Biosynthesis of silver nanoparticles using red algae Portieria hornemannii and its antibacterial activity against fish pathogens.

In the present study, red algae Portieria hornemannii was scrutinized for the productive synthesis of silver nanoparticles. These biosynthesized nanoparticles were characterized by visible color change and ultraviolet visible spectrophotometry that indicated the formation of nanoparticles at the absorbance of 418 nm. Fourier transforms infrared spectroscopy, X-ray Diffraction analysis was further carried out to study the functional groups and crystalline nature of the substance. Scanning electron microscopy and Transmission electron microscopy exposed the shape of the silver nanoparticles, which was found to be spherical. Energy dispersive X-ray spectroscopy confirmed the presence of the metal silver. Stability of nanoparticles was analyzed using Zeta potential. The synthesized nanoparticles were found to be active against the fish pathogens Vibrio harveyii, Vibrio parahaemolyticus, Vibrio alginolyticus and Vibrio anguillarum. The highest activity was found against the pathogens V. harveyii and V. parahaemolyticus. We have established an environmental friendly synthesis of Silver nanoparticles which can be used as an alternative to commercially available antibiotics in the treatment of fish diseases.

A simple filter paper-based method for transporting and storing Enterocytozoon hepatopenaei DNA from infected Litopenaeus vannamei tissues.

The microsporidian parasite Enterocytozoon hepatopenaei (EHP) causes a significant negative impact in shrimp aquaculture. A diagnostic procedure for detecting EHP in shrimp was developed, but transportation of the infected shrimp samples from the farm / hatcheries to the laboratory is burdensome and preservation of the tissues is problematic. Here, we developed a simple method of transporting nucleic acid without preservatives using the Flinders Technology Associates filter card (FTA matrix card; Whatman). DNA can be stored and extracted without the need for centrifugation and hazardous chemicals. EHP infected shrimp homogenate was spotted on a FTA matrix card and stored at room temperature. Storage stability was confirmed by analysis at different time points and we efficiently recovered DNA up to 6 months post spotting. The recovery efficiency of FTA-DNA was compared with the existing DNA extraction methods DNeasy® Blood & Tissue kit method and Guanidine hydrochloride method. The efficiency of extraction and sensitivity of the DNA in the FTA card confirmed that recovery of EHP-DNA from the FTA matrix was superior to with other methods.

Experimental horizontal transmission of Enterocytozoon hepatopenaei in post-larvae of whiteleg shrimp, Litopenaeus vannamei.

Enterocytozoon hepatopenaei (EHP) is a microsporidian parasite that causes hepatopancreatic microsporidiosis (HPM) in penaeid shrimp. HPM was observed in several countries, including Thailand and India; it has become a prominent pathogen in shrimp culture. Based on observations on EHP infection in the wild, the route of transmission has been hypothesized. Identification of artificial EHP infection procedures can facilitate our understanding of EHP transmission. Experimental transmission of EHP was attempted using the immersion and oral infections of infection. In the immersion mode, post-larvae (PL) were exposed to an EHP tissue homogenate (0.2%) by immersion for 48 hr. Experimental samples were collected at various time points, and infection was confirmed using polymerase chain reaction, haematoxylin and eosin staining, transmission electron microscopy and modified trichrome staining. All test results revealed successful EHP transmission. Similar results were obtained through oral infection (oral infection). Innate immune gene expression patterns during infection were analysed; prophenoloxidase, crustin and superoxide dismutase were upregulated at 6, 6 and 48 hr post-challenge, respectively. Experimental infection procedures facilitate the development of diagnostic and prevention strategies. This is the first study demonstrating the experimental transmission of EHP in shrimp PL.

Enhancement of immune response and resistance against Vibrio parahaemolyticus in kuruma shrimp (Marsupenaeus japonicus) by dietary supplementation of ß-1,4-mannobiose.

