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Marine molluscan cell lines, required for virus screening and cultivation, form essential tools for developing health management strategies for these animals in the blue economy. Moreover, they are also crucial to develop cultivated seafood. As there is no valid marine molluscan cell line, primary cell cultures are relied upon for all investigations. A sound protocol for generating primary cell cultures from molluscs is entailed, but existing protocols often involve heavy antibiotic usage and depuration that invariably affect gene expression and cell health. This work presents an easy‐to‐adopt, time‐saving protocol using non‐depurated mollusc Crassostrea madrasensis, which requires only initial antibiotic treatment and minimal exposure or no use of antibiotics in the cell culture medium. The important experimental considerations for arriving at this protocol have been elucidated. Accordingly, sodium hypochlorite and neomycin sulfate were chosen for disinfecting tissues. The study is the first to use shrimp cell culture medium (SCCM) as a cell culture medium for molluscan cell culture. Despite being osmoconformers, the oysters exhibited stable intracellular osmotic conditions and pH, which, when provided in vitro, promoted effective cardiomyocyte formation. The cell viability could be enhanced using 10% fetal bovine serum (FBS), but healthy cell culture could also be obtained using SCCM without FBS. The optimized culture conditions allowed for regular beating cardiomyocyte clusters that could be retained for a month. Limited cell proliferation, as shown by the BrdU assay, demands further interventions, such as possibly producing induced pluripotent stem cells. The optimized protocol and culture conditions also align with some requirements for producing cultivated meat from marine molluscs. [ABSTRACT FROM AUTHOR]

Cell line development from shrimp is not a novel venture as researchers across the globe have been trying to have crustacean cell lines over 30 years. The reason for not attaining a crustacean or precisely a shrimp cell line is believed to be the replicative senescence and the inability to maintain telomere length in vitro. Moreover, spontaneous in vitro transformations do not happen in shrimp cells. Oncogenic induction in primary cell culture is one of the ways to attain in vitro transformation by way of disrupting the mechanisms which involve cellular senescence. In this context, a recombinant baculovirus with shrimp viral promoter IHHNV-P2 was used for the transduction aimed at immortalization. An oncogene, H-ras, was successfully amplified and cloned in to the baculoviral vector, downstream to shrimp viral promoter IHHNV-P2 and upstream to GFP. Recombinant baculovirus with H-ras was generated and used for transduction into shrimp lymphoid cells during early dividing stage. Accordingly, fibroblast-like primary cell culture got developed, and H-ras and GFP expression could be confirmed. The study suggests that the simple method of incubating recombinant baculovirus with minced tissue enables in vitro transduction during early dividing stage of the cells, and the transduction efficiency gets enhanced by adding 5 mM sodium butyrate to the culture medium. [ABSTRACT FROM AUTHOR]

Shrimp progressively gets more attention among marine invertebrates from researchers all over the world due to it being a healthy food as well as having economic importance. There were a lot of attempts to develop a continuous cell line from shrimp but none successful. In this context a novel hybrid cell line named ' Pm LyO- Sf 9' could be developed by fusing shrimp lymphoid organ cells with Sf 9 cells after to metabolic blocking of Sf 9 cells using puromycin and actinomycin D and effecting the fusion by way of PEG application. The cells are maintained and multiplied in a mixture of SCCM and TNM-FH having osmolality 550 mOsm kg−1 and pH 6.8. Transmission electron microscopy of the hybrid cells revealed the presence of two nuclei during the initial stages and a single nucleus subsequently. The cell line is with shrimp and Sf 9 genomic components and shrimp specific protein and is susceptible to WSSV. Shrimp elongation factor, Sf9 beta-actin, shrimp STAT and peroxinectin could be expresses through RT-PCR in the cell line. This is the first successful report of a hybrid cell line with shrimp genomic components and envisaged to be recognized a model system for multitudes of biomedical research in vitro. The cell line is in the National Cell Line Repository of ICAR – National Bureaue of Fish Genetic Resources, Lucknow, India. • A novel hybrid cell line ' Pm LyO- Sf 9' developed by fusing shrimp lymphoid organ cells with Sf 9 cells. • Morphologically, hybrid cells are bi- and tri-polar with tail like extension. • Electron microscopy analysis revealed two distinct nuclei in early stages. • Shrimp and Sf9 genomic components were separately amplified. • Shrimp immune genes peroxinectin and STAT expression confirmed in hybrid cell line. [ABSTRACT FROM AUTHOR]

