We report an effective gene knockdown technique in rainbow trout embryos using additional RNA components combined with ribozymes (R
zs). Chimeric R
zs (tR
zCs) containing tRNA
Val, R
z against GFP, ...and a constitutive transport element were microinjected into transgenic embryos. tR
zCs induced greater gene interference than R
zs alone. Control tR
zCs did not affect unpaired bases of target RNA, and the tR
zC did not interfere with non-relevant gene expression, suggesting that the tR
zC-mediated gene-interference effects were sequence-specific. Furthermore, the tR
zC-containing expression vector specifically suppressed target GFP expression in transgenic trout. tR
zCs enhance R
z cleavage and could therefore be powerful tools for studying unknown gene function in vertebrates.
A mucoadhesive chitosan polymer-based nanoplatform has been increasingly recognized as an effective mucosal vaccine delivery system for fish. The present study aimed to investigate the effectiveness ...of immersion vaccination with a chitosan polymer-based nanovaccine to elicit an immune response in serum and mucus of red tilapia and evaluate its protective efficacy after immersion challenge with a heterogenous strain of Aeromonas veronii UDRT09. Six hundred red tilapia (22 ± 1.8 g) were randomly allocated into four experimental groups: control, empty-polymeric nanoparticle (PC), formalin-killed vaccine (FKV), and chitosan polymer-based nanovaccine (CS-NV) in triplicate. The specific IgM antibody levels and their bactericidal activity were assessed in serum and mucus for 28 days after immersion vaccination and followed by immersion challenge with A. veronii. The immersion vaccine was found to be safe for red tilapia, with no mortalities occurring during the vaccination procedure. The specific IgM antibody levels and bactericidal activity against A. veronii in both serum and mucus were significantly higher in red tilapia vaccinated with CS-NV compared to the FKV and control groups at all time points. Furthermore, the serum lysozyme activity, ACH50, and total Ig levels demonstrated a significant elevation in the groups vaccinated with CS-NV compared to the FKV and control groups. Importantly, the Relative Percentage Survival (RPS) value of the CS-NV group (71 %) was significantly higher than that of the FKV (15.12 %) and PC (2.33 %) groups, respectively. This indicates that the chitosan polymer-based nanovaccine platform is an effective delivery system for the immersion vaccination of tilapia.
•The chitosan polymer-based immersion nanovaccine improved both systemic and mucosal immune responses in fish vaccinated against A. veronii.•The chitosan polymer-based immersion nanovaccine exhibited a noteworthy augmentation in specific IgM antibody levels and bactericidal activity against A. veronii both in serum and mucus.•The serum lysozyme activity, ACH50, and total Ig levels increased substantially in the group receiving the chitosan polymer-based immersion nanovaccine.•The chitosan-polymer-based immersion nanovaccine increased relative percent survival (RPS) against a heterogenous strain of A. veronii.
Sensory characteristics and microbiological quality of Nile tilapia cooked with various sous-vide (SV) conditions including 50 and 60°C, for 30, 45 and 60 min (S5-30, S5-45, S5-60, S6-30, S6-45 and ...S6-60) and their changes during storage (4°C) were investigated, compared with control (cooking with boiling water). The result found that increasing temperature and time of SV accelerated protein degradation, both myofibrillar protein and connective tissue. This affected to the lower water-holding capacity (WHC) and shear force of samples treated with severe SV conditions (S6-45 and S6-60) (P<0.05). SV technique may less contribution to the flavor formation, particularly lipid oxidation products, since there were no differences in total volatile base nitrogen (TVB-N) content and thiobarbituric acid reactive substances (TBARS) value among all samples (P>0.05). However, SV cooked samples had higher sensory scores than control, both at day 0 (before storage) or throughout the storage time. This suggested the potential to improve consumer acceptability by this technique. In this study, SV at 60°C, 60 min seem to be the optimal condition for tilapia fillet, which can preserve at 4℃ for at least 6 weeks without any spoilage.
Nutritional programming is a concept proposed to be applied in the field of fish nutrition to improve the use of new diets in aquaculture. This study aimed to investigate for the first time the ...effects of a glucose injection into the yolk at the alevin stage on intermediary metabolism and growth in adult Nile tilapia (Oreochromis niloticus) at 32–37 weeks later in the life. The early stimulus was performed through direct microinjection of 2 M glucose into yolk sacs of Nile tilapia alevin. Subsequently, in adult tilapia, the long-term effects of glucose stimulus on growth performance, blood metabolites, chemical composition in the liver and muscle, expression of genes involved in glucose transport and metabolism (glycolysis and gluconeogenesis) and related pathways (amino acid catabolism and lipogenesis) were investigated. Our results showed that, even though early glucose injection had no effect on growth performance in adult fish, very few significant effects on glucose metabolism were observed. Furthermore, to evaluate the potential metabolic programming after a dietary challenge, a 2 × 2 factorial design with two early stimuli (0.85% NaCl or 2 M glucose) and two different dietary carbohydrate intakes (medium-carbohydrate diet, CHO-M; high-carbohydrate diet, CHO-H) was performed between weeks 33 and 37. As expected, compared with the CHO-M diet, the CHO-H diet led to decreased growth performance, higher glyceamia and triglyceridemia, higher glycogen and lipid levels in the liver as well as down-regulation of gluconeogenesis and amino acid catabolism gene expressions. More interestingly, although early glucose injection had no significant effect on growth performance, it enhanced the capacities for lipogenesis, glycolysis and gluconeogenesis, particularly in fish that were fed the CHO-H diet. Thus, the nutritional programming of tilapia linked to glucose injection into the yolk of alevins is always visible at the adult stage albeit less intense than what we previously observed in juvenile.
