Comparative analysis of gut microbiome in Pangasionodon hypopthalmus and Labeo catla during health and disease.

The present study was conducted to study the composition of gut microbiome in the advanced fingerling and fingerling stage of striped pangasius catfish and catla during healthy and diseased conditions. Healthy pangasius and catla fishes were obtained from commercial farms and injected with the LD50 dose of A. hydrophila. The intestinal samples from the control and injected group were collected and pooled for 16 s metagenomic analysis. Community analysis was performed by targeting the 16 s rRNA gene to explore and compare the gut microbiota composition of these fishes. The operational taxonomic units (OTUs) consisted of four major phyla: Bacteroidia, Proteobacteria, Firmicutes, and Actinobacteria. Alpha and beta diversity indices were carried out to understand the diversity of microbes within and between a sample. While comparing the advanced fingerling and fingerling stage gut microbiome of Pangasius catfish, the dominance of Proteobacteria was found in fingerlings, whereas Firmicutes and Bacteroides were found in advanced fingerlings. In catla, Proteobacteria and Bacteroides were predominant. Taxonomic abundance of the microbiota in control and diseased Pangasius and catla fishes at phylum, class, order, family, genus, and species levels were also depicted. The present study is the first of its kind, and it will help to identify the diversity of novel potential bacterial species involved in disease protection of fishes. It can lead to the development of sustainable prophylactic measures against (re-)emerging bacterial diseases in aquaculture.

Insights into genomic variations in rice Hsp100 genes across diverse rice accessions.

MAIN CONCLUSION: The Hsp101 gene is present across all sequenced rice genomes. However, as against Japonica rice, Hsp101 protein of most indica and aus rice contain insertion of glutamic acid at 907th position. The understanding of the heat stress response of rice plants is important for worldwide food security. We examined the presence/absence variations (PAVs) of heat shock proteins (Hsps)/heat shock transcription factor (Hsf) genes in cultivated rice accessions. While 53 Hsps/Hsfs genes showed variable extent of PAVs, 194 genes were the core genes present in all the rice accessions. ClpB1/Hsp101 gene, which is critically important for thermotolerance in plants, showed 100% distribution across the rice types. Within the ClpB1 gene sequence, 40 variation sites consisting of nucleotide polymorphisms (SNPs) and short insertion/deletions (InDels) were discerned. An InDel in ClpB1 leading to an in-frame insertion of 3 nucleotides (TCC) thereby an additional amino acid (glutamic acid) at 907th amino acid position was noted in most of the indica and aus as against japonica rice types. Three rice types namely Moroberekan (japonica), IR64 (indica) and N22 (aus) were further analyzed to address the question of ClpB1 genomic variations and its protein levels with the heat tolerance phenotype. The growth profiling analysis in the post heat stress (HS) period showed that N22 seedlings were most tolerant, IR64 moderately tolerant and Moroberekan highly sensitive. Importantly, the ClpB1 protein sequences of these three rice types showed distinct differences in terms of SNPs. As the ClpB1 protein levels accumulated post HS were generally higher in Moroberekan than N22 seedlings in our study, it is proposed that some additional gene loci in conjunction with ClpB1 regulate the overall rice heat stress response.

Tools and techniques for rational designing of antimicrobial peptides for aquaculture.

Fisheries and aquaculture industries remain essential sources of food and nutrition for millions of people worldwide. Indiscriminate use of antibiotics has led to the emergence of antimicrobial-resistant bacteria and posed a severe threat to public health. Researchers have opined that antimicrobial peptides (AMPs) can be the best possible alternative to curb the rising tide of antimicrobial resistance in aquaculture. AMPs may also help to achieve the objectives of one health approach. The natural AMPs are associated with several shortcomings, like less in vivo stability, toxicity to host cell, high cost of production and low potency in a biological system. In this review, we have provided a comprehensive outline about the strategies for designing synthetic mimics of natural AMPs with high potency. Moreover, the freely available AMP databases and the information about the molecular docking tools are enlisted. We also provided in silico template for rationally designing the AMPs from fish piscidins or other peptides. The rationally designed piscidin (rP1 and rp2) may be used to tackle microbial infections in aquaculture. Further, the protocol can be used to develop the truncated mimics of natural AMPs having more potency and protease stability.

Lethal dose and histopathological alterations induced by Aeromonas salmonicida in experimentally challenged common carp, Cyprinus carpio.