White spot syndrome virus (WSSV) is the most devastating pathogen found in shrimp aquaculture. The lack of certified continuous/established cell lines from penaeid shrimp restricts in vitro studies on the viruses to bring out effective prophylactic and therapeutic measures. In this context, a novel hybrid cell line named, Pm LyO- Sf 9, consisting of shrimp and Sf9 genomes has been established and employed to study WSSV susceptibility and multiplication. The hybrid cells were exposed to the shrimp virus WSSV and cytopathic effects (CPE) such as (a) enlargement of cells, (b) cessation cell division, (c) granulation of cytoplasm, (d) rounding off of cells, shortening and disappearance of tail-like structures and (e) detachment from the flask. Expression of immediate early genes such as ie 1, dnapol, rr1, tk-tmk, and pk 1could be confirmed indicating that viral DNA replication in the Pm LyO- Sf 9 took place followed by the expression of late genes such as VP-28, VP-26, VP-15 and VP-19. Electron micrograph of WSSV infected cells demonstrated marginated dense zones in the nucleus with clumped chromatin, and the mid zone with virus-like particles. However, neither discrete virus particles nor the culture supernatant having infectivity could be observed suggesting that virions were not getting formed in the cells. This is the first report of the susceptibility of Pm LyO- Sf 9 to WSSV, and the ' Pm LyO- Sf 9 - WSSV Complex' formed, defined as the infected status of Pm LyO- Sf 9 with WSSV, could be of use for unraveling at molecular level the mechanism of viral entry, replication impediments and cellular apoptosis. [ABSTRACT FROM AUTHOR]

Diversity studies of endophytic assemblages are emerging challenges, which unveil novel phenotypes producing interesting chemical entities and a better understanding of their ecological significance. In the present investigation, we selected an extremely complex and unique environment supporting unexplored endophytes, 'Macroalgae of Kerala coast, India'. Unlike terrestrial flora and mangroves, reports displaying endophytic assemblages of marine flora remain limited, especially from India. The main goal of this study was to expose hidden endophytic fungi from macroalgae and examination of their bioactive potential. An ecological investigation of four red, four green and three brown algae resulted in 133 fungal taxa with 29 distinct morphospecies. Aspergillus and Penicillium were found to be the dominant genera. Penicillium chrysogenum was the sole fungi that contributed 11% of the entire endophytic community. Antimicrobial activity against various aquaculture/human pathogens revealed that around 59% of endophytes inhibited at least one of the pathogens screened. The maximum number of isolates (37%) inhibited Escherichia coli tailed by Aspergillus fumigatus (27%). Antimicrobial profile of fungal endophytes endorses them as a potential source of bioactive molecules that can be explored to find a solution for drug resistance in microbial pathogens. [ABSTRACT FROM AUTHOR]

To mitigate the toxicity of ammonia in aquaculture systems, marine and brackish water ammoniaoxidizing bacterial consortia have been developed and are used for activation of nitrifying bioreactors integrated to recirculating aquaculture systems. To shed more light on to these biological entities, diversity of both the consortia were analyzed based on random cloning of 16S rRNA gene and ammonia-oxidizing bacterial specific amoA gene sequences. The dendrograms of representative clones on the basis of amplified ribosomal DNA restriction analysis generated 22 and 19 clusters for marine and brackish water nitrifying consortia, respectively. Phylogenetic analysis demonstrated the presence of various autotrophic nitrifiers belonging to α-, β- and γ-Proteobacteria, anaerobic ammonia oxidizers, heterotrophic denitrifiers, Bacteroidetes, and Actinobacteria. Distribution patterns of the organisms within the two consortia were determined using the software Geneious and diversity indices were investigated using Mega 5.0, VITCOMIC and Primer 7. The abundance of ammonia oxidizers was found in the order of 2.21±0.25 x 109 copies/g wet weight of marine consortium and 6.20±0.23 x 107 copies/g of brackish water consortium. Besides, marine ammoniaoxidizing consortium exhibited higher mean population diversity and Shannon Wiener diversity than the brackish water counterparts. [ABSTRACT FROM AUTHOR]

