Decoding the molecular phylogenetics of ornamental catfishes (siluriformes) of North East India using DNA barcoding approach.

BACKGROUND: Catfishes (order Siluriformes) are among the most diverse and widely distributed fish groups in the world. They are not only used for human consumption but are also a major part of the ornamental fish trade. Being a Biodiversity Hotspot, the North Eastern Region of India is home to a diverse population of ornamental fishes. Catfishes contain a humongous number of species; in this study, the authors have tried to elucidate the phylogenetic relationship of some important ornamental catfishes found in North East India using DNA barcodes. METHODS AND RESULTS: In this study, we have tried to explore the phylogenetic history of 13 species (41 specimens) of ornamental catfishes spanning 12 genera and 9 families of Siluriformes using DNA barcoding. Pairwise genetic distances using Kimura 2-Parameter (K2P) were calculated at intra-specific and inter-specific levels. A Neighbor-Joining tree was constructed to understand the phylogenetic relationship among the nine different catfish families. All the specimens under this study clustered with their respective species under the same family and formed three sub-clades. However, Olyra longicaudata, belonging to the Bagridae family, did not cluster with other species from the same family. In this study, the authors have suggested a revision of the classification of O. longicaudata back to its original family, Olyridae. CONCLUSIONS: In this study, the maximum intraspecific genetic distance of 0.03 and the minimum interspecific genetic distance of 0.14 were observed among the species. Therefore, it is evident that there is a barcoding gap among the species, which helped in the correct identification of the species. Thus, DNA barcoding helped complement the phenetic approach and also revealed a different phylogenetic relationship among the catfishes belonging to the Bagridae family.

Proteomics analysis of differentially abundant proteins in the rohu kidney infected with Edwardsiella tarda.

Edwardsiella tarda (Et) is a zoonotic gram-negative pathogen with a diverse host range, including fish. However, the in-depth molecular mechanisms underlying the response of Labeo rohita (rohu) kidney to Et are poorly understood. A proteomic and histopathological analysis was performed for the rohu kidney after Et infection. The histopathology of the infected rohu kidney showed vacuolation and necrosis. After LC-MS/MS analysis, ~1240 proteins were identified with ≥2 unique peptides. A total of 96 differentially abundant proteins (DAPs) were observed between the control and Et infected group (ET). Metascape and STRING analysis were used for the gene ontology (GO), and protein-protein interaction network (PPI) for the significant pathways of DAPs. In PPI, low-abundant proteins were mapped to metabolic pathways and oxidative phosphorylation (cox5ab, uqcrfs1). High-abundance proteins were mapped to ribosomes (rplp2), protein process in the ER (hspa8), and immune system (ptgdsb.1, muc2). Our label-free proteomic approach in the rohu kidney revealed abundant enriched proteins involved in vesicle coat (ehd4), complement activation (c3a.1, c9, c7a), phagosome (thbs4, mapk1), metabolic reprogramming (hao1, glud1a), wound healing (vim, alox5), and the immune system (psap) after Et infection. A targeted proteomics approach of multiple reaction monitoring (MRM) validated the DAPs (nprl3, ambp, vmo1a, hspg2, muc2, hao1 and glud1a) between control and ET. Overall, the current analysis of histology and proteome in the rohu kidney provides comprehensive data on pathogenicity and the potential immune proteins against Et.

Development and characterization of a new muscle cell culture system from Clarias magur (Hamilton, 1822).

The cell line has been used as a novel in vitro tool for executing several studies in life sciences. The current study aimed to develop and characterize a muscle cell culture system derived from Clarias magur. The primary muscle cell cultures derived from the caudal peduncle muscle have been successfully sub cultured up to 13 passages to establish a new muscle cell culture system known as CMM. At a temperature of 28 °C, L-15 medium supplemented with 20% FBS produced the maximum growth of muscle cells. However, muscle cells were optimized to grow at 10% FBS. To enhance the proliferation capacity of the CMM cells, a growth-promoting factor bFGF (10 ng/ml) was added, thereby reducing the time interval of passages for the subsequent cultures. DNA barcoding of the CMM cell culture system authenticated the species of origin. The cell culture system was successfully cryopreserved by a slow freezing procedure at - 80 °C with a revival efficiency of 60%.

Development of a cell line from skeletal trunk muscle of the fish Labeo rohita.

