Experimental evolution for improved post-infection survival selects for increased disease resistance in Drosophila melanogaster.

Disease resistance (defined as the host capacity to limit systemic infection intensity) and disease tolerance (defined as the host capacity to limit infection-induced damage) are two complementary defense strategies that help the hosts maximize their survival and fitness when infected with pathogens and parasites. In addition to the underlying physiological mechanisms, existing theory postulates that these two strategies differ in terms of the conditions under which each strategy evolves in the host populations, their evolutionary dynamics, and the ecological and epidemiological consequences of their evolution. Here we explored if one or both of these strategies evolve when host populations are subjected to selection for increased post-infection survival. We experimentally evolved Drosophila melanogaster populations, selecting for the flies that survived an infection with the entomopathogen Enterococcus faecalis. We found that the host populations evolved increased disease resistance in response to selection for increased survival. This was despite the physiological costs associated with increased resistance, expression of which varied with the phase of infection. We did not find evidence of any change in disease tolerance in the evolved host populations.

Modeling the effects of prebiotic interventions on luminal and mucosa-associated gut microbiota without and with Clostridium difficile challenge in vitro.

Prebiotics can modulate the gut microbial community composition and function for improved (gut) health and increase resilience against infections. In vitro models of the gut facilitate the study of intervention effects on the gut microbial community relevant to health. The mucosa-associated gut microbiota, which thrives in close contact with the host plays a pivotal role in colonization resistance and health. Therefore, we here introduce the Mi-screen, an experimental approach implementing a 96-well plate equipped with a mucus agar layer for the additional culturing of mucosa-associated microbiota in vitro. In this study, we screened the effects of 2'-Fucosyllactose (2'-FL), fructooligosaccharides (FOS), and inulin within a complex microbiota without and with infection with the C. difficile strains ATCC 43599 (Ribotype 001) or ATCC BAA-1870 (Ribotype 027). We analyzed the microbial community composition and short-chain fatty acid levels after 48 h of incubation. The inclusion of an additional substrate and surface in the form of the mucus agar layer allowed us to culture a microbial richness ranging between 100-160 in Chao index, with Shannon indices of 5-6 across culture conditions, indicative of a microbial diversity of physiological relevance. The mucus agar layer stimulated the growth of characteristic mucosa-associated bacteria such as Roseburia inulinovorans. The prebiotic interventions affected luminal and mucosal microbial communities cultured in vitro and stimulated short-chain fatty acid production. FOS, inulin and 2'-FL promoted the growth of Bifidobacterium adolescentis within the mucosa-associated microbiota cultured in vitro. When spiking the untreated conditions with pathogenic C. difficile, the strains thrived within the luminal and the mucosal sample types, whereas prebiotic treatments exhibited inhibitory effects on C. difficile growth and prevented colonization. In conclusion, the Mi-screen facilitates the screening of luminal and mucosa-associated gut microbial community dynamics in vitro and therefore fills an important gap in the field of in vitro modeling.

Autochthonous Bacilli and Fructooligosaccharide as Functional Feed Additives Improve Growth, Feed Utilisation, Haemato-Immunological Parameters and Disease Resistance in Rohu, Labeo rohita (Hamilton).

The effects of Bacillus spp. (7 Log CFU g-1 feed) and fructooligosaccharide (FOS, 1%) as functional feed additives, either alone or in combination, were evaluated in a study on rohu, Labeo rohita fingerlings. The fish were fed different diets for 90 days, including a control diet and diets supplemented with FOS, B. licheniformis, B. methylotrophicus or synbiotic formulations of these. The results showed that the combination of B. licheniformis and FOS significantly improved weight gain, feed utilisation and protease activity compared to the other groups. Overall, the groups supplemented with probiotics and synbiotics (B. licheniformis + FOS or B. methylotrophicus + FOS) showed improvements in haematology, serum biochemistry and immune parameters compared to the control group. After 90 days of experimental feeding, the fish were challenged with pathogenic Aeromonas hydrophila, and data on haematology, immunity and stress parameters were collected. The results indicated that the application of Bacillus spp. and FOS boosted immunity and resistance to physiological stress in the fish. The highest post-challenge survival rate was observed in fish fed a diet with B. licheniformis and FOS, indicating the potential of this particular combination of functional feed additives to enhance growth, immunity and disease resistance in L. rohita.