: Gene knock-down technology using antisense molecules has many applications for studying gene function, disrupting undesirable genetic traits, as well as providing effective therapy for a number of ...viral diseases. Encouraged by these applications, we developed a gene knock-down technique to interfere with gene expression using transgenic rainbow trout expressing the green fluorescent protein (GFP) gene as a model. One of the antisense morpholino phosphorodiamidate oligonucleotides (AMOs) used in this study (AtGFP-1) was 25 nucleotides in length and localized against codons 2 to 8 of GFP messenger RNA. Microinjection of AtGFP-1 into the blastodisc of fertilized eggs decreased the level of GFP gene expression in a dose-dependent manner, A comparison of the effects of various doses of AtGFP-1 suggested that 10 ng of AtGFP-1 was the optimal concentration in that it interfered with specific gene expression without being strongly toxic to trout embryos. Conversely, morpholino phosphorodiamidate oligonucleotides with the inverted AtGFP-1 sequence, which cannot bind to the target mRNA, did not inhibit GFP gene expression. AtGFP-1 did not affect the expression of nontargeted genes such as the skeletal muscle actin and foreign lacZ genes. These results also indicate that AtGFP-1 interfered with the expression of only the targeted gene. Western blot and reverse transcriptase polymerase chain reaction analyses revealed that the amount of GFP protein drastically decreased whereas the mRNA level was not affected by AtGFP-1, suggesting that AtGFP-1 blocked specific gene function at the translational level. Further, this gene inhibition persisted until the hatching stage. Another AMO, which was localized against the junction region between the 5? untranslated region and the starting codon of GFP mRNA (AtGFP-2), also caused inhibition effects. Thus AMOs can have potent and specific gene knock-down effects in trout embryos. This technology may be useful for examining the roles of selected genes and disrupting their expression during embryonic development of salmonid fish.
So far, there are a number of fish genome projects, including experimental and economically important fish that provide available DNA sequence information. However, the function of a gene cannot be ...deduced only by its DNA sequence. Therefore, a technique with which to investigate the function of the fish gene is needed. Gene knockdown (GKD), or antisense technology, is now being used as a powerful technique to study gene functions in living organisms. GKD effects result from the introduction of an antisense molecule into living cells. The antisense agents bind to target messenger RNA, thus inactivating the target gene expression. The appropriately spatial inhibitory effects on protein production from corresponding gene resulted in the phenotypic change. Therefore, the function of the gene can be understood. To date, there are a number of antisense molecules that can affect efficient GKD in fish. These include antisense oligonucleotides, small interfering RNA, and ribozyme. These antisense molecules cause specific gene inhibitor effects with different mechanisms. The various antisense mechanism types facilitate a number of GKD applications with various approaches in animals. In this review, we demonstrate the characteristics of each antisense molecule, its mechanism, and its application, especially for gene functional analysis in fish.
The influence of 17α-methyltestosterone (MT) on growth responses, biological parameters and the expression of genes involved in the GH–IGF pathway of the hypothalamic–pituitary–liver–gonadal axis ...were investigated in female, male, and sex-reversed Nile tilapia to evaluate the relationship between sex and MT-induced changes in these parameters. Female fish had a lower growth rate than male and sex-reversed fish, and MT increased growth performance and duodenal villi in females. Most but not all biological parameters of sex-reversed fish were similar to those of male fish. Male fish had higher red blood cell counts and hemoglobin levels than female and sex-reversed fish, suggesting that these hematological indices reflect a higher metabolic rate in male fish. Greater blood triglyceride levels indicated the vitellogenin process in female fish. MT increased the alternative complement activity in female fish (P<0.05). Sex and MT had no significant effects on the hypothalamic mRNAs of GHRH and PACAP. Although not statistically significant, females tended to have higher GH mRNA levels than male and sex-reversed fish. Additionally, MT tended to decrease and increase GH mRNA levels in female and male fish, respectively. There were significant differences among sexes in the expression of GHR, and IGF mRNAs at the peripheral level in the liver and gonads. Females had lower hepatic GHRs and higher ovarian GHRs than male and sex-reversed fish. While the mRNA levels of IGF-1 were lower in the ovary, the levels of IGF-2 were higher compared with those in testes. A significant correlation between GHRs and IGFs was demonstrated in the liver and gonad (except for IGF-1). Multiple regression analysis showed a significant relationship between GH mRNA and both GHRs and IGFs in the liver and gonad. MT exerted androgenic and, to some extent, estrogenic effects on several physiological parameters and GH–IGF action.