Aeromonas salmonicida is the obligate pathogen of fishes having zoonotic potential. It is reported to cause considerable losses in world aquaculture. The current study has successfully demonstrated the induction of histopathological lesions in experimentally infected common carp. In the current study, the lethal concentration (LD50-96 h) of typical A. Salmonicida for common carp was found to be 1.5 × 107CFU mL-1. About 40% and 60% fish mortalities occurred after 72 h in the groups inoculated with 107 and 108 CFU mL-1 bacterial suspension, respectively. The fish challenged with A. salmonicida showed symptoms like abnormal swimming behaviour, lethargy, intra-abdominal fluid, haemorrhages on the ventral side of the body, vent and fins. The signs proceeded with the death of fish. In the histological sections, severe pathological alterations were reported in the tissue sections of internal organs. The microscopic observation showed sinusoidal and large blood vessel congestion in the liver, profuse haemorrhage, necrosis and infiltration of blood cells in the internal organs. The tubular architecture was lost with the infiltration of leucocytes in the kidney. In gills, more intense and prominent lamellar fusion was observed with leucocytic infiltration, telangiectasia and hyperplasia of lamellar epithelial cells. In summary, we have experimentally induced the typical A. salmonicida infection in common carp. The study will provide a research foundation for further studies on the host-pathogen interaction, therapeutics and epidemiology of A. salmonicida.

Development of species-specific IgM antibodies and elevation of mucosal immune response in Labeo rohita using recombinant bicistronic nano DNA vaccine priming.

Immunoglobulin (IgM) is the primary immunoglobulin essential for defense mechanisms in fish. It is difficult to reliably quantify IgM because a lack of standardization in methodology and limited availability of commercially reagents. In the present study, a polyclonal antibody was developed for the specific detection and quantification of IgM in Labeo rohita. Recombinant bicistronic NanoDNA plasmid (RBND Vac) encoding the glyceraldehyde-3-phosphate dehydrogenase gene of Edwarsiella tarda conjugated with poly (lactic-co-glycolic acid) - Chitosan (PLGA-Chit) was developed and its potential as a DNA vaccine, to prevent the infection of E. tarda in L. rohita was investigated. Two treatment groups [T1 - (PLGA-Chit-NPs-pDNA), T2 - (PLGA-NPs-pDNA) and one control group (T0 - 1 × PBS)] were utilized. Polyclonal antibody was developed to estimate IgM titers in the serum and mucosal associated tissues (MAT) using Enzyme-linked Immunosorbent Assay (ELISA) technique. Additionally, immune gene expression was studied using qRT-PCR. Vaccinated groups also exhibited a significant increase in the total serum protein, globulin concentration and relatively less mortality was observed in T1 group. IgM level in serum and mucosal tissues (skin, gill and gut) increased significantly days post vaccination compared to control group, also non-specific immune parameters (myeloperoxidase and lysozyme levels) showed significant improvement in vaccinated fish. Furthermore, histopathological examination confirmed minor damage in physiological structure of kidney and liver tissues in vaccinated fish. Knowledge of the immunoglobulin in L. rohita primed with RBND Vac complex provides the better protection against E. tarda. The normal physiology findings of this study will aid in monitoring changes in the health status of fish, when the animals undergo vaccination by immersion method.

A novel myxozoan parasite, Ellipsomyxa boleophthalmi sp. nov. (Myxozoa: Ceratomyxidae) in the brackishwater fish, Boleophthalmus dussumieri Valenciennes, 1837 (Perciformes: Gobiidae) from India.

A novel myxozoan parasite is identified and described from mudskipper, Boleophthalmus dussumieri, collected from a brackishwater ecosystem in Maharashtra, India. Ellipsomyxa boleophthalmi sp. nov. was found in the gallbladder of 58 of 60 fish examined (96.7%). The parasite formed disporous plasmodia that varied in size and shape, and the thin-walled, ellipsoidal and elongated myxospores measured 9.0-10.7 × 6.0-7.8 µm. The two, spherical polar capsules measured 2.7 µm in diameter and enclosed 3-4 coils of polar tubules. Histological observations of infected gallbladder revealed the attachment of disporous plasmodial stages of the parasite to the gallbladder wall with fine pseudopodia. Under the scanning electron microscope (SEM), the myxospores showed a distinct central sutural line and two distinct depressions on the opposite sides at the openings of polar capsules. SEM also revealed the engulfment of microvilli of gallbladder wall by pseudopodia of the plasmodial stages. Analysis of the partial fragment of the SSU rDNA region (1386 bp) showed less than 98% sequence similarity with the other reported Ellipsomyxa spp. In the phylogenetic tree, the present species formed as a distinct subclade within the major clade of Ellipsomyxa spp. The unique morphological and morphometric features of the myxospore, together with the molecular analysis, allowed us to conclude that the present myxozoan is a new species and is named Ellipsomyxa boleophthalmi sp. nov., after the generic name of the host. This is the first report on the occurrence of the genus Ellipsomyxa in B. dussumieri.