Investigation of dietary exogenous protease and humic substance on growth, disease resistance to Flavobacterium covae and immune responses in juvenile channel catfish (Ictalurus punctatus).

Many bacterial pathogens impact the US catfish industry, and disease control can be challenging for producers. Columnaris disease in channel catfish, Ictalurus punctatus, is primarily caused by Flavobacterium covae (formerly F. columnare). Immunostimulants may enhance nonspecific immune responses and offer an alternative to antibiotic treatments in catfish. Furthermore, dietary protein sources and inclusions are also essential to fish health and nutrition and may enhance overall fish performance in pond culture. The current project evaluated two immunostimulants: a protease complex (PC) and a humic substance (HS) derived from a reed-sedge peat product. A 60-day trial examined the effects of five diets on growth performance, immune response and resistance to experimental F. covae infection in channel catfish. Diets included a high-quality fishmeal diet (32%; CF32), a high-protein soy-based diet (32%; C32), a low-protein soy-based diet (28%; C28; predominately used in industry), a low-protein soy diet supplemented with C28 + PC at 175 g metric ton-1 and C28 + HS in a low-protein diet at 23 g metric ton-1 . Following feeding for 60 d, juvenile channel catfish were sampled for growth performance and baseline health indicators (n = 3; body mucus, blood for sera, kidney and spleen). A subset of fish was then subjected to an immersion-based in vivo challenge trial with F. covae (ALG-00-530; 106 CFU mL-1 exposure). At 60d post-initiation, there were no dietary differences in the relative growth rate (p = .063) or thermal growth coefficient (p = .055), but the 32% diets generally appeared to perform best. Post-challenge, the C32 group's mortality was higher than the C28 + PC (p = .006) and C28 + HS diets (p = .005). Although not significant, the C28 and CF32 groups also demonstrated higher mortality compared to both PC and HS diets. Sera lysozyme concentration was found to increase following pathogen challenge (p < .001) and in comparison with mock-challenged catfish (p < .001). Elevated expression levels of proinflammatory cytokines (il-1ß, il-8, tnf-α and tgf-ß) were observed at trial midpoint and post-infection when compared to 60d. The C28 treatment was found to have lower tnf-α expression than the C28 + PC (p = .042) and C28 + HS (p = .042) groups following exposure to F. covae. These challenge data suggest that the immunostimulants (PC and HS) in plant-based protein may be beneficial in protecting against F. covae when offered in low-protein channel catfish diets.

Probiotics enhance resistance to Streptococcus iniae in Nile tilapia (Oreochromis niloticus) reared in biofloc systems.

Biofloc technology is a rearing technique that maintains desired water quality by manipulating carbon and nitrogen and their inherent mixture of organic matter and microbes. Beneficial microorganisms in biofloc systems produce bioactive metabolites that may deter the growth of pathogenic microbes. As little is known about the interaction of biofloc systems and the addition of probiotics, this study focused on this integration to manipulate the microbial community and its interactions within biofloc systems. The present study evaluated two probiotics (B. velezensis AP193 and BiOWiSH FeedBuilder Syn 3) for use in Nile tilapia (Oreochromis niloticus) culture in a biofloc system. Nine independent 3785 L circular tanks were stocked with 120 juveniles (71.4 ± 4.4 g). Tilapia were fed for 16 weeks and randomly assigned three diets: a commercial control diet or a commercial diet top-coated with either AP193 or BiOWiSH FeedBuilder Syn3. At 14 weeks, the fish were challenged with a low dose of Streptococcus iniae (ARS-98-60, 7.2 × 107 CFU mL-1 , via intraperitoneal injection) in a common garden experimental design. At 16 weeks, the fish were challenged with a high dose of S. iniae (6.6 × 108 CFU mL-1 ) in the same manner. At the end of each challenge trial, cumulative per cent mortality, lysozyme activity and expression of 4 genes (il-1ß, il6, il8 and tnfα) from the spleen were measured. In both challenges, the mortalities of the probiotic-fed groups were significantly lower (p < .05) than in the control diet. Although there were some strong trends, probiotic applications did not result in significant immune gene expression changes related to diet during the pre-trial period and following exposure to S. iniae. Nonetheless, overall il6 expression was lower in fish challenged with a high dose of ARS-98-60, while tnfα expression was lower in fish subjected to a lower pathogen dose. Study findings demonstrate the applicability of probiotics as a dietary supplement for tilapia reared in biofloc systems.