Labeo rohita is a widely cultivated tropical freshwater carp and found in rivers of South Asian region. A new cell line, designated LRM, has been developed from the muscle tissue of L. rohita. Muscle cells were subcultured up to 38 passages in a Leibovitz's-15 (L-15) supplemented with 10% FBS (Fetal Bovine Serum) and 10 ng/ml bFGF. The LRM cells exhibited fibroblastic morphology with a doubling time of 28 h, and a plating efficiency of 17%. A maximum growth rate was observed for LRM cells at 28 °C, 10% FBS and 10 ng/ml bFGF. A cytochrome C oxidase subunit I (COI) gene sequence was used to authenticate the developed cell line. Chromosome analysis revealed 50 diploid chromosomes. The fibroblastic characteristics of the of the LRM cells was confirmed by immunocytochemistry. The expression of MyoD gene in LRM cells was analyzed by quantitative PCR in comparison with passages 3, 18 and 32. The expression of MyoD was higher at passage 18 compared to the passages 3 and 32. The LRM cells attached properly onto the 2D scaffold and the expression of the F-actin filament protein was confirmed by phalloidin staining followed by counter staining with DAPI to observe the distribution of the muscle cell nuclei and the cytoskeleton protein. A revival rate of 70-80% was achieved when the LRM cells were cryopreserved at - 196 °C using liquid nitrogen. This study would further contribute to understanding the in vitro myogenesis and progress toward cultivated fish meat production.

Functional characterization of Labeo rohita muscle cell line for in vitro research.

BACKGROUND: Labeo rohita represents the most dominant fish species in Indian aquaculture and the fish cell lines have been used as an excellent in vitro platform for performing various biological research. METHODS AND RESULTS: The LRM cell culture developed from the muscle tissue of L. rohita was used to study the in vitro applications. The developed muscle cells were maintained in a Leibovitz's-15 (L-15) supplemented with 10% FBS (Fetal Bovine Serum) and 10 ng/ml bFGF at 28 oC temperature. The LRM cells showed fibroblastic-like morphology and was authenticated by sequencing mitochondrial gene 16S rRNA. The expression of myogenic regulatory factors (MRFs) was studied in different stages of LRM cells; however, the expression patterns varied at different passages. The MEF2A, Mrf-4, and Myogenin expressions were higher in passage 25, while the expression of MyoD was maximum in passage 15, and the expression of Myf-5 was highest in passage 1. The transfection efficiency of LRM cells revealed 14 % of the GFP expression with a pmaxGFP vector DNA. The LRM cells were susceptible to the extracellular products prepared from Aeromonas hydrophilla and Edwardsiella tarda. The acute cytotoxicity of six heavy metals (Hg, Cd, Zn, Cu, Pb, Ni) was assessed in LRM cells by a dose-dependent manner in comparison to IC50 values obtained from MTT and NR assays. A revival rate of 70-75% was achieved when the LRM cells were cryopreserved at - 196 °C using liquid nitrogen. CONCLUSION: The developed muscle cells serve as an functional in vitro tool for toxicological and biotechnological studies.

Proteomic analysis of liver tissue reveals Aeromonas hydrophila infection mediated modulation of host metabolic pathways in Labeo rohita.

Aeromonas hydrophila (Ah) is a Gram-negative bacterium and a serious global pathogen causing Motile Aeromonas Septicaemia (MAS) in fish leading to global loss in aquaculture. Investigation of the molecular alterations of host tissues such as liver could be a powerful approach to identify mechanistic and diagnostic immune signatures of disease pathogenesis. We performed a proteomic analysis of Labeo rohita liver tissue to examine the protein dynamics in the host cells during Ah infection. The proteomic data was acquired using two strategies; discovery and targeted proteomics. Label-free quantification was performed between Control and challenged group (AH) to identify the differentially expressed proteins (DEPs). A total of 2525 proteins were identified and 157 were DEPs. DEPs include metabolic enzymes (CS, SUCLG2), antioxidative proteins, cytoskeletal proteins and immune related proteins (TLR3, CLEC4E). Pathways like lysosome pathway, apoptosis, metabolism of xenobiotics by cytochrome P450 were enriched by downregulated proteins. However, upregulated proteins majorly mapped to innate immune system, signaling of B cell receptor, proteosome pathway, ribosome, carbon metabolism and protein processing in ER. Our study would help in exploring the role of Toll-like receptors, C-type lectins and, metabolic intermediates like citrate and succinate in Ah pathogenesis to understand the Ah infection in fish. SIGNIFICANCE: Bacterial diseases such as motile aeromonas septicaemia (MAS) are among the most serious problems in aquaculture industry. Small molecules that target the metabolism of the host have recently emerged as potential treatment possibilities in infectious diseases. However, the ability to develop new therapies is hampered due to lack of knowledge about pathogenesis mechanisms and host-pathogen interactions. We examined alterations in the host proteome during MAS caused by Aeromonas hydrophila (Ah) infection, in Labeo rohita liver tissue to find cellular proteins and processes affected by Ah infection. Upregulated proteins belong to innate immune system, signaling of B cell receptor, proteosome pathway, ribosome, carbon metabolism and protein processing. Our work is an important step towards leveraging host metabolism in targeting the disease by providing a bigger picture on proteome pathology correlation during Ah infection.