Antibacterial and antioomycete activities of a novel designed RY12WY peptide against fish pathogens.

In the present study, we have designed and synthesized a short compositionally simple peptide RY12WY having potent antimicrobial activity. The molecular docking study results showed that peptide has a strong affinity towards two protein targets of A. sobria; aerolysin and outer membrane protein (OMP). The MIC values ranged from 0.98 to 500 µM and MBC values ranged from 4 to 650 µM against the selected bacterial and fungal pathogens. The intense antimicrobial activity of RY12WY is reported against A. sobria, A. hydrophila, E. tarda, S. aureus, V. parahaemolyticus, P. aeruginosa and E.coli at low concentration.The peptide also showed good activity against A. salmonicida and S. parasitica zoospores. The peptide retained its antimicrobial activity at higher temperatures. Besides, it was active in the presence of physiological salts and serum.The peptide showed negligible haemolytic activity at 125 µM and HC50 was found to be 1437.10 µM. The DNA binding assay indicated that peptide can bind with the genetic material of the bacteria and may inhibit its replication. The bacterial viability assay reported that the peptide interferes with bacterial membrane integrity. To conclude, the results suggest that RY12WY could be a promising therapeutic agent in aquaculture and has possible application in food processing industry which warrants higher temperatures.

Tissue specific expression profile of some immune related genes in Labeo rohita to Edwardsiella tarda infection.

Rohu (Labeo rohita), an Indian Major Carp (IMC) is an economically important aquaculture species in India. Inspite of the technological advances, infectious diseases caused by viruses, bacteria and parasites have been a major limiting factor in the development and profitability of fish farms. At present, information regarding the immune status of the Indian major carps is limited. This lack of knowledge is a major impediment for establishment of effective preventive measures against broad spectrum of infectious agents. The present study was undertaken to examine the modulation of few immune-regulatory genes: IgHC, NOD 1, TLR 22, iNOS and IL-1ß during experimental infection of E. tarda in L. rohita to understand their role in pathogenesis. Rohu fingerlings were intra-peritoneally injected with Edwardsiella tarda (LD50 dose of 8.7 × 104 CFU/fish) and sampled for three immunologically important organs (kidney, liver and spleen) at different time intervals (zero hour or pre-challenge and 6 h, 12 h, 24 h, 48 h and 96 h post challenge). For absolute quantification of genes by real time RT-PCR, all the genes transcript were amplified from Poly I:C induced rohu lymphocytes and cloned in pTZ57R/T plasmid. Standard curves for each gene was generated from serially diluted plasmid bearing respective genes. Evaluation of copy number of different genes present in the tissue showed that the expression of IgHC, iNOS and IL-1ß was highest in kidney followed by spleen and least in liver. While for NOD 1 and TLR 22 gene, liver showed higher expression than kidney and spleen. Further, the expression of IgHC, INOS, TLR 22, NOD 1 and IL-1ß genes significantly differed (P < 0.05) in the E. tarda challenged fish when compared with pre-challenged control fish. Among the five genes we studied, the basal expression of TLR 22 gene was highest. The result also depicts that iNOS and NOD 1 are immediate responsive genes as their expression reached maximum level at 6-24 h post infection (hpi) after which the expression declined. In contrast, TLR 22 and IgHC gene transcript showed enhanced expression during the late phase of with maximum expression observed after 48 hpi and 96 hpi respectively. IL-1ß, being the exception, showed high expression both at 24 hpi and 96 hpi. From this study, we conclude that these five immune genes have a definite role to play in the defense mechanism of host (L. rohita) against E. tarda.

Studies on expression pattern of toll-like receptor 5 (TLR5) in Edwardsiella tarda infected Pangasianodon hypophthalmus.