Glutaredoxin 2 in the mud crab Scylla paramamosain: Identification and functional characterization under hypoxia and pathogen challenge.

Glutaredoxin (Grx) is a glutathione-dependent oxidoreductase that plays a key role in antioxidant defense. In this study, a novel Grx2 gene (SpGrx2) was identified from the mud crab Scylla paramamosain, which consists of a 196 bp 5' untranslated region, a 357 bp open reading frame, and a 964 bp 3' untranslated region. The putative SpGrx2 protein has a typical single Grx domain with the active center sequence C-P-Y-C. The expression analysis revealed that the SpGrx2 mRNA was most abundant in the gill, followed by the stomach and hemocytes. Both mud crab dicistrovirus-1 and Vibrioparahaemolyticus infection as well as hypoxia could differentially induce the expression of SpGrx2. Furthermore, silencing SpGrx2 in vivo affected the expression of a series of antioxidant-related genes after hypoxia treatment. Additionally, SpGrx2 overexpression significantly increased the total antioxidant capacity of Drosophila Schneider 2 cells after hypoxia, resulting in a reduction of reactive oxygen species and malondialdehyde content. The subcellular localization results indicated that SpGrx2 was localized in both the cytoplasm and the nucleus of Drosophila Schneider 2 cells. These results indicate that SpGrx2 plays a crucial role as an antioxidant enzyme in the defense system of mud crabs against hypoxia and pathogen challenge.

Comparison of injection and immersion challenges of Renibacterium salmoninarum strains in Rainbow Trout.

OBJECTIVE: Renibacterium salmoninarum is a pathogenic gram-positive bacterium and is the causative agent of bacterial kidney disease (BKD), a malady that mainly impacts salmonid species. Experimental challenges were conducted to assess the virulence and challenge route for select R. salmoninarum strains (CK-90 and ATCC 33739) in Rainbow Trout Oncorhynchus mykiss. METHODS: The CK-90 strain was intracoelomically injected (100 µL) at a high dose containing 4.80 × 106 CFU/g of fish (optical density at 525 nm [OD525 ] = 1.779) and a low dose containing 6.86 × 105 CFU/g of fish (OD525  = 1.077); alternatively, fish were immersed in a solution containing 4.5 × 107 CFU/mL of fish (OD525  = 0.886). The ATCC 33739 strain (originating from Brook Trout Salvelinus fontinalis) was also included and intracoelomically injected at 3.58 × 105 CFU/g of fish (OD525  = 1.431) to discern differences in virulence between the strains. RESULT: Clinical signs of BKD manifested at approximately 10 d postchallenge, and mortalities began at 19 days postchallenge. To confirm infection and quantify R. salmoninarum antigen load, an enzyme-linked immunosorbent assay (ELISA) was conducted using kidney tissue collected after the challenge. Rainbow Trout that were challenged with CK-90 by injection (both high- and low-dose groups) exhibited significantly higher mortality than fish that were injected with ATCC 33739 or those that were exposed to CK-90 via immersion challenge. The R. salmoninarum p57 (57-kDa protein) antigen was confirmed via ELISA. Antigen load for fish injected with CK-90 (high dose: OD405  = 0.71; low dose: OD405  = 0.66) was significantly higher than that for fish injected with ATCC 33739 (OD405  = 0.34). The CK-90 strain (both high and low doses) was more virulent than ATCC 33739, which caused no mortalities over the 28-days trial. Although there were no mortalities among ATCC 33739 fish, the ELISA confirmed that the R. salmoninarum antigen infiltrated kidney tissue in those fish. CONCLUSION: The immersion challenge methodology for R. salmoninarum CK-90 was ineffective for inducing mortalities at the examined dose.

