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Purpose: Viruses are the largest genetic repository and most diverse host-associated replicating organisms in a wide range of ecosystems in the entire biosphere. Although playing crucial roles in numerous processes such as nutrient cycles, viral shunt, and population control within marine ecosystems, viral communities continue to be the least explored biological entities, particularly in extreme marine habitats. Therefore, details about the viral diversity and their metabolic potential from Arctic sediments has great importance. Methods: Sediment samples were collected from two Arctic fjords, Kongsfjorden and Krossfjorden, in Svalbard. Viral nucleic acids were extracted from both fjord sediments after a pre-processing step. Illumina Novaseq based shotgun sequencing was performed and bioinformatic analysis was done to explore viral communities and their functional potential. Results: An array of DNA and RNA viruses especially bacteriophages along with viruses infecting algae, plants, molluscs, shrimps, fishes, amphibians, reptiles, birds, humans, and other mammals were detected from both fjords. The double-stranded DNA viruses were the most abundant (Siphoviridae, Podoviridae, Myoviridae, and Phycodnaviridae), followed by single-stranded DNA (Inoviridae) and RNA (Retroviridae and Betaflexiviridae) viruses from both metavirome. Functional analysis explored genes encoding virus structures, enzymes for phage replication, integration and excision and proteins related to phage regulation of gene expression. Conclusions: Fjord metavirome analysis revealed the occurrence of virus groups endemic to Arctic freshwater and marine habitats along with an extra pool of unclassified or unassigned virus reads. Viruses infecting a variety of bacterial groups, and other higher trophic levels in fjord environments were explored. Functional annotation revealed the abundance of phage-related structural genes and metabolic genes. Comparative analysis revealed the abundance of dsDNA viruses from Caudovirales (Myoviridae, Podoviridae and Siphoviridae) in all the analysed Arctic samples in different proportions. The present study enhances our understanding of viral diversity and their metabolic potential in Arctic fjord sediments. [ABSTRACT FROM AUTHOR]

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]

Conventional selective breeding in aquaculture has been effective in genetically enhancing economic traits like growth and disease resistance. However, its advances are restricted by heritability, the extended period required to produce a strain with desirable traits, and the necessity to target multiple characteristics simultaneously in the breeding programs. Genome editing tools like zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) are promising for faster genetic improvement in fishes. CRISPR/Cas9 technology is the least expensive, most precise, and well compatible with multiplexing of all genome editing approaches, making it a productive and highly targeted approach for developing customized fish strains with specified characteristics. As a result, the use of CRISPR/Cas9 technology in aquaculture is rapidly growing, with the main traits researched being reproduction and development, growth, pigmentation, disease resistance, trans-GFP utilization, and omega-3 metabolism. However, technological obstacles, such as off-target effects, ancestral genome duplication, and mosaicism in founder population, need to be addressed to achieve sustainable fish production. Furthermore, present regulatory and risk assessment frameworks are inadequate to address the technical hurdles of CRISPR/Cas9, even though public and regulatory approval is critical to commercializing novel technology products. In this review, we examine the potential of CRISPR/Cas9 technology for the genetic improvement of edible fish, the technical, ethical, and socio-economic challenges to using it in fish species, and its future scope for sustainable fish production. [ABSTRACT FROM AUTHOR]

Despite the attempts that have started since the 1960s, not even a single cell line of marine molluscs is available. Considering the vast contribution of marine bivalve aquaculture to the world economy, the prevailing viral threats, and the dismaying lack of advancements in molluscan virology, the requirement of a marine molluscan cell line is indispensable. This synthetic review discusses the obstacles in developing a marine molluscan cell line concerning the choice of species, the selection of tissue and decontamination, and cell culture media, with emphasis given on the current decade 2010–2020. Detailed accounts on the experiments on the virus cultivation in vitro and molluscan cell immortalization, with a brief note on the history and applications of the molluscan cell culture, are elucidated to give a holistic picture of the current status and future trends in molluscan cell line development. [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]

The rapid growth of industrialization and urbanization results in deterioration of freshwater systems around the world, rescinding the ecological balance. Among many factors that lead to adverse effects in aquatic ecology, metals are frequently discharged into aquatic ecosystems from natural and anthropogenic sources. Metals are highly persistent and toxic substances in trace amounts and can potentially induce severe oxidative stress in aquatic organisms. In this study, adverse effects of the two metal elements zinc (maximum concentration of 167.25 mg/L) and mercury (104.2 mg/L) were examined using Chlorella vulgaris under acute and chronic exposure period (48 h and 7 days, respectively). The metal-induced adverse effects have been analyzed through photosynthetic pigment content, total protein content, reactive oxygen species (ROS) generation, antioxidant enzymatic activities, namely catalase and superoxide dismutase (SOD) along with morphological changes in C. vulgaris. Photosynthetic pigments were gradually reduced (~32–100% reduction) in a dose-dependent manner. Protein content was initially increased during acute (~8–12%) and chronic (~57–80%) exposure and decreased (~44–56%) at higher concentration of the two metals (80%). Under the two metal exposures, 5- to 7-fold increase in ROS generation indicated the induction of oxidative stress and subsequent modulations in antioxidant activities. SOD activity was varied with an initial increase (58–129%) followed by a gradual reduction (~3.7–79%), while ~1- to 12-fold difference in CAT activity was observed in all experimental condition (~83 to 1605%). A significant difference was observed in combined toxic exposure (Zn+Hg), while comparing the toxic endpoint data of individual metal exposure (Zn and Hg alone). Through this work, lethal effects caused by single and combined toxicity of zinc and mercury were assessed, representing the significance of appropriate monitoring system to trim down the release of metal contaminants into the aquatic ecosystems. [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]

We have used the cyclopoid copepod Paracyclopina nana to understand responses to the temperature shift. To determine the changes of molecular mechanisms, which would lead to physiological changes in P. nana, we investigated mRNA expression involved in lipogenesis, the area of lipid droplets, the ratio of fatty acids, and life parameters (growth and fecundity) in response to temperature changes (15, 20, and 30 °C) with comparative control at 25 °C. Setting the temperature 25 °C as a standard point, there were increases in mRNA expression, the area of the lipid droplets, and fatty acid composition at temperatures below the standard (15 °C), while all the markers mentioned above significantly decreased at higher temperatures than the standard (25 °C). Through fecundity and growth rate experiments, daily nauplii production was reduced and growth retardation was observed at both 15 and 20 °C, but no noticeable differences in the two parameters were observed at 30 °C compared to the control (25 °C). This study provides a better understanding of the effects of temperature on lipogenesis in copepods. [ABSTRACT FROM AUTHOR]

It has been realized that shrimp cell immortalization may not be accomplished without in vitro transformation by expressing immortalizing gene in cells. In this process, efficiency of transgene expression is confined to the ability of vectors to transmit gene of interests to the genome. Over the years, unavailability of such vectors has been hampering application of such a strategy in shrimp cells. We report the use of recombinant baculovirus mediated transduction using hybrid promoter system for transgene expression in lymphoid cells of Penaeus monodon. Two recombinant baculovirus vectors with shrimp viral promoters (WSSV-Ie1 and IHHNV-P2) were constructed (BacIe1-GFP and BacP2-GFP) and green fluorescent protein (GFP) used as the transgene. The GFP expression in cells under the control of hybrid promoters, PH-Ie1 or PH-P2, were analyzed and confirmed in shrimp cells. The results indicate that the recombinant baculovirus with shrimp specific viral promoters (hybrid) can be employed for delivery of foreign genes to shrimp cells for in vitro transformation. [ABSTRACT FROM AUTHOR]