The CYP2 genes are the largest and most diverse cytochrome P450 (CYP) subfamily in vertebrates. We have identified nine co-localized CYP2 genes (∼55 kb) in a new cluster in the genome of the highly resilient ecotoxicological fish model Kryptolebias marmoratus . Molecular characterization, temporal and tissue-specific expression pattern, and response to xenobiotics of these genes were examined. The CYP2 gene clusters were characterized and designated CYP2N22 - 23 , CYP2AD12 , and CYP2P16-20 . Gene synteny analysis confirmed that the cluster in K. marmoratus is similar to that found in other teleost fishes, including zebrafish. A gene duplication event with diverged catalytic function was observed in CYP2AD12 . Moreover, a high level of divergence in expression was observed among the co-localized genes. Phylogeny of the cluster suggested an orthologous relationship with similar genes in zebrafish and Japanese medaka. Gene expression analysis showed that CYP2P19 and CYP2N20 were consecutively expressed throughout embryonic development, whereas CYP2P18 was expressed in all adult tissues, suggesting that members of each CYP2 gene family have different physiological roles even though they are located in the same cluster. Among endocrine-disrupting chemicals (EDCs), benzo[α]pyrene (B[α]P) induced expression of CYP2N23 , bisphenol A (BPA) induced CYP2P18 and CYP2P19 , and 4-octylphenol (OP) induced CYP2AD12 , but there was no significant response to 4-nonylphenol (NP), implying differential catalytic roles of the enzyme. In this paper, we identify and characterize a CYP2 gene cluster in the mangrove killifish K. marmoratus with differing catalytic roles toward EDCs. Our findings provide insights on the roles of nine co-localized CYP2 genes and their catalytic functions for better understanding of chemical-biological interactions in fish. [ABSTRACT FROM AUTHOR]

To evaluate the effects of ultraviolet B (UV-B) radiation at the developmental, reproductive, and molecular levels in aquatic invertebrates, we measured UV-B-induced acute toxicity, impairments in developmental and reproductive traits, and UV-B interaction with the entire family of cytochrome P450 ( CYP ) genes in the intertidal benthic copepod Tigriopus japonicus . We found a significant, dose-dependent reduction ( P < 0.05) in the survival of T. japonicus that began as a developmental delay and decreased fecundity. The 48 h LD10 and LD50 were 1.35 and 1.84 kJ/m 2 , and the CYP inhibitor (PBO) elevated mortality, confirming the involvement of CYP genes in UV-B induced toxicity. Low-dose UV-B (1.5 kJ/m 2 ) induced developmental delays, and higher doses (6–18 kJ/m 2 ) caused reproductive impairments in ovigerous females. The significant up-regulation of CYP genes belonging to clans 2/3/MT/4/20 in T. japonicus exposed to UV-B (12 kJ/m 2 ) confirmed molecular interaction between UV-B and CYP genes. Moreover, orphan CYPs, such as CYP20A1 , provide good insight on the deorphanization of invertebrate CYPs . Overall, these results demonstrate the involvement of UV-B radiation in the expression of all the CYP genes in T. japonicus and their susceptibility to UV-B radiation. This will provide a better understanding of the mechanistic effects of UV-B in copepods through the predicted AhR-mediated up-regulation of CYP genes. [ABSTRACT FROM AUTHOR]

Brominated flame retardant, 2, 2′, 4, 4′-tetrabromodiphenyl ether (BDE-47), has received grave concerns as a persistent organic pollutant, which is toxic to marine organisms, and a suspected link to endocrine abnormalities. Despite the wide distribution in the marine ecosystem, very little is known about the toxic impairments on marine organisms, particularly on invertebrates. Thus, we examined the adverse effects of BDE-47 on life history trait (development), oxidative markers, fatty acid composition, and lipid accumulation in response to BDE-47-induced stress in the marine copepod Paracyclopina nana . Also, activation level of mitogen-activated protein kinase (MAPK) signaling pathways along with the gene expression profile of de novo lipogenesis (DNL) pathways were addressed. As a result, BDE-47 induced oxidative stress ( e.g . reactive oxygen species, ROS) mediated activation of extracellular signal-regulated kinase (ERK) and c-Jun-N-terminal kinase (JNK) signaling cascades in MAPK pathways. Activated MAPK pathways, in turn, induced signal molecules that bind to the transcription factors (TFs) responsible for lipogenesis to EcR , SREBP , ChREBP promoters. Also, the stress stimulated the conversion of saturated fatty acids (SFAs) to polyunsaturated fatty acids (PUFAs), a preparedness of the organism to adapt the observed stress, which could be correlated with the elongase and desaturase gene ( e.g . ELO3 , Δ5-DES , Δ9-DES ) expressions, and then extended to the delayed early post-embryonic development and increased accumulation of lipid droplets in P. nana . This study will provide a better understanding of how BDE-47 effects on marine invertebrates particularly on the copepods, an important link in the marine food chain. [ABSTRACT FROM AUTHOR]