MEK homologue is involved in immune response by regulating antimicrobial peptides expression in Chinese mitten crab Eriocheir sinensis.

MEK activates the phosphorylation of downstream molecules involved in various immune responses. In this study, an MEK homologue gene in Chinese mitten crab Eriocheir sinensis (designated as EsMEK) was investigated. EsMEK mRNA was constitutively expressed in all tissues with higher expression in hepatopancreas, hemocytes, and gills. EsMEK protein was mainly localized in the cytoplasm. Lipopolysaccharide (LPS) and Aeromonas hydrophila challenge significantly increased the mRNA levels of EsMEK in hemocytes. In addition, the mRNA expression level of some antimicrobial peptides (AMPs), including EsWAP, EsDWD1, and EsALF decreased significantly due to the inhibition of EsMEK by specific dsRNA in LPS-challenged crabs. Downstream pathway analysis revealed that the phosphorylation of EsERK decreased prominently after EsMEK inhibition. These results suggested that EsMEK played an important role in regulating the expression of antimicrobial peptides in E. sinensis through MEK-ERK pathway.

Adverse effects of dietary virgin (nano)microplastics on growth performance, immune response, and resistance to ammonia stress and pathogen challenge in juvenile sea cucumber Apostichopus japonicus (Selenka).

It has been well documented that micro- and nanoplastics are emerging pollutants in aquatic environments, and their potential toxic effects has attracted widespread concerns. Here, we evaluated the adverse effects of dietary polystyrene nanoplastics and microplastics (PS-N/MPs) on growth performance, oxidative stress induction, immune response, ammonia detoxification, and bacterial pathogen resistance of sea cucumber Apostichopus japonicus. After collection and acclimation, sea cucumbers were randomized into 3 groups (i.e., control, 100 nm PS-NPs and 20 µm PS-MPs at 100 mg kg-1 diet) for 60-day feeding experiment. Every group contained 360 sea cucumbers which were equally divided into 3 aquaria as biological triplicates. The results showed that the specific growth rate and final weight of the sea cucumbers fed with diets containing PS-N/MPs were significantly lower than those of control group. Dietary virgin PS-N/MPs significantly increased the reactive oxygen species production and malondialdehyde content in coelomic fluid, causing oxidative stress and damage to the growth and development of A. japonicus. During the experiment, 100 nm PS-NPs significantly induced the depletion in cellular and humoral immune parameters. The calculated IBR values based on multi-level biomarkers revealed the size-dependent toxic differences of PS-NPs > PS-MPs. The relative expression levels of GDH and GS mRNA showed first rise and then fall trends after exposure to ammonia, and 100 nm PS-NPs had a more profound impact on suppressing ammonia detoxification compared with 20 µm PS-MPs. Moreover, the expression of Hsp90, Hsp70, CL, TLR, and CASP2 genes were all down-regulated by ammonia exposure. Taken together of IBR results, ammonia stress test and pathogen challenge, we deduced that dietary 100 nm PS-NPs are more potentially hazardous than 20 µm PS-MPs. These findings provide valuable information for understanding the size-dependent toxic effects of PS-N/MPs and early risk warning on marine invertebrates.

In vitro screening and in vivo colonization pilot model of Lactobacillus plantarum LP5 and Campylobacter coli DSPV 458 in mice.