TLR5 is one of the important PRR (pathogen recognition receptors) and plays a fundamental role in pathogen recognition and activation of innate immune responses. It recognizes bacterial flagellin and stimulates the production of proinflammatory cytokines, through signalling via the adaptor protein MyD88. In this study, we characterized partial TLR5 (soluble form) gene from Pangasianodon hypophthalmus and analysed its expression profile upon challenge by Edwardsiella tarda. Bioinformatic analysis of gene sequence revealed a putative protein of 266 amino acids with four Leucine rich repeats. Quantitative expression analysis of TLR 5S showed its wide distribution in various organs and tissues. However, significant expression of TLR5S was observed in liver and spleen at 12 h (∼207.8 fold, p < 0.05). Significant upregulation was observed in kidney at 72 h.p.i. (50 folds, p < 0.05) indicating that the kidney provides longer protection almost till the activation of the adaptive immune system. This study enriches the knowledge of TLR5S in boosting the innate immunity against bacterial invasion in fish.

Cellular metabolic, stress, and histological response on exposure to acute toxicity of endosulfan in tilapia (Oreochromis mossambicus).

Endosulfan is one of the most hazardous organochlorines pesticides responsible for environmental pollution, as it is very persistent and shows bio-magnification. This study evaluated the impact of acute endosulfan toxicity on metabolic enzymes, lysozyme activities, heat shock protein (Hsp) 70 expression, and histopathology in Tilapia (Oreochromis mossambicus). Among the indicators that were induced in dose dependent manner were the enzymes of amino acid metabolism (serum alanine aminotransferase and aspartate aminotransferase), carbohydrate metabolism (serum lactate dehydrogenase), pentose phosphate pathway (Glucose-6-phosphate dehydrogenase) as well as lysozyme and Hsp70 in liver and gill, while liver and gill Isocitrate dehydrogenase (TCA cycle enzyme) and marker of general energetics (Total adenosine triphosphatase) were inhibited. Histopathological alterations in gill were clubbing of secondary gill lamellae, marked hyperplasia, complete loss of secondary lamellae and atrophy of primary gill filaments. Whereas in liver, swollen hepatocyte, and degeneration with loss of cellular boundaries were distinctly noticed. Overall results clearly demonstrated the unbalanced metabolism and damage of the vital organs like liver and gill in Tilapia due to acute endosulfan exposure.

Expression studies on NA+/K(+)-ATPase in gills of Penaeus monodon (Fabricius) acclimated to different salinities.

The decapod crustacean Penaeus monodon survives large fluctuations in salinity through osmoregulation in which Na+/K(+)-ATPase (NKA) activity in the gills plays a central role. Adult P. monodon specimens were gradually acclimatized to 5, 25 and 35 per thousand salinities and maintained for 20 days to observe long-term alterations in NKA expression. Specific NKA activity assayed in gill tissues was found to be 3 folds higher at 5 per thousand compared to 25 per thousand (isosmotic salinity) and 0.48 folds lower at 35 per thousand. The enzyme was immunolocalized in gills using mouse α-5 monoclonal antibody that cross reacts with P. monodon NKA α-subunit. At 5 per thousand the immunopositive cells were distributed on lamellar tips and basal lamellar epithelium of the secondary gill filaments and their number was visibly higher. At both 25 per thousand and 35 per thousand NKA positive cells were observed in the inter-lamellar region but the expression was more pronounced at 25 per thousand. Gill architecture was normal at all salinities. However, the 1.5 fold increase in NKA α-subunit mRNA at 5 per thousand measured by quantitative RT-PCR (qRT-PCR) using EF1α as reference gene was not statistically significant. The study confirms the osmoregulating ability of P. monodon like other crustaceans at lower salinities. It is likely that significant increase in NKA transcript level happens at an earlier time point. At higher salinities all three methods record only marginal or no change from isosmotic controls confirming the hypothesis that the animal largely osmoconforms in hyperosmotic environment.

Structural and functional characterization of haemoglobin genes in Labeo catla: Insights into hypoxic adaptation and survival.

Haemoglobin (HB) protein comprises four subunits: two identical α-subunits (HBA) and two identical ß-subunits (HBB), encoded by the HBA and HBB genes. In this investigation, 5'/3' RACE PCR (Rapid Amplification of cDNA Ends) was used to obtain complete coding sequences (CDSs) of both the genes from farmed Labeo catla. The resulting CDSs were 432 base pairs and 447 base pairs for HBA and HBB, respectively, corresponding to 143 and 148 amino acids. Phylogenetic analysis revealed close relationships with other cyprinids, with Labeo rohita being the closest relative. Functional analysis and protein structure prediction were conducted using bioinformatics tools. Expression profiling of both genes was checked in various tissues under control (C) and hypoxic (H) conditions. Notably, under hypoxia, HBA and HBB genes were significantly upregulated (P < 0.05) initially, followed by a return to normal expression levels. Similar trends were observed for Hif1α (Hypoxia-inducible factor one alpha) and EPO (Erythropoietin) genes. Additionally, haematological indices also significantly increased corresponding to the gene expressions. However, with the decrease in the expression of these genes an onset of mortality was observed in the hypoxia (H) treated groups. The results of the current study explored the role of haemoglobin genes in adaptation to the hypoxic condition.