Supplementation of prebiotic carbohydrates can act as a potent immunomodulator and have the efficacy to induce immune-related genes which are involved in host defense. Pure ß-1,4-mannobiose (MNB) showed activation of prophenoloxidase system of shrimp hemocytes in vitro. The resistance of kuruma shrimp Marsupenaeus japonicus against Vibrio parahaemolyticus was examined after the shrimp were fed with 0 (control), 0.02, 0.2, and 2% MNB supplemented diets. The results showed significantly higher survival rates in MNB supplemented shrimp than those of the control one from 2 to 12 days post challenge. In another experiment, the hemocyte count, ROS production, phagocytic, phenoloxidase and bactericidal activities, and expression of immune-related genes were investigated in the control and MNB supplemented groups at day 1, 4, 6, 8 and 11 of the feeding. These immune parameters were significantly enhanced in MNB supplemented groups. Furthermore, the gene expression analysis showed that transcripts of lysozyme, crustin, penaeidin and TNF were significantly up-regulated in hemolymph, lymphoid organs and intestines of MNB treated shrimp. Overall, the results provided evidence that MNB supplementation could improve the immune response and increase shrimp resistance against V. parahaemolyticus infection.

Assessment of microplastic contamination in commercially available fishes.

Plastics have widespread applications for human use, but their disposal poses a significant threat to living organisms and these plastics end up in the marine environment. They will be fragmented into small pieces as a result of ultraviolet exposure, climatic changes, and temperature changes; Microplastics (MPs) are plastics that are less than 5 mm in size. The level of MP (Microplastic) pollution in commercially harvested fish from different habitant in Vellore, India is currently unknown. Therefore, this study aimed to determine the presence and characteristics of ingested or inhaled MPs in marine and freshwater fishes highly consumed by the local population. Fish gills and gastrointestinal tracts were aseptically dissected and digested (30% hydrogen peroxide), then filtered and examined under a microscope for the presence of MPs. Further analysis was performed on the samples using Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy (ATR-FTIR) and Scanning Electron Microscopy (SEM) with Energy Dispersive X-Ray (EDAX). Of the samples analysed, a total of 875 MPs were recovered from 32 fishes, with 478 from marine fishes and 397 from freshwater fishes. The most common colours of the MPs were blue and black, while stereo microscopy analysis revealed that the majority of MPs were fibers (91%), followed by fragments (8%) and a small number of films. The ATR-FTIR analysis identified polyvinyl alcohol (39.76%), polyethylene (16.51%), methylcellulose (12.84%) and styrene (9.07%), as the predominant types of MPs in the fish samples. This study highlights the significant impact of MP pollution on marine ecosystems. The research provides insight into the nature and extent of MPs in fish from both marine and freshwater habitats, with an aim for policies and interventions aimed to reduce plastic pollution in the locality.

Oral Administration of Carotenoid-Rich Dunaliella salina Powder Inhibits Colon Carcinogenesis via Modulation of Wnt/ß-catenin Signaling Cascades in a Rat Model.

The present study aims to investigate the oral therapeutic and molecular role of carotenoid-rich Dunaliella salina powder (DSP) against 1,2-dimethylhydrazine (DMH)-triggered colon carcinogenesis. In this study, thirty six male Wistar rats were categorized into six distinct groups (G1-G6): G1 group with no intervention, G2 group received only DSP (1000 mg/kg), G3 group received only DMH carcinogen (20 mg/kg), and G4-G6 group received both DMH and DSP at various phases (pre-initiation, post-initiation and entire phases) for 32 weeks. Body weight, tumor incidence, tumor volume, histopathological examination, antioxidants, and detoxification enzymes activities were analyzed in the experimental rats. In addition, the protein expression profile of components involved in the Wnt/ß-catenin signaling pathway was determined by western blot analysis. Matrix metalloproteinases (MMP-7 and MMP-9), proliferation marker (PCNA), and pro-apoptotic (Bcl-2 and Bax) proteins were analyzed using immunohistochemistry. Colorimetric assay was used to determine the levels of anti-inflammatory (iNOS and COX-2) and apoptotic proteins (Caspase-3 and Caspase-9). Results showed that concomitant administration of DSP with DMH significantly reduced tumor progression and prevented colon carcinogenesis in rats. However, treatment with DSP before or after DMH exposure did not significantly prevent colon carcinogenesis. DMH and DSP treatment group showed increased activities of antioxidant enzymes with significant reduction in the oxidative stress. Additionally, the detoxification enzymes and colonic histopathology of those rats were restored to that of control rats. The administration of DSP to rats exposed to DMH exhibited antitumor effects via inhibition of the Wnt/ß-catenin signaling pathway with induced apoptosis through the Bcl-2/Bax/caspases signaling cascades. Moreover, the same group also showed significant anti-inflammatory activity via regulating iNOS and COX-2 biomarkers. Our findings revealed molecular chemopreventive activity of carotenoid-rich DSP through regulating Wnt/beta-catenin and intrinsic apoptotic pathways. Thus, DSP is propound to function as a potent antioxidant, anti-proliferative, and anti-inflammatory therapeutic agent against colon carcinogenesis.