The objective of this work was to determine the antibacterial effect of Lactobacillus plantarum strains of pork origin against Campylobacter coli strains, and to conduct experimental colonization pilot models in mice for both microorganisms. Inhibition assays allowed evaluation and selection of L. plantarum LP5 as the strain with the highest antagonistic activity against C. coli and with the best potential to be used in in vivo study. Adult 6-week-old female Balb/cCmedc mice were lodged in two groups. The treated group was administered with 9.4 log10CFU/2 times/wk of L. plantarum LP5. L. plantarum LP5 was recovered from the feces and cecum of the inoculated mice. However, when bacteria stopped being administered, probiotic counts decreased. Experimental colonization with C. coli was carried out in five groups of mice. All animals were treated with antibiotics in their drinking water to weaken the indigenous microbiota and to allow colonization of C. coli. Four groups were administered once with different C. coli strains (DSPV458: 8.49 log10CFU; DSPV567: 8.09 log10CFU; DSPV570: 8.46 log10CFU; DSPV541: 8.86 log10CFU, respectively). After 8 h, mice inoculated with different C. coli strains were colonized because the pathogen was detected in their feces. L. plantarum LP5 tolerated the gastrointestinal conditions of murine model without generating adverse effects on the animals. C. coli DSPV458 colonized the mice without causing infection by lodging in their digestive tract, thus generating a reproducible colonization model. Both models combined could be used as protection murine models against pathogens to test alternative control tools to antibiotics.

Assessment of Flavobacterium psychrophilum-associated mortality in Atlantic salmon (Salmo salar) and brook trout (Salvelinus fontinalis).

Salmonid diseases caused by infections of Flavobacterium psychrophilum, the causative agent of bacterial coldwater disease, remain difficult to manage as novel, pathogenic strains continue to emerge in aquaculture settings globally. To date, much of the research regarding treatment options and vaccine development has focused on rainbow trout (Oncorhynchus mykiss), but other inland-reared salmonids are also impacted by this Gram-negative bacterium. As such, Atlantic salmon (Salmo salar) and brook trout (Salvelinus fontinalis) were injection-challenged with a variety of previously reported F. psychrophilum strains isolated from disease diagnostic cases in salmonids, as well as a standard and well-studied F. psychrophilum strain (CSF 259-93) known to be virulent in rainbow trout. In three separate virulence assessments (Trials A, B and C), strains US063 (isolated from lake trout; Salvelinus namaycush) and US149 (isolated from Atlantic salmon) caused a significantly higher cumulative per cent mortality (CPM) relative to other strains in Atlantic salmon (p <.001 for all trials), with US149 causing significantly greater mortality than US063 in Trials A (CPM 97% vs. 65%, p =.008) and B (CPM 96% ± 2.3% vs. 81.33% ± 4.8%, p =.014). Trial C used a lower dose (1.86 × 108  CFU/mL) for US149, resulting in a lower mortality (78.67% ± 9.33%) relative to Trials A and B. CSF259-93 did not cause significant mortality in any trials. In brook trout, the strain 03-179 (originally isolated from steelhead trout; Oncorhynchus mykiss) was significantly more virulent than any other (CPM 100% ± 0%, p <.001), followed by US063 (73% ± 3.8%) and US149 (40% ± 6.1%,) respectively. Again, CSF259-93 did not cause significant mortality relative to a mock challenge treatment. Results provide information about the applicability of strain selection in F. psychrophilum virulence testing in Atlantic salmon and brook trout, demonstrating the high virulence of US063 and US149 for these salmonid species. This information is applicable for the development of therapeutics and vaccines against F. psychrophilum infections and demonstrates the reproducibility of the experimental challenge model.

Yarrowia lipolytica N6-glucan protects goat leukocytes against Escherichia coli by enhancing phagocytosis and immune signaling pathway genes.

Immunostimulant and protective effects of Yarrowia lipolytica glucans against important pathogens, such as Escherichia coli, have not been investigated in goats and other ruminants. This study aimed to characterize Y. lipolytica N6-glucan (Yl-glucan) and its possible role in immunological signaling pathway activation and immunoprotection against E. coli in goat leukocytes. Characterization analyses showed that Y. lipolytica content had a mix of ß and α-D-glucans, molecular weight of 3301.53 kDa and low solubility after the heat treatment. The stimulation of goat leukocytes with Yl-glucan induced protection against E. coli challenge. Remarkably, Yl-glucan and E. coli interaction increased gene expression of dectin-1 and TLR-2 receptors, signaling pathway Syk/NFκB, and cytokines, such as TNF-α and IL-10. As a consequence of signaling activation, phagocytosis, and nitric oxide production enhanced killing of pathogens. Altogether, Y. lipolytica-glucan demonstrated to possess an immunoprotective potential against E. coli through innate immune response modulation in goat leukocytes.