The goal of this study was to investigate the effects of dietary supplementation with β-glucan and synbiotics (i.e., microencapsulated Bacillus subtilis and Pediococcus acidilactici) on immune gene ...expression and immune parameters of Pacific white shrimp (Litopenaeus vannamei) as well as modulation of immune parameters when shrimp were exposed to ammonia stress. Shrimp were fed one of four treatment diets basal diet (C), β-glucan-containing diet (β-glu), β-glucan plus B. subtilis-containing diet (β-glu + Bs), and β-glucan plus P. acidilactici-containing diet (β-glu + Pa) for 90 days. The microencapsulated bacteria colonized in the proximal and central parts of the shrimp intestine. The β-glu diet increased expression of lipopolysaccharide and β-1,3-glucan-binding protein (LGBP), and the β-glu + Bs diet induced expression of the serine protease and prophenoloxidase genes. In addition, β-glu + Pa increased the LGBP transcription. However, the mRNA level of peroxinectin did not differ significantly among the experimental groups. Shrimp fed the β-glu and β-glu + Pa diets had higher superoxide dismutase activity (SOD) compared to shrimp fed the basal diet. In addition, the β-glu + Bs and β-glu + Pa diets increased phenoloxidase activity (PO). However, there were no significant differences in total hemocyte count (THC) and lysozyme activity among the treatment diets. Ammonia stress led to increased THC and decreased SOD activity, but it seemed to have no adverse effects on PO and lysozyme activity. Thus, both β-glucan and synbiotic supplementation had beneficial effects on several immune parameters, and the effect of dietary synbiotics was superior to that of β-glucan only for PO activity.
This study shows the effects of dietary activated charcoal (AC) on health status, intestinal morphology and fillet geosmin content of Nile tilapia prior to harvesting (2 and 4 weeks). Four dietary ...treatments (each diet in six replicates) were formulated to incorporate AC at levels of 0, 10, 20 and 30 g kg⁻¹ of the dry diet. Fish were reared in hapas, which were located in earthen ponds. There were not significant differences in growth performances among experimental treatments. The moisture and protein content in the fillet decreased and increased, respectively, as the incorporation level of AC increased. The hematological indices and several immune parameters did not differ significantly among treatment groups. Among the fifteen blood chemicals parameters examined, the significant reductions in protein and cholesterol and the changes in blood minerals were observed in fish fed dietary AC ≥20 g kg⁻¹. Dietary AC tended to increase the height of intestinal villi and goblet cell number. Dietary AC also influenced the reduction in geosmin in the fish fillet. Taken together, these findings indicate that AC (at 10 g kg⁻¹ diet) could be used as feed supplement for Nile tilapia prior harvesting to reduce geosmin without negative effects.
The Vago interferon-like protein participates in the interplay between interferon regulatory factors and the expression of immune-responsive genes. Vago was initially perceived to participate only in ...the antiviral activation through JAK/STAT pathway. However, certain isoforms of Vago can stimulate antimicrobial responses. Here we identify Vago isoforms in Fenneropenaeus merguiensis (FmVagos) and how they function in antiviral and antibacterial responses against highly invasive pathogens, including white spot syndrome virus (WSSV) and Vibrio parahaemolyticus (VP
). Three isoforms of FmVagos were identified: FmVago4, FmVago5a, and FmVago5b, and expressed throughout tissues of the shrimp. During infection, FmVago4, FmVago5a, and FmVago5b, were up-regulated after WSSV and VP
challenges at certain time points. Pre-injection of purified recombinant FmVago4 (rVago4), FmVago5a (rVago5a), and FmVago5b (rVago5b) proteins could significantly reduce the mortality of shrimp upon WSSV infection, while the increase of survival rate of VP
-infected shrimp was observed only in rVago4 treatment. The immunity routes that FmVagos might instigate in response to the pathogens were examined by qRT-PCR, revealing that the JAK/STAT pathway was activated after introducing rVago4, rVago5a, and rVago5b, while the Toll/IMD pathway and proPO system, combined with PO activity, were provoked only in the rVago4-treated shrimp. Our finding suggests cross-talk between Vago's antiviral and antimicrobial responses in shrimp immunity. These findings complement previous studies in which Vago and its specific isoform could promote viral and bacterial clearance in shrimp.
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