Engineered multi-walled carbon nanotubes (MWCNTs) have received widespread applications in a broad variety of commercial products due to low production cost. Despite their significant commercial applications, CNTs are being discharged to aquatic ecosystem, leading a threat to aquatic life. Thus, we investigated the adverse effect of CNTs on the marine copepod Paracyclopina nana . Additional to the study on the uptake of CNTs and acute toxicity, adverse effects on life parameters ( e.g . growth, fecundity, and size) were analyzed in response to various concentrations of CNTs. Also, as a measurement of cellular damage, oxidative stress-related markers were examined in a time-dependent manner. Moreover, activation of redox-sensitive mitogen-activated protein kinase (MAPK) signaling pathways along with the phosphorylation pattern of extracellular signal-regulated kinase (ERK), p38, and c-Jun-N-terminal kinases (JNK) were analyzed to obtain a better understanding of molecular mechanism of oxidative stress-induced toxicity in the copepod P. nana . As a result, significant inhibition on life parameters and evoked antioxidant systems were observed without ROS induction. In addition, CNTs activated MAPK signaling pathway via ERK, suggesting that phosphorylated ERK ( p -ERK)-mediated adverse effects are the primary cause of in vitro and in vivo endpoints in response to CNTs exposure. Moreover, ROS-independent activation of MAPK signaling pathway was observed. These findings will provide a better understanding of the mode of action of CNTs on the copepod P. nana at cellular and molecular level and insight on possible ecotoxicological implications in the marine environment. [ABSTRACT FROM AUTHOR]

Antimicrobial peptides (AMPs) derived from histone proteins form an important category of peptide antibiotics. Present study deals with the molecular and functional characterization of a 27-amino acid histone H2A derived AMP from the Indian White shrimp, Fenneropenaeus indicus designated as Fi- Histin. This peptide displayed distinctive features of AMPs such as amphiphilic alpha helical structure and a net charge of +6. The synthetic peptide exhibited significant antimicrobial activity against Gram-negative and Gram-positive bacteria especially against V. vulnificus , P. aeruginosa, V. parahaemolyticus, V. cholera and S. aureus. Disruption of cell membrane and cell content leakage were observed in peptide treated V. vulnificus using scanning electron microscopy. The synthetic peptide Fi- His 1-21 exhibited DNA binding activity and found to be non-haemolytic at the tested concentrations. Peptide was also found to possess anticancer activity against NCI–H460 and HEp-2 cell lines with an IC 50 of 22.670 ± 13.939 μM and 31.274 ± 24.531 μM respectively. This is the first report of a histone H2A derived peptide from F. indicus with a specific antimicrobial activity and anticancer activity, which could be a new candidate for future applications in aquaculture and medicine. • A histone H2A derived AMP Fi -Histin was identified & characterized from F. indicus. • Fi- His 1-21 showed notable activity against V. vulnificus , P. aeruginosa & S. aureus. • Fi- His 1-21 treated V. vulnificus revealed an altered membrane structure. • Fi- His 1-21 exhibited anticancer activity against NCI–H460 and HEp-2 cell lines. • Fi- His 1-21 is a prospective therapeutant for bacterial infections and cancer therapy. [ABSTRACT FROM AUTHOR]

• TEPIE contains heavy load of metal pollutants. • TEPIE critically affects in vivo and biochemical endpoints in C. vulgaris. • Gradual reduction of total protein and chlorophyll contents were in a concentration (effluent) dependent manner. • Concentration-dependent increases were observed in ROS with SOD and CAT activities. The microalga Chlorella vulgaris is one of the prominent and most widely distributed green microalgae found in aquatic environments, often used in toxicity tests due to its sensitivity to various pollutants. To examine the toxicity of metals found in the effluent discharges from an electroplating industry, physicochemical parameters in the microalga C. vulgaris were measured. pH, turbidity, total dissolved solids, color, and the concentrations of metals such as chromium (1.97 mg/L), mercury (104.2 mg/L), and zinc (167.25 mg/L) were found exceeding the permissible limits. Several endpoints such as total protein content, reactive oxygen species (ROS) production, photosynthetic pigment contents, and antioxidant enzymatic activities, including those of superoxide dismutase (SOD) and catalase (CAT), were measured in C. vulgaris in response to treated electroplating industrial effluent (TEPIE). In addition, concentration-dependent morphological changes were also observed in response to TEPIE. Under both acute and chronic TEPIE exposure, increase in the ROS level was observed indicating increased production of ROS in C. vulgaris cells. The total protein and chlorophyll contents were found to be gradually decreasing in an effluent concentration-dependent manner. Moreover, lower concentrations of effluent stimulated the antioxidant enzyme systems. A concentration-dependent increase was observed in both SOD and CAT enzymatic activities. The results indicated toxic impairments by the effluent on the function of C. vulgaris in response to both acute and chronic exposure, indicating an urgent need of proper treatment processes/modification of the existing one of TEPIE, with continuous monitoring of the discharge of the pollutants into the aquatic ecosystems using biological assays. [ABSTRACT FROM AUTHOR]