Intermediary metabolic response and gene transcription modulation on the Sub-Antarctic notothenioid Eleginops maclovinus (Valenciennes, 1930) injected with two strains of Piscirickettsia salmonis.

Pathogen interactions with cultured fish populations are well studied, but their effects on native fishes have not been characterized. In Chile, the disease caused by bacterial species Piscirickettsia salmonis represents one of the main issues and is considered to be one of the important pathogens in the field of aquaculture. They have been found to infect native fish. Therefore, it is necessary to understand the impact of P. salmonis on native species of local commercial value, as well as the potential impact associated with the emergence of antibiotic-resistant strains of P. salmonis. Due to this purpose, the native fish Eleginops maclovinus was used in our study. Fish were randomly distributed in tanks and intraperitoneally inoculated with two strains of P. salmonis. No mortality was recorded during the experiment. Cortisol, glucose and total α-amino acid levels increased in fish injected with AUSTRAL-005 strain compared to sham-injected and LF-89-inoculated fish. Moreover, results showed an increase in the activity of carbohydrates and lipids metabolism in liver; and an increase in the carbohydrates, lipids and total α-amino acid metabolism in muscle after injection with AUSTRAL-005. Our results suggest that P. salmonis modulates the physiology of E. maclovinus and the physiological impact increase in the presence of the antibiotic-resistant strain AUSTRAL-005.

Cloning and identification of antimicrobial peptide, hepcidin from freshwater carp, Catla catla on pathogen challenge and PAMPs stimulation.

Hepcidin, a cationic cysteine-rich antimicrobial peptide (AMP) acts in hormone regulation and iron homeostasis in the host body. However, the biological property of hepcidin in immune reaction remains unexplored. In aquatic milieu, environmental and pathogenic stressors cause detrimental infections, which are defended by various immunological cells and antimicrobial peptides. In this study, hepcidin gene has been cloned from freshwater carp, Catla catla. The partially cloned hepcidin consists of 200 bp nucleotide sequence encoding 66 amino acids. Nucleotide sequence showed 97% and 91% similarity with Labeo rohita and Cyprinus carpio, respectively. Expression profile revealed significant up-regulation (P ≤ 0.0001) in liver as compared to other tissues in different conditions. In Aeromonas hydrophila challenged C. catla, liver showed higher expression level of hepcidin at 72 h as compared to other tissues. In skin, hepcidin expression showed significant upraise during 24 h in Streptococcus uberis infection. In Argulus sp. infected fishes, up-regulation of hepcidin expression was noted in liver, intestine and skin. The inactivated viral antigen-stimulated fishes, a substantial rise in liver was observed implying hepcidin as an important molecule in combating the pathogenic infections in freshwater carp, C. catla. Fishes stimulated with pathogen-associated molecular patterns (PAMPs) triggered the increased expression of hepcidin mRNA in liver, kidney and skin. This study indicates the presence of hepcidin as antimicrobial peptide in neutralizing the pathogenic infection in fishes.

A forty year journey: The generation and roles of NO in plants.

In this year there is the 40th anniversary of the first publication of plant nitric oxide (NO) emission by Lowell Klepper. In the decades since then numerous milestone discoveries have revealed that NO is a multifunctional molecule in plant cells regulating both plant development and stress responses. Apropos of the anniversary, these authors aim to review and discuss the developments of past concepts in plant NO research related to NO metabolism, NO signaling, NO's action in plant growth and in stress responses and NO's interactions with other reactive compounds. Despite the long-lasting research efforts and the accumulating experimental evidences numerous questions are still needed to be answered, thus future challenges and research directions have also been drawn up.

Molecular characterization and expression of toll-like receptor 5 genes from Pelteobagrus vachellii.

