in ovary. Investigation of MaRspo1 expression levels throughout the early development stage (10-60 days post hatching) under three temperature treatments (25 °C, 28 °C, and 31 °C) revealed significantly differential expression of MaRspo1 among the three temperature groups, with decreased MaRspo1 expression in the high-temperature (31 °C) group. The results of DNA methylation analysis indicated that exposure to high temperature during early development can increase the average promoter methylation level of MaRspo1 in both females and males. Taken together, the results of this study provide the basis for the further investigation of the molecular mechanism of Rspo1 in response to temperature.

Molecular characterization, expression and functional analysis of IRAK1 and IRAK4 in Nile tilapia (Oreochromis niloticus).

Interleukin-1 receptor-associated kinase 1 (IRAK1) and IRAK4 are critical signalling mediators and play pivotal roles in the innate immune and inflammatory responses mediated by TLR/IL-1R. In the present study, two IRAK family members, OnIRAK1 and OnIRAK4, were identified in the Nile tilapia Oreochromis niloticus with a conserved N-terminal death domain and a protein kinase domain, similar to those of other fishes and mammals. The gene structures of OnIRAK1 and OnIRAK4 are organized into fifteen exons split by fourteen introns and ten exons split by nine introns. OnIRAK1 and OnIRAK4 were broadly expressed in all of the tissues tested, with the highest expression levels being observed in the blood and the lowest expression levels being observed in the liver. Both genes could be detected from 2 d post-fertilization (dpf) to 8 dpf during embryonic development. Moreover, the expression levels of OnIRAK1 and OnIRAK4 were clearly altered in all five tissues after Streptococcus agalactiae infection in vivo and could be induced by LPS, Poly I: C, S. agalactiae WC1535 and △CPS in Nile tilapia macrophages. The overexpression of OnIRAK1 and OnIRAK4 in 293T cells showed that they were both distributed in the cytoplasm and could significantly increase NF-κB activation. Interestingly, after transfection, OnIRAK1 significantly upregulated OnMyd88-induced NF-κB activation, while OnIRAK4 had no effect on OnMyd88-induced NF-κB activation. Co-immunoprecipitation (Co-IP) assays showed that OnMyd88 did not interact with either OnIRAK1 or OnIRAK4 and that OnIRAK1 did not interact with OnIRAK4. Taken together, these findings suggest that OnIRAK1 and OnIRAK4 could play important roles in TLR/IL-1R signalling pathways and the immune response to pathogen invasion.

MHC Class IIB gene polymorphisms associated with resistance/susceptibility to Streptococcus agalactiae in Nile tilapia Oreochromis niloticus.

Genetic variation in the major histocompatibility complex (MHC) Class IIB was tested in Nile tilapia Oreochromis niloticus, and the association between the MHC IIB alleles and disease resistance was also studied. F3 fry offspring (n = 1200) from 12 full-sib families were challenged with Streptococcus agalactiae, which caused significantly different mortalities in different Nile tilapia families (11.00-81.10%). Twenty fry (F1) from each of the 12 families were selected to study the polymorphisms of the MHC Class IIB gene using PCR followed by cloning and sequencing methods. The results showed that the size of the amplified fragment was 770-797 bp. Thirty-seven sequences from 240 individuals revealed 22 different alleles, which belonged to 9 major allele types. Up to 63.58% of nucleotide positions were variable, while the proportion of the amino acid variable positions was up to 68.73%. According to the survival rate of offspring (F3) from 12 full-sib families, we deduced that the alleles Orni-DAB*0107, Orni-DAB*0201 and Orni-DAB*0302 were highly associated with resistance to S. agalactiae, while the allele Orni-DAB*0701 was associated with susceptibility to S. agalactiae. In addition, our previous study found that the allele Orni-DAB*0201 was more frequently distributed in the disease-resistant groups. Therefore, the allele Orni-DAB*0201 could be used as an S. agalactiae resistance-related MHC marker in molecular marker-assisted selective breeding programs for S. agalactiae-resistant Nile tilapia.

Effects of Lactococcus lactis subsp. lactis JCM5805 on colonization dynamics of gut microbiota and regulation of immunity in early ontogenetic stages of tilapia.