Deciphering of the Dual oxidase (Nox family) gene from kuruma shrimp, Marsupenaeus japonicus: full-length cDNA cloning and characterization.

In many physiological processes, including the innate immune system, free radicals such as nitric oxide (NO) and reactive oxygen species (ROS) play significant roles. In humans, 2 homologs of Dual oxidases (Duox) generate hydrogen peroxide (H(2)O(2)), which is a type of ROS. Here, we report the identification and characterization of a Duox from kuruma shrimp, Marsupenaeus japonicus. The full-length cDNA sequence of the M. japonicus Dual oxidase (MjDuox) gene contains 4695 bp and was generated using reverse transcriptase-polymerase chain reaction (RT-PCR) and random amplification of cDNA ends (RACE). The open reading frame of MjDuox encodes a protein of 1498 amino acids with an estimated mass of 173 kDa. In a homology analysis using amino acid sequences, MjDuox exhibited 69.3% sequence homology with the Duox of the red flour beetle, Tribolium castaneum. A transcriptional analysis revealed that the MjDuox mRNA is highly expressed in the gills of healthy kuruma shrimp. In the gills, MjDuox expression reached its peak 60 h after injection with WSSV and decreased to its normal level at 72 h. In gene knockdown experiments of free radical-generating enzymes, the survival rates decreased during the early stages of a white spot syndrome virus (WSSV) infection following the knockdown of the NADPH oxidase (MjNox) or MjDuox genes. In the present study, the identification, cloning and gene knockdown of the kuruma shrimp MjDuox are reported. Duoxes have been identified in vertebrates and some insects; however, few reports have investigated Duoxes in crustaceans. This study is the first to identify and clone a Dual oxidase from a crustacean species.

Facile Synthesis and Characterization of Cupric Oxide Loaded 2D Structure Graphitic Carbon Nitride (g-C3N4) Nanocomposite: In Vitro Anti-Bacterial and Fungal Interaction Studies.

The active and inexpensive catalyst cupric oxide (CuO) loaded foliar fertilizer of graphitic carbon nitride (g-C3N4) is investigated for biological applications due to its low cost and easy synthesis. The synthesized CuO NPs, bulk g-C3N4, exfoliated g-C3N4, and different weight percentages of 30 wt%, 40 wt%, 50 wt%, 60 wt%, and 70 wt% CuO-loaded g-C3N4 are characterized using different analytical techniques, including powder X-ray diffraction, scanning electron microscopy, energy dispersive X-ray analysis, and ultraviolet-visible spectroscopy. The nanocomposite of CuO NPs loaded g-C3N4 exhibits antibacterial activity against Gram-positive bacteria (Staphylococcus aureus and Streptococcus pyogenes) and Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa). The 20 µg/mL of 70 wt% CuO/g-C3N4 nanocomposite showed an efficiency of 98% for Gram-positive bacteria, 80% for E. Coli, and 85% for P. aeruginosa. In the same way, since the 70 wt% CuO/g-C3N4 nanocomposite showed the best results for antibacterial activity, the same compound was evaluated for anti-fungal activity. For this purpose, the fungi Fusarium oxysporum and Trichoderma viride were used. The anti-fungal activity experiments were not conducted in the presence of sunlight, and no appreciable fungal inhibition was observed. As per the literature, the presence of the catalyst g-C3N4, without an external light source, reduces the fungal inhibition performance. Hence, in the future, some modifications in the experimental conditions should be considered to improve the anti-fungal activity.

Molecular cloning and characterization of the NADPH oxidase from the kuruma shrimp, Marsupenaeus japonicus: early gene up-regulation after Vibrio penaeicida and poly(I:C) stimulations in vitro.