Toll-like receptor 5 (TLR5) is an important pathogen recognition receptor (PRR) that recognizes the flagellin protein of pathogenic bacteria and plays a fundamental role in activating the innate immune response. In this study, full-length pvTLR5m (membrane) and pvTLR5s (soluble) genes were cloned from darkbarbel catfish Pelteobagrus vachellii, and their expression and that of downstream genes were analyzed following exposure to the Aeromonas hydrophila pathogen. The 3009 bp pvTLR5m cDNA includes a 2652 bp open reading frame (ORF) encoding 884 amino acids. The 2422 bp pvTLR5s cDNA includes a 1944 bp ORF encoding a predicted protein of 648 amino acids. The genes are most closely related to TLR5m (75%) and TLR5s (69%) from Ictalurus punctatus, respectively, and both have a typical TLR structure. Both genes were constitutively expressed in all examined tissues, and most abundantly in the head kidney and spleen. Following pathogen challenge, pvTLR5m and pvTLR5s expression was increased significantly (P <0.05) and peaked at 24 and 12 h post-exposure in the liver, 24 and 12 h in the head kidney, and 48 and 24 h in the spleen, respectively. The downstream genes interleukin-1ß (IL-1ß), IL-12 and tumor necrosis factor-alpha (TNF-α) were significantly up-regulated following pathogen exposure in spleen, and the NF-kB inhibitor (IκB) was down-regulated. These findings indicated that pvTLR5 may play an important role in the immune responses to A. hydrophila. These results provide new insight to elucidate the immune signalling pathways of fish TLR.

Suppressor of cytokine signaling 6 can enhance epidermal growth factor receptor signaling pathway in Bombyx mori (Dazao).

The SOCS (Suppressor of cytokine signaling) family members are a potential negative regulator of cytokine signaling pathway and play a key role to maintain immunological functions in animals. SOCS-6 is an important member of the SOCS family, however the functions of this gene have rarely been explored among eukaryotes. Herein, we cloned and expressed SOCS-6 gene from Bombyx mori (Dazao) (BmSOCS-6), and anti-rabbit antibodies were prepared using purified recombinant BmSOCS-6 protein. Under normal physiological conditions, the BmSOCS-6 expression was observed at varied levels in six tissues, with most greatly expressed in fat body and hemocytes. After immune challenge with viral, fungal and bacterial pathogens, the BmSOCS-6 showed distinctly varied expression patterns in tissue, time and microbe dependent manner. By contrast, recombinant BmSOCS-6 protein strongly enhanced the expression of epidermal growth factor receptor (EGFR) pathway related genes, while the depletion of BmSOCS-6 by double stranded RNA suppressed their production. Altogether we concluded that BmSOCS-6 may improve the efficiency of EGFR signaling pathway in B. mori (Dazao).

Under control: how a dietary additive can restore the gut microbiome and proteomic profile, and improve disease resilience in a marine teleostean fish fed vegetable diets.

BACKGROUND: The constant increase of aquaculture production and wealthy seafood consumption has forced the industry to explore alternative and more sustainable raw aquafeed materials, and plant ingredients have been used to replace marine feedstuffs in many farmed fish. The objective of the present study was to assess whether plant-based diets can induce changes in the intestinal mucus proteome, gut autochthonous microbiota and disease susceptibility of fish, and whether these changes could be reversed by the addition of sodium butyrate to the diets. Three different trials were performed using the teleostean gilthead sea bream (Sparus aurata) as model. In a first preliminary short-term trial, fish were fed with the additive (0.8%) supplementing a basal diet with low vegetable inclusion (D1) and then challenged with a bacteria to detect possible effects on survival. In a second trial, fish were fed with diets with greater vegetable inclusion levels (D2, D3) and the long-term effect of sodium butyrate at a lower dose (0.4%) added to D3 (D4 diet) was tested on the intestinal proteome and microbiome. In a third trial, the long-term effectiveness of sodium butyrate (D4) to prevent disease outcome after an intestinal parasite (Enteromyxum leei) challenge was tested. RESULTS: The results showed that opposed forces were driven by dietary plant ingredients and sodium butyrate supplementation in fish diet. On the one hand, vegetable diets induced high parasite infection levels that provoked drops in growth performance, decreased intestinal microbiota diversity, induced the dominance of the Photobacterium genus, as well as altered the gut mucosal proteome suggesting detrimental effects on intestinal function. On the other hand, butyrate addition slightly decreased cumulative mortality after bacterial challenge, avoided growth retardation in parasitized fish, increased intestinal microbiota diversity with a higher representation of butyrate-producing bacteria and reversed most vegetable diet-induced changes in the gut proteome. CONCLUSIONS: This integrative work gives insights on the pleiotropic effects of a dietary additive on the restoration of intestinal homeostasis and disease resilience, using a multifaceted approach.