The administration of probiotics during early ontogenetic stages can be an effective way to manipulate the gut microbiota of animals. Specifically, the administration of probiotics can enhance gut-colonization success and regulate the immune response. In this study, the effects of early contact with probiotic Lactococcus lactis subsp. lactis JCM5805 on the gut microbial assembly of larvae Nile tilapia were examined. The effects of JCM5805 on IFNα expression through the TLR7 and TLR9-dependent signal transduction pathway as well as larval disease resistance were studied. Three days postfertilization, embryos were randomly allocated into nine 30 L tanks with a concentration of 20 eggs L-1. Triplicate tanks were performed for each treatment. Treatments included a control group (C), a low probiotic concentration group (T1), where JCM5805 was added to the water at 1 × 104 cfu ml-1, and a high probiotic concentration group (T2), where JCM5805 was added to the water at 1 × 108 cfu ml-1. Probiotics were administered continuously for 15 days. qPCR was used to analyze transcript levels of the TLR7, TLR9, MyD88, IRF7 and IFNα genes using RNA extracted from whole embryos on day 5 and 10, and from the intestine of larvae on day 15. Transcription of these genes was also measured in the intestine, liver and spleen of larvae one month after the cessation of probiotic administration. The results showed that MyD88 and IRF7 were significantly elevated on days 5 and 10 in the T2 group. TLR9 and IFNα were also significantly elevated on days 5, 10 and 15 during probiotic application of T2 (P < 0.05). One month after the cessation of probiotics administration, no significant difference was observed in the expression of these genes (P > 0.05). The larvae were fed probiotics for 15 days and were infused with Streptococcus agalactiae strain WC1535 at a final concentration of 1 × 106 cfu ml-1. The survival rate of T2 was significantly higher than that of the C group (P < 0.05). Microbial characterization by Illumina HiSeq sequencing of 16S rRNA gene amplicons showed the significantly higher presence of JCM5805 in the guts of T2 after 15 days of probiotic continuous application. Although JCM5805 was below the detection level after the cessation of probiotic for 5 days, the gut microbiota of the exposed tilapia larvae in T2 remained clearly different from that of the control treatment after the cessation of probiotic administration. These data indicated that a high concentration of the probiotic strain JCM5805 upregulated the expression of IFNα via the TLR7/TLR9-Myd88 pathway and enhanced disease resistance of larvae. JCM5805 was only transiently detected and thus was not included in the stable larval microbiota. The early microbial exposure of tilapia larvae affects the gut microbiota at later life stages. However, whether the upregulation of related genes is related to the presence of JCM5805 strain in the intestine requires further verification.

Capsular polysaccharide of Streptococcus agalactiae is an essential virulence factor for infection in Nile tilapia (Oreochromis niloticus Linn.).

Streptococcus agalactiae (Group B Streptococcus, GBS) is associated with diverse diseases in aquatic animals. The capsule polysaccharide (CPS) encoded by the cps gene cluster is the major virulence factor of S. agalactiae; however, limited information is available regarding the pathogenic role of the CPS of serotype Ia piscine GBS strains in fish. Here, a non-encapsulated mutant (Δcps) was constructed by insertional mutagenesis of the cps gene cluster. Mutant pathogenicity was evaluated in vitro based on the killing of whole blood from tilapia, in vivo infections, measuring mutant survival in tilapia spleen tissues and pathological analysis. Compared to wild-type (WT) GBS strain, the Δcps mutant had lower resistance to fresh tilapia whole blood in vitro (p < 0.01), and more easily cleared in tilapia spleen tissue, and was highly attenuated in tilapia and zebrafish. Additionally, compared to the Δcps mutant, numerous GBS strains and severe tissue necrosis were observed in the tilapia spleen tissue infected with WT strains. These results indicated that the CPS is essential for GBS pathogenicity and may serve as a target for attenuation in vaccine development. Gaining a better understanding of the role, the GBS pathogenicity in fish will provide insight into related pathogenesis and host-pathogen interactions.

MHC class IIA polymorphisms and their association with resistance-susceptibility to Streptococcus agalactiae in Nile tilapia, Oreochromis niloticus.

The association between major histocompatibility complex (MHC) class IIA polymorphisms and the severity of infection by Streptococcus agalactiae was investigated using 40 susceptible and 40 resistant individuals of Nile tilapia Oreochromis niloticus. Twenty-five alleles were identified from 80 individuals, which belong to 22 major allele types. High polymorphism of mhcIIa gene and at least two loci were discovered in O. niloticus. In peptide-binding region (PBR) and non-PBR, the ratio of nonsynonymous substitution (dN) to synonymous substitution (dS) was 1.294 (>1) and 1.240 (>1), suggesting that the loci are evolving under positive balancing selection. Association analysis showed that the allele, orni-daa*0501, was significantly associated with resistance to S. agalactiae, while the alleles, orni-daa*1101, orni-daa*1301, orni-daa*1401 and orni-daa*1201, were associated with susceptibility to S. agalactiae. To confirm these correlations, another independent challenge experiment was performed in the Huizhou population of the O. niloticus. The frequency distribution showed that the orni-daa*1101 allele was significantly more frequent in the Huizhou-Susceptible group (HZ-SG) than in the Huizhou-Resistant group (HZ-RG) (P < 0.05), which was consistent with the first challenge. However, orni-daa*0501 did not present in HZ-SG and HZ-RG and the distribution frequencies of the orni-daa*1201, orni-daa*1301 and orni-daa*1401 alleles were not significantly more frequent in HZ-SG than in HZ-RG. These results indicate that the orni-daa*1101 allele confers susceptibility to S. agalactia infection. These results suggest that the diversity of exon 2 of mcaIIa alleles could be used to explore the association between disease susceptibility or resistance and the multiformity of mcaIIa and to achieve the molecular-assisted selection of O. niloticus with enhanced disease resistance.