Establishment and characterization of a new fibroblast-like cell line from the skin of a vertebrate model, zebrafish (Danio rerio).

BACKGROUND: The available fully sequenced genome and genetic similarities compared to humans make zebrafish a prominent in vitro vertebrate model for drug discovery & screening, toxicology, and radiation biology. Zebrafish also possess well developed immune systems which is ideal for studying infectious diseases. Fish skin confers immunity by serving as a physical barrier against the invading pathogens in the aquatic habitat. Therefore in vitro models from the skin tissue of zebrafish help to study the physiology, functional genes in vitro, wound healing, and pathogenicity of microbes. Hence the study aimed to develop and characterize a skin cell line from the wild-type zebrafish Danio rerio. METHODS AND RESULTS: A novel cell line designated as DRS (D. rerio skin) was established and characterized from the skin tissue of wild-type zebrafish, D. rerio, by the explant technique. The cells thrived well in the Leibovitz's -15 medium supplemented with 15% FBS and routinely passaged at regular intervals. The DRS cells mainly feature fibroblast-like morphology. The culture conditions of the cells were determined by incubating the cells at varying concentrations of FBS and temperature; the optimum was 15% FBS and 28 °C, respectively. Cells were cryopreserved and revived with 70-75% viability at different passage levels. Two extracellular products from bacterial species Aeromonas hydrophila and Edwardsiella tarda were tested and found toxic to the DRS cells. Mitochondrial genes, namely COI and 16S rRNA PCR amplification and partial sequencing authenticated the species of origin of cells. The modal diploid (2n) chromosome number of the cells was 50. The cell line DRS was found to be free from mycoplasma. The cells were transfected with pMaxGFP plasmid and tested positive for green fluorescence at 24-48 h post-transfection. CONCLUSION: The findings from this study thus confirm the usefulness of the developed cell line in bacterial susceptibility and transgene expression studies.

Ecological Monitoring and Omics: A Comprehensive Comparison of Workflows for Mass Spectrometry-Based Quantitative Proteomics of Fish (Labeo rohita) Liver Tissue.

Introduction: The liver is highly sensitive to the environmental factors. Liver tissue, particularly from fish, is often used as a biological target in ecological monitoring, disease research, and stress response studies. Labeo rohita (rohu) is a fish with a significant role in the global aquaculture economy. Methods: Bottom-up proteomics relies on efficient sample preparation for performing mass spectrometric analysis of the liver tissue. Optimization of protein solubilization and digestion strategies is the key step to obtain reliable data for a successful proteomics experiment. Because the goal of extraction is to acquire the optimum protein quality and yield, the first step should be to choose an appropriate extraction method based on the type of sample. Solubilization buffers containing sodium dodecyl sulfate (SDS) or urea, and digestion methods such as filter-aided sample preparation (FASP), suspension trap (S-Trap) and in-solution are often used in proteomics but are in need of comparative evaluation with an eye to protocol optimization. Experiment: We applied two different solubilization buffers (one containing SDS, and other containing urea) and three digestion methods (FASP, S-Trap, and in-solution) to the proteomic analysis of the fish (L. rohita) liver tissue. Label-free quantification analysis was performed to analyze the similarities and differences in the results with each method. Gene ontology-based functional analysis was performed for the identified proteome across the experimental conditions to overview their protein classes, molecular functions, and biological processes. Results: SDS lysis followed by S-Trap digestion outperformed the other combinations of lysis and digestion in terms of higher protein coverage, consistency in the results and repeatability. Filter-based methods provided comparatively better results than in-solution digestion. Discussion: This protocol presents new insights on ways to optimize discovery and targeted proteomic analyses of liver tissue using the fish L. rohita as a case study. Other tissues can also be evaluated in the future drawing from the results in this study. This would help the scientific community with hypothesis-driven studies on topics ranging from basic biology to applied aquaculture research and ecological monitoring. This is particularly relevant in the current era of ecological crises and environmental pollution, where advances and optimization in research protocols can contribute to in-depth studies of ecosystems and planetary health.

Cellular Aquaculture: Prospects and Challenges.

Aquaculture plays an important role as one of the fastest-growing food-producing sectors in global food and nutritional security. Demand for animal protein in the form of fish has been increasing tremendously. Aquaculture faces many challenges to produce quality fish for the burgeoning world population. Cellular aquaculture can provide an alternative, climate-resilient food production system to produce quality fish. Potential applications of fish muscle cell lines in cellular aquaculture have raised the importance of developing and characterizing these cell lines. In vitro models, such as the mouse C2C12 cell line, have been extremely useful for expanding knowledge about molecular mechanisms of muscle growth and differentiation in mammals. Such studies are in an infancy stage in teleost due to the unavailability of equivalent permanent muscle cell lines, except a few fish muscle cell lines that have not yet been used for cellular aquaculture. The Prospect of cell-based aquaculture relies on the development of appropriate muscle cells, optimization of cell conditions, and mass production of cells in bioreactors. Hence, it is required to develop and characterize fish muscle cell lines along with their cryopreservation in cell line repositories and production of ideal mass cells in suitably designed bioreactors to overcome current cellular aquaculture challenges.

The PeptideAtlas of a widely cultivated fish Labeo rohita: A resource for the Aquaculture Community.

Labeo rohita (Rohu) is one of the most important fish species produced in world aquaculture. Integrative omics research provides a strong platform to understand the basic biology and translate this knowledge into sustainable solutions in tackling disease outbreak, increasing productivity and ensuring food security. Mass spectrometry-based proteomics has provided insights to understand the biology in a new direction. Very little proteomics work has been done on 'Rohu' limiting such resources for the aquaculture community. Here, we utilised an extensive mass spectrometry based proteomic profiling data of 17 histologically normal tissues, plasma and embryo of Rohu to develop an open source PeptideAtlas. The current build of "Rohu PeptideAtlas" has mass-spectrometric evidence for 6015 high confidence canonical proteins at 1% false discovery rate, 2.9 million PSMs and ~150 thousand peptides. This is the first open-source proteomics repository for an aquaculture species. The 'Rohu PeptideAtlas' would promote basic and applied aquaculture research to address the most critical challenge of ensuring nutritional security for a growing population.

Organ-Based Proteome and Post-Translational Modification Profiling of a Widely Cultivated Tropical Water Fish, Labeo rohita.

Proteomics has enormous applications in human and animal research. However, proteomic studies in fisheries science are quite scanty particularly for economically important species. Few proteomic studies have been carried out in model fish species, but comprehensive proteomics of aquaculture species are still scarce. This study aimed to perform a comprehensive organ-based protein profiling of important tissue samples for one of the most important aquaculture species,Labeo rohita.Deep proteomic profiling of 17 histologically normal tissues, blood plasma, and embryo provided mass-spectrometric evidence for 8498 proteins at 1% false discovery rate that make up about 26% of the total annotated protein-coding sequences in Rohu. Tissue-wise expression analysis was performed, and the presence of several biologically important proteins was also verified using a targeted proteomic approach. We identified the global post-translational modifications (PTMs) in terms of acetylation (N-terminus and lysine), methylation (N-terminus, lysine, and arginine), and phosphorylation (serine, threonine, and tyrosine) to present a comprehensive proteome resource. An interactive web-based portal has been developed for an overall landscape of protein expression across the studied tissues of Labeo rohita (www.fishprot.org). This draft proteome map of Labeo rohita would advance basic and applied research in aquaculture to meet the most critical challenge of providing food and nutritional security to an increasing world population.

Functional Aspects of Fish Mucosal Lectins-Interaction with Non-Self.

Mucosal surfaces are of key importance in protecting animals against external threats including pathogens. In the mucosal surfaces, host molecules interact with non-self to prevent infection and disease. Interestingly, both inhibition and stimulation of uptake hinder infection. In this review, the current knowledgebase on teleost mucosal lectins' ability to interact with non-self is summarised with a focus on agglutination, growth inhibition, opsonisation, cell adhesion, and direct killing activities. Further research on lectins is essential, both to understand the immune system of fishes, since they rely more on the innate immune system than mammals, and also to explore these molecules' antibiotic and antiparasitic activities against veterinary and human pathogens.