Tumor necrosis factor receptor-associated factor 3 from Anodonta woodiana is an important factor in bivalve immune response to pathogen infection.

Tumor necrosis factor receptor-associated factor 3 (TRAF3) is a multifunctional adaptor protein in innate and acquired immune system that plays a key role in the regulation of the RIG-I-like receptor (RLR) and Toll-like receptor (TLR) signaling pathway in mammals. However, the immune function of TRAF3 homologs in freshwater mollusks is not well understood. In this study, we identified a bivalve TRAF3 gene (AwTRAF3) from Anodonta woodiana and investigated its potential roles during immune challenges. The present AwTRAF3 encoded a polypeptide of 562 amino acids with predicted molecular mass of 64.5 kDa and PI of 7.9. Similar to other reported TRAF3s, AwTRAF3 contained a RING finger domain, two TRAF domains with zinc finger domains, a coiled coli region and a conserved C-terminal meprin and TRAF homology (MATH) domain. Quantitative real-time PCR (qRT-PCR) analysis revealed that AwTRAF3 mRNA was broadly expressed in all of the examined tissues, with high expression in hepatopancreas, gill and heart. In addition, immune challenge experiments directly showed that transcript levels of AwTRAF3 in hepatopancreas were significantly regulated upon bacterial (Vibrio alginolyticus and Staphylococcus aureus) and viral (poly (I:C)) challenges, respectively. Moreover, GFP-tagged AwTRAF3 fusion protein was found to be located primarily in the cytoplasm in HEK293T cells. Altogether, these data provided the first experimental demonstration that freshwater mollusks possess a functional TRAF3 that was involved in the innate defense against bacterial and viral infection.

Molecular cloning, characterization and expression of immunoglobulin D on pathogen challenge and pathogen associated molecular patterns stimulation in freshwater carp, Catla catla.

The primordial immunoglobulin class, IgD, was the first non-IgM isotype discovered in teleosts. The crucial roles of IgM and IgZ in imparting systemic and mucosal immunity, respectively, in various fish species have been widely established. However, the putative function of a unique IgD isotype during pathogenic invasions has not been well explored. The present study reports the existence of an IgD ortholog in freshwater carp, Catla catla, and further evaluates its differential expression profile in response to bacterial, parasitic and viral antigenic exposure and pathogen associated molecular patterns (PAMPs) stimulation. The IgD of C. catla (CcIgD) cDNA sequence was found to encode 226 amino acids and confirmed homology with heavy chain delta region of Cyprinidae family members. Phylogenetic analysis of CcIgD exhibited greatest similarity with Ctenopharyngodon idella. qRT-PCR analysis revealed significant upregulation (P < 0.001) of IgD gene expression in kidney with respect to other tissues at 24 hr post-Aeromonas hydrophila challenge. CcIgD gene expression in skin was enhanced following Streptococcus uberis infection and in blood following Argulus infection and inactivated rhabdoviral antigen stimulation. Further, the treatment of bacterial and viral products (PAMPs) also triggered significant (P < 0.05) increases in CcIgD mRNA expression in kidney. These findings indicate the functional importance of teleost IgD in orchestrating tissue specific neutralization of antigens on stimulation with different pathogens and PAMPs.