Free radicals such as nitric oxide (NO) and reactive oxygen species (ROS) are involved in many physiological processes. In humans, there are 5 homologs of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (Noxes) that generate superoxide (O(2)(-)), which can dismute to produce ROS, and play significant roles in innate immunity and cell proliferation. Though Noxes have been identified in vertebrates (humans and fishes) and some insects, there are very few reports investigating Noxes in crustaceans. In the present study, we describe the entire cDNA sequence (4216 bp) of Marsupenaeus japonicus (kuruma shrimp) Nox (MjNox) generated using reverse transcriptase-polymerase chain reaction (RT-PCR) and random amplification of cDNA ends (RACE). The open reading frame of MjNox encodes a protein of 1280 amino acids with an estimated mass of 146 kDa that has 46.8% sequence homology with the Nox gene of the fruit fly, Drosophila melanogaster. Highly conserved amino acid sequences were observed in the NADPH binding domain. Transcriptional analysis revealed that MjNox mRNA is highly expressed in the lymphoid organ, hepatopancreas and hemocytes of the healthy kuruma shrimp. In the hemocytes, MjNox expression reached its peak 4 h after stimulation with either Vibrio penaeicida or poly(I:C) and decreased to its normal level after 12 h.This study is the first to identify and clone a Nox family member (MjNox) from a crustacean species.

Class B scavenger receptor, Croquemort from kuruma shrimp Marsupenaeus japonicus: Molecular cloning and characterization.

The scavenger receptor, Croquemort is a member of the CD36 superfamily comprising transmembrane proteins involved in the recognition of polyanionic ligands. Various researchers have proved that members of the CD36 superfamily are involved in immunity and developmental processes. In the present study, we report a cDNA encoding the kuruma shrimp, Marsupenaeus japonicus Croquemort scavenger receptor (MjSCRBQ) obtained from a cDNA library of lymphoid organ by RACE amplification. The full-length cDNA of 2098 bp consists an open reading frame of 1596 nucleotides that translates into a 532-amino acid putative protein, with a 5' untranslated region of 323 bp and 3' UTR of 153 bp. The MjSCRBQ is constitutively expressed in gills, heart, hemolymph, hepatopancreas, intestine, lymphoid organ, muscle, nerve, and stomach and at high levels in the brain. Expression analysis in lymphoid organs of shrimp infected with white spot syndrome virus (WSSV) revealed high levels of MjSCRBQ 72 and 120 h post-infection. The MjSCRBQ contains putative functional domains including transmembrane domains and a CD36 domain. Multiple alignments of MjSCRBQ amino acid sequences showed significant identity with Drosophila melanogaster SCRBQ (31%), Salmo salar SCRBQ (29%), Homo sapiens SCRBQ (28%) and Rattus norvegicus SCRBQ (30%). In a phylogenetic analysis, MjSCRBQ was identified in the invertebrate scavenger receptor cluster. This is the first report in crustaceans of the identification and characterization of a Croquemort scavenging receptor.

A novel gene of tumor necrosis factor ligand superfamily from kuruma shrimp, Marsupenaeus japonicus.

A tumor necrosis factor (TNF) gene has been isolated and characterized in kuruma shrimp, Marsupenaeus japonicus, providing the first conclusive evidence for the existence of the TNF ligand in shrimp. The kuruma shrimp TNF (MjTNF) cDNA was composed of 1868 bp with a 262 bp 5'-untranslated region (UTR) and a 220 bp 3'-UTR, which was translated into a protein of 462 amino acid residues that included a predicted transmembrane domain of 23 amino acid residues (Trp20-Val42) and the TNF family signature (Pro321-Leu448). Homology analysis of MjTNF showed 30.7% and 26.7% identities with fruit fly (Drosophila melanogaster) Eiger and human (Homo sapiens) ectodysplasin A, respectively. The MjTNF gene was constitutively expressed in unstimulated organs of shrimp such as the muscle, stomach, brain and gill. In lymphoid organ cells, an enhanced expression of the MjTNF gene was observed following stimulation with peptidoglycan and polycytidylic acid. A high expression level of MjTNF was observed in vivo 2 h and 4 h after stimulation with lipopolysaccharide and Vibrio penaeicida, respectively. These observations suggest that MjTNF plays a role in the innate immune defense in kuruma shrimp. The discovery of shrimp TNF will allow a more complete and concrete understanding of shrimp inflammatory responses.