Ultrastructural, molecular and haemato-immunological changes: Multifaceted toxicological effects of microcystin-LR in rohu, Labeo rohita.

No water body is resilient to afflicts of algal bloom, if goes unmanaged. With the increasing trend of intensification, eutrophication and climate change, Labeo rohita (rohu) is highly anticipated to suffer from the deleterious effects of bloom and eventually its toxins. A comprehensive study was conducted to understand the toxicopathological effects of microcystin-LR (MC-LR) in rohu following intraperitoneal injection of 96 h-LD50 dose i.e., 713 µg kg-1. Substantial changes in micro- and ultrastructural level were evident in histopathology and transmission electron microscope (TEM) study. The haematological, biochemical, cellular and humoral innate immune biomarkers were significantly altered (p < 0.05) in MC-LR treated fish. The mRNA transcript levels of IL-1ß, IL-10, IgM and IgZ in liver and kidney tissues were significantly up-regulated in 12 hpi and declined in 96 hpi MC-LR exposed fish. The relative mRNA expression of caspase 9 in the liver and kidney indicates mitochondrial-mediated apoptosis which was strongly supported by TEM study. In a nutshell, our study illustrates for the first time MC-LR induced toxicological implications in rohu displaying immunosuppression, enhanced oxidative stress, pathophysiology, modulation in mRNA transcription, genotoxicity, structural and ultrastructural alterations signifying it as a vulnerable species for MC-LR intoxication.

Bioaccumulation of arsenic in fish (Labeo rohita) in presence of periphyton: ameliorative effect on oxidative stress, physiological condition, immune response and risk assessment.

The present study explores the use of periphyton to ameliorate toxic properties of arsenic (As) to Labeo rohita and also assesses the human food safety aspects. Fish were introduced to arsenite [As(III)] contaminated water (0.3 and 3 mg/L) along with periphyton. Biochemical, physiological and immunological parameters, including gene expression, were assessed after 30 days of exposure. Periphyton incorporation significantly improved (p < 0.05) the adverse effects of As on respiration, NH3 excretion and brain AChE activity by reducing oxidative stress and As bioaccumulation. The presence of periphyton in As(III) exposed fish (3 mg/L) increased the immune response (Immunoglobulin M and Complement C3) in the serum and the regulation of the respective immune genes in the anterior kidney was found to be similar to the control. A speciation study using LC-ICP-MS confirmed the high accumulation of As by periphyton (5.0-31.9 µg/g) as arsenate [As (V)], resulting in a lower amount of As in fish muscle. The calculated human health risk indices, Target Hazard Quotient (THQ) and Target Cancer risk (TCR) indicate that fish grown in periphyton-treated water may lower the human health risks associated with As. The study signifies the importance of periphyton-based aquaculture systems in As contaminated regions for safe fish production with enhanced yield.

Ontogeny and tissue specific expression profiles of recombination activating genes (RAGs) during development in Nile tilapia, Oreochromisniloticus.

Recombination activating genes (RAGs) mediates the process of rearrangement and somatic recombination (V(D)J) to generate different antibody repertoire. Studies on the expression pattern of adaptive immune genes during ontogenic development are crucial for the formulation of fish immunization strategy. In the present study, Nile tilapia was taken to explore the relative expression profile of RAG genes during their developmental stages. The developmental stages of Nile tilapia, i.e., unfertilized egg, 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 and 30 days post-hatch (dph) and kidney, blood, gill, liver and spleen tissues from adult fish were collected and the cDNA synthesis was carried out. Gene specific primers for RAG-1 and RAG-2 of Nile tilapia were designed and their annealing temperature (Tm) was optimized by gradient PCR. Consequently, PCR was performed to confirm the specific amplification of RAG-1 and RAG-2 genes. Quantitative real-time PCR (qRT-PCR) gene expression of RAG-1 and RAG-2 were noticed in all the developmental stages; however, a significant increase was observed after 12 dph and peaked at 24 dph, followed by a gradual decrease until 30 dph. Tissue-specific gene expression profiling revealed that the highest expression of RAG-1 and RAG-2 was observed in the kidney, followed by spleen, gill, liver and blood. The findings of the study explored the suitable timing of lymphoid maturation that could be technically used for the adoption of strategies to improve disease resistance of fish larvae for mitigating larval mortality.

Arabidopsis plants overexpressing additional copies of heat shock protein Hsp101 showed high heat tolerance and endo-gene silencing.

Hsp101 chaperone is vital for survival of plants under heat stress. We generated transgenic Arabidopsis thaliana (Arabidopsis) lines with extra copies of Hsp101 gene using diverse approaches. Arabidopsis plants transformed with rice Hsp101 cDNA driven by Arabidopsis Hsp101 promoter (IN lines) showed high heat tolerance while the plants transformed with rice Hsp101 cDNA driven by CaMV35S promoter (C lines) were like wild type plants in heat stress response. Transformation of Col-0 plants with 4633 bp Hsp101 genomic fragment (GF lines) from A. thaliana containing both its coding and the regulatory sequence resulted in mostly over-expressor (OX) lines and a few under-expressor (UX) lines of Hsp101. OX lines showed enhanced heat tolerance while the UX lines were overly heat sensitive. In UX lines, silencing of not only Hsp101 endo-gene was noted but also transcript of choline kinase (CK2) was silenced. Previous work established that in Arabidopsis, CK2 and Hsp101 are convergent gene pairs sharing a bidirectional promoter. The elevated AtHsp101 protein amount in most GF and IN lines was accompanied by lowered CK2 transcript levels under HS. We observed increased methylation of the promoter and gene sequence region in UX lines; however, methylation was lacking in OX lines.

Hepatic microsporidiosis of mudskipper, Boleophthalmus dussumieri Valenciennes, 1837 (Perciformes: Gobiidae), due to Microgemma sp.

The present study reports a case of hepatic microsporidiosis caused by Microgemma sp. in brackishwater fish, Boleophthalmus dussumieri (Valenciennes, 1837) (n = 60), from the west coast of India. An eight-month study from September 2017 to April 2018 revealed a prevalence of 11.7% for this parasite. The microsporidian showed tissue-specific infection and did not reveal any gross pathology in infected fish. Small whitish cysts containing microspores of size 0.3-0.5 mm were observed in the liver of fish. The range of pyriform microsporidian spore size varied from 2.9-3.77 × 1.85-2.67 µm. Scanning electron microscopy of the spores showed a distinct groove on the anterior end of the spore for polar tube extrusion. Polymerase chain reaction (PCR) amplification of the DNA extracted from the microsporidian-infected liver tissue using primers targeting small ribosomal subunit DNA (SSU rDNA) yielded ~ 1340 bp amplicon and the genetic distance analysis showed a 0.2% variation with the reported M. tilanpasiri. Accordingly, in the phylogenetic tree, the present species of Microgemma clustered with M. tilanpasiri. Even though, the morphomeristic characters of the present Microgemma sp. was marginally different from the reported M. tilanpsasiri; the SSU rDNA showed considerably higher similarity with M. tilanpasiri. Thus, we report the species of Microgemma as Microgemma aff. tilanpasiri from a new host. This is the first report of a microsporidian from B. dussumieri and the first record of the genus Microgemma from India.

Antimicrobial activity of an artificially designed peptide against fish pathogens.

Antimicrobial peptides (AMPs) are considered alternatives to classical antibiotics and may become an excellent candidate for tackling antimicrobial resistance in aquaculture. Designing novel antimicrobial peptides for curbing antimicrobial resistance in aquaculture is paramount in one health approach. In this study, a short and compositionally simple peptide, KK16, was designed. KK16 is amphipathic with a net charge of + 6. Molecular docking results revealed that KK16 has a strong affinity towards two virulence proteins of Aeromonas sobria; aerolysin and outer membrane protein (omp). The peptide was synthesised using Fmoc-chemistry, and its antimicrobial efficacy was evaluated in vitro against A.sobria, A. salmonicida, Edwardsiella tarda, A. hydrophila, Vibrio parahaemolyticus, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus epidermidis and methicillin-resistant S. aureus. The KK16 AMP showed potent activity against the tested bacterial pathogens as revealed by the MIC and MBC, ranging from 7.81 to 500 µM, and 15-900 µM, respectively. Moreover, the peptide was stable at higher temperatures and retained its activity in presence of serum and salt. The peptide displayed less haemolytic and cytotