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Antimicrobial peptides (AMPs), derived from a variety of proteins such as ribosomal proteins, play a pivotal role in the innate immune system. However, information regarding ribosomal protein-derived AMPs is currently limited and their mechanisms of action remain poorly defined. Here we identified and characterized the antibacterial activity of amphioxus RPL27 (BjRPL27) and its core functional region located at residues 51–72 (termed BjRPL27 51-72). We found that BjRPL27 expression was upregulated in the hepatic caecum following bacterial infection. Both the recombinant protein rBjRPL27 and the synthetic peptide BjRPL27 51-72 effectively killed the Gram-positive bacterium Staphylococcus aureus and the Gram-negative bacterium Aeromonas hydrophila via a combined action of disrupting cell membrane integrity, inducing membrane depolarization, and increasing intracellular reactive oxygen species (ROS) production. Additionally, the sequence of BjRPL27 51-72 was highly conserved among all eukaryotic RPL27s, implying an ancient origin for the antibacterial activity of the RPL27 family. In vivo assays showed that BjRPL27 51-72 not only efficiently protected zebrafish from A. hydrophila infection, but also killed the bacterium S. aureus on the skin wound of rats. Furthermore, neither BjRPL27 nor BjRPL27 51-72 exhibited hemolytic activity towards human red blood cells, making them promising lead molecules for designing novel AMPs. These findings highlight the potential of BjRPL27 51-72 as a novel AMP with selective bactericidal properties. • Bjrpl27 gene expression was up-regulated in amphioxus during bacterial infection. • BjRPL27 showed bactericidal activity against Gram-negative and -positive bacteria. • A peptide, termed BjRPL27 51-72 , was identified as a novel AMP. • BjRPL27 51-72 could disrupt bacterial cell membrane and induce ROS production. • Neither BjRPL27 nor BjRPL27 51-72 exhibited hemolytic activity. [ABSTRACT FROM AUTHOR]

• BjGHRH interacted directly with BjGHRHR. • BjGHRH activated the cAMP/PKA signal pathway via binding to BjGHRHR. • BjGHRH increased the expression of growth hormone like gene in amphioxus. Growth hormone-releasing hormone (GHRH) has been widely shown to stimulate growth hormone (GH) production via binding to GHRH receptor GHRHR in various species of vertebrates, but information regarding the functional roles of GHRH and GHRHR in the protochordate amphioxus remains rather scarce. We showed here that two mature peptides, BjGHRH-1 and BjGHRH-2, encoded by BjGHRH precursor, and a single BjGHRHR protein were identified in the amphioxus Branchiostoma. japonicum. Like the distribution profiles of vertebrate GHRHs and GHRHRs, both the genes Bjghrh and Bjghrhr were widely expressed in the different tissues of amphioxus, including in the cerebral vesicle, Hatschek's pit, neural tube, gill, hepatic caecum, notochord, testis and ovary. Moreover, both BjGHRH-1 and BjGHRH-2 interacted with BjGHRHR, and triggered the cAMP/PKA signal pathway in a dose-dependent manner. Importantly, BjGHRH-1 and BjGHRH-2 were both able to activate the expression of GH-like gene in the cells of Hatschek's pit. These indicate that a functional vertebrate-like GHRH-GHRHR axis had already emerged in amphioxus, which is a seminal innovation making physiological divergence including reproduction, growth, metabolism, stress and osmoregulation possible during the early evolution of vertebrates. [ABSTRACT FROM AUTHOR]

Enzyme activities and gene expression of a number of innate immune parameters in the serum, mucus and skin of Atlantic salmon ( Salmo salar ) were investigated after challenge with a pathogenic strain of Aeromonas salmonicida ( A . salmonicida ). Fish were injected in the dorsal muscle with either 100 μl bacterium solution, about 3.05 × 10 7 CFU/ml A . salmonicida , or 100 μl 0.9% NaCl (as control group) and tissue samples were collected at days 0, 2, 4 and 6 post-injection. Lysozyme (LSZ) and alkaline phosphatase (AKP) activities in serum, mucus and skin, and LSZ and AKP mRNA expression in skin of the challenged fish were higher than those of the control at most of the experimental time, with significant differences at several time points ( P < 0.05), indicating the involvement of LSZ and AKP in the innate immunity of Atlantic salmon to A . salmonicida . Superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities in mucus and skin, along with the SOD, POD and CAT mRNA expression in skin significantly decreased at day 4 and 6, indicating the decreased antioxidant capacity of the challenged fish. Glutamate pyruvate transaminase (GPT) and glutamic oxalacetic transaminase (GOT) activities in serum, mucus and skin of the challenged group were all higher than those of the control after the injection, and at several time points significant differences were found between the two groups，suggesting organs of fish were impaired after the pathogen infection. The changes of the GPT and GOT activities could be used as potential biomarkers for the impairment of physiological functions caused by the pathogen infection. Identified biomarkers of the immune responses will contribute to the early-warning system of the disease. So this study will not only provide a theoretical basis for vaccine development, but also provide basic data for the establishment of early warning systems for diseases caused by A . salmonicida in Atlantic salmon rearing. [ABSTRACT FROM AUTHOR]

Antimicrobial peptides (AMPs), characterized by their cationic nature and amphiphilic properties, play a pivotal role in inhibiting the biological activity of microbes. Currently, only a fraction of the antimicrobial potential within the ribosomal protein family has been explored, despite its extensive membership and resemblance to AMPs. Herein we demonstrated that amphioxus RPL17 (BjRPL17) exhibited not only upregulated expression upon bacterial stimulation but also possessed bactericidal capabilities against both Gram-negative and -positive bacteria through combined action mechanisms including interaction with cell surface molecules LPS, LTA, and PGN, disruption of cell membrane integrity, promotion of membrane depolarization, and induction of intracellular ROS production. Furthermore, a peptide derived from residues 127–141 of BjRPL17 (termed BjRPL17-1) showed antibacterial activity against Staphylococcus aureus and its methicillin-resistant strain via the same mechanism observed for the full-length protein. Additionally, the rpl17 gene was highly conserved in Metazoa, hinting it may play a universal role in the antibacterial defense system in different animals. Importantly, neither BjRPL17 nor peptide BjRPL17-1 exhibited toxicity towards mammalian cells thereby offering prospects for designing novel AMP agents based on these findings. Collectively, our results establish RPL17 as a novel member of AMPs with remarkable evolutionary conservation. • BjRPL17 was significantly up-regulated during bacterial infection. • BjRPL17 showed potent bactericidal activity against Gram-negative and -positive bacteria. • BjRPL17 could disrupt bacterial cell membrane and induce intracellular ROS production. • BjRPL17-1, a peptide derived from residues 127-141 of BjRPL17, inhibited the growth of S. aureus and MRSA. • Neither BjRPL17 nor BjRPL17-1 exhibited toxicity towards mammalian cells. [ABSTRACT FROM AUTHOR]

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, On IRAK1 and On IRAK4, 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 On IRAK1 and On IRAK4 are organized into fifteen exons split by fourteen introns and ten exons split by nine introns. On IRAK1 and On IRAK4 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 On IRAK1 and On IRAK4 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 On IRAK1 and On IRAK4 in 293T cells showed that they were both distributed in the cytoplasm and could significantly increase NF-κB activation. Interestingly, after transfection, On IRAK1 significantly upregulated On Myd88-induced NF-κB activation, while On IRAK4 had no effect on On Myd88-induced NF-κB activation. Co-immunoprecipitation (Co-IP) assays showed that On Myd88 did not interact with either On IRAK1 or On IRAK4 and that On IRAK1 did not interact with On IRAK4. Taken together, these findings suggest that On IRAK1 and On IRAK4 could play important roles in TLR/IL-1R signalling pathways and the immune response to pathogen invasion. • On IRAK1 and On IRAK4 gene were identified in Nile tilapia and were upregulated after S. agalactiae challenge. • On IRAK1 and On IRAK4 induced by LPS, Poly I: C, wild and mutant type S. agalactiae in in vitro. • On Myd88 did not interact with either On IRAK1 or On IRAK4, and On IRAK1 did not interact with On IRAK4. • Overexpression of On IRAK1 and On IRAK4 significantly increased NF-κB activation. • On IRAK1 significantly upregulated the NF-κB activity induced by On Myd88. [ABSTRACT FROM AUTHOR]

Abstract 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. Highlights • JCM5805 was used in early ontogenetic stages of tilapia for the first time. • JCM5805 upregulated the expression of IFNα via the TLR7 / TLR9 - Myd88 pathway. • JCM5805 enhanced disease resistance of tilapia larvae. • The early microbial exposure affects the gut microbiota at later life stages. • The JCM5805 couldn't colonize in the intestine of tilapia larvae. [ABSTRACT FROM AUTHOR]

The present study aimed to evaluate the individual and combined effects of Lactobacillus rhamnosus (LR) JCM1136 and Lactococcus lactis subsp. lactis (LL) JCM5805 on the growth, intestinal microbiota, intestinal morphology, immune response and disease resistance of juvenile Nile tilapia ( Oreochromis niloticus ). A total of 720 apparently healthy juvenile Nile tilapia (0.20 ± 0.05 g) were randomly divided into four equal groups. Fish were fed with a basal diet (CK) supplemented with JCM1136 (LR), JCM5805 (LL), and JCM1136 + JCM5805 (LR+LL) at 1 × 10 8 CFU/g basal diet for 6 weeks, followed by a basal diet for 1 week. After 6 weeks of feeding, the LL treatment significantly increased the growth and feed utilization of Nile tilapia when compared with the CK. Light microscopy and transmission electron microscopy images of the midgut revealed that probiotic supplementation significantly increased gut microvilli length and microvilli density compared to CK. The transcript levels of several key immune-related genes in the mid-intestine and liver of fish were analyzed by means of quantitative polymerase chain reaction (qPCR) at the end of the sixth week. The results showed the following: when compared to CK group, fish in LR had significantly increased transcript levels of IFN-γ, lyzc, hsp70 and IL-1β in the intestine; LL fish showed significantly increased expressions of TNF-α, IFN-γ, lyzc, hsp70 and IL-1β in the intestine and liver; and intestine lyzc, hsp70 and IL-1β and liver TNF-α, IFN-γ, hsp70 and IL-1β were significantly increased in LR+LL fish. Following a 6-week period of being fed probiotics or a control diet, the tilapia were challenged with an intraperitoneal injection of 20 μl of the pathogenic Streptococcus agalactiae (WC1535) (1 × 10 5  CFU/ml). The survival rates of the probiotic-fed groups were significantly higher than that of the CK group, and the LL group had the highest survival rate. High-throughput sequencing revealed a significantly higher presence of JCM5805 in the guts of LL fish during the period of probiotic application, but this was no longer detected in all LL samples 1 week post cessation of probiotic administration. Cessation of probiotic administration led to disorders of individual gut microbes within the LR and LL groups. Statistical analysis (LEfSe) demonstrated that three phyla, namely, Bacteroidetes, Fusobacteria and Actinobacteria were enriched in the CK group, while the abundance of Proteobacteria was greater in the probiotic-fed fish. At the genus level, Plesiomonas , which includes potential pathogens of fish, were significantly decreased in the probiotic-fed groups. In contrast, a significant increase of Rhizobium and Achromobacter , which can produce a variety of enzymes with cellulolytic and pectolytic activity, were observed in fish fed with probiotics, indicating that dietary probiotics were helpful in the propagation of some probiotic bacteria. Our data revealed that JCM1136 and JCM5805, as a feed additive at 10 8  CFU/g feed, could improve intestinal morphology, enhance immune status and disease resistance, and affect the gut microbiota of tilapia; thus, these additives could be used as probiotics for juvenile Nile tilapia. JCM5805 was more effective than JCM1136 or the mixture of the two for promoting the growth, enhancing the immune status and disease resistance of tilapia. [ABSTRACT FROM AUTHOR]