Molecular cloning and characterization of the nitric oxide synthase gene from kuruma shrimp, Marsupenaeus japonicus.

Nitric oxide (NO) signaling is involved in many physiological processes in vertebrates and invertebrates. In crustaceans, nitric oxide synthase (NOS) plays a significant role in the regulation of the nervous system and in innate immunity. Here, we describe the entire cDNA sequence (4616 bp) of the kuruma shrimp Marsupenaeus japonicus NOS (Mj NOS) generated using the reverse transcriptase-polymerase chain reaction (RT-PCR) and 5'- and 3'- rapid amplification PCRs of cDNA ends from brain and gill mRNAs. The open reading frame of Mj NOS encoded a protein of 1187 amino acids with an estimated mass of 134 kDa, and had an 82.3% sequence homology with the NOS gene of the land crab Gecarcinus lateralis. Highly conserved amino acid sequences in heme and tetrahydrobiopterin were observed in the oxygenase domain. FMN, FAD and NADPH were found in the reductase domain. Mj NOS mRNA was constitutively expressed in the brain, gill, intestine, thoracic ganglion and testis of the kuruma shrimp. When Vibrio penaeicida was injected into the kuruma shrimp, Mj NOS was expressed in the brain, gill, heart, lymphoid organ, intestine and thoracic ganglion. Mj NOS expression in the gill reached its peak 12 h and decreased to its normal level 24 h after V. penaeicida injection.

Truncated Thioredoxin Peptides Serves as an Efficient Fusion Tag for Production of Proinsulin.

BACKGROUND: Insulin is a peptide hormone used for regulating blood glucose levels. Human insulin market is projected to grow at a rate of 12.5% annually. To meet the needs of patients, a cost effective insulin manufacturing strategy has to be developed. This can be achieved by selecting a competent host, ideal fusion tag and streamlined downstream process. OBJECTIVE: In this article, we have demonstrated that selecting a right fusion partner for expression of toxic proteins like insulin, plays a major role in increasing the recombinant protein yield. METHODS: In this article, we have focused on identifying a peptide tag fusion partner for expressing proinsulin by truncating thioredoxin tag. Truncations were carried out from both Amino and Carboxy terminus of the protein and efficiency of truncated sequences was evaluated by expressing it with proinsulin gene. FCTRX (1-15) sequence fused to proinsulin was processed further to establish downstream protocol for purification. RESULTS: Thioredoxin tag was truncated appropriately by considering the fusion tag: protein ratio. A couple of sequences ranging 10 - 15 amino acids were identified based on its in silico properties. Of these FCTRX (1-15) showed increased expression and stability of fusion protein. 156 mg of purified insulin was generated from 1g of inclusion body after enzymatic conversion and chromatographic steps. CONCLUSION: As a result of the current study, it was concluded that FCTRX (1-15) peptide has advantageous attributes to be considered as an ideal fusion tag for expression of proinsulin. This can be further explored by expressing it with other proteins.

Real-time reverse transcription loop-mediated isothermal amplification for rapid detection of yellow head virus in shrimp.

A real-time reverse transcription loop-mediated isothermal amplification (real-time RT-LAMP) method was applied for detecting the replicase polyprotein-encoding gene of yellow head virus (YHV) in shrimp, Penaeus monodon. It is a novel, gene-specific assay that amplifies nucleic acid with high specificity, sensitivity and rapidity under isothermal conditions using a set of six specially designed primers that recognize eight distinct sequences of the target gene. This method works with even low copies of DNA and is based on magnesium pyrophosphate turbidity detection by an inexpensive photometer for quantitative analysis. A user-friendly protocol was developed with optimal conditions standardized at 63 degrees C for 60 min. With this protocol, the assay sensitivity was 10 times higher than the widely used YHV nested RT-PCR system. Cross-reactivity analysis using other shrimp virus DNA/cDNA and YHV-negative shrimp demonstrated high specificity of the assay. The real-time RT-LAMP method was performed also for an internal control gene, EF-1alpha, to compare with the expressions of the YHV gene in different organs of infected shrimp, and the resulting standard curves showed high correlation coefficient values. These results suggest that this assay is applicable widely as a new quantitative detection method in the pursuit of YHV-free shrimp culture.