In this study, Illumina Miseq sequencing of 16S rRNA gene amplicon was performed on sediments collected from Krossfjorden, Arctic for analyzing the bacterial community structure. Metagenome contained 15,936 sequences with 5,809,491 bp size and 53% G+C content. Metagenome sequence information are now available at NCBI under the Sequence Read Archive (SRA) database with accession no. SRP159159. Taxonomic hits distribution from MG-RAST analysis revealed the dominance of Alpha- and Gamma-subdivisions of Proteobacteria (88.89%) along with Bacteriodetes (8.89%) and Firmicutes (2.22%). Predominant species were Alteromonadales bacterium TW-7 (24%), Pseudoalteromonas haloplanktis (20%) and Pseudoalteromonas spp. SM9913 (18%). MG-RAST assisted analysis also detected the presence of a variety of marine taxa like Bacteriodes, Pseudovibrio, Marinobacter, Idiomarina, Teredinibacter, etc. which take part in key ecological functions and biogeochemical activities of Arctic fjord ecosystems.

Endophytic fungi have currently been acknowledged as the most promising source of bioactive compounds for drug discovery, and considerable progress has been made in exploring their diversity, species richness, and bioprospecting. Fungal endophytes from unique environmental settings offer a pool of potentially useful medicinal entities. Owing to the constant stresses imposed on macroalgae by marine environments, it is believed that algae and their associated endophytic symbionts represent a good source of structurally diverse bioactive secondary metabolites. Despite the proven significance of active metabolites of algal endophytes, little have been exploited. This review highlights the latest discoveries in algicolous endophytic research, with particular focus on the bioactive metabolites from algal endophytes. Compounds are classified according to their reported biological activities, like anticancer, antibacterial, antifungal, and antioxidant properties. Present experimental evidence suggests that a majority of the bioactive metabolites were reported from Phaeophyceae followed by Rhodophyceae and Chlorophyceae. An intensive search for newer and more effective bioactive metabolites has generated a treasure trove of publications, and this review partially covers the literature published up to 2016.

Self-assembled magnetic colloidal suspensions are sought after by material scientists owing to its huge application potential. The biomedical applications of colloidal nanoparticles necessitate that they are biocompatible, non-interacting, monodispersed and hence the synthesis of such nanostructures has great relevance in the realm of nanoscience. Silica-coated superparamagnetic iron oxide nanoparticles based ferrofluids were prepared using polyethylene glycol as carrier fluid by employing a controlled co-precipitation technique followed by a modified sol-gel synthesis. A plausible mechanism for the formation of stable suspension of SiO2-coated Iron Oxide nanoparticles with a size of about 9 nm dispersed in polyethylene glycol (PEG) is proposed. Core-shell nature of the resultant SiO2-Iron Oxide nanocomposite was verified using transmission electron microscopy. Fourier transform-infrared spectroscopy studies were carried out to understand the structure and nature of chemical bonds. The result suggests that Iron Oxide exist in an isolated state inside silica matrix. Moreover, the presence of silanol bonds establishes the hydrophilic nature of silica shell confirming the formation of stable ferrofluid with PEG as carrier fluid. The magnetic characterization reveals the superparamagnetic behavior of the nanoparticles with a rather narrow distribution of blocking temperatures. These properties are not seen in ferrofluids prepared from Iron Oxide nanoparticles without SiO2 coating. The latter suggests the successful tuning of the inter-particle interactions preventing agglomeration of nanoparticles. Cytotoxicity studies on citric acid coated water based ferrofluid and silica-coated PEG-based ferrofluid were evaluated by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium chloride assay and it shows an enhanced compatibility for silica modified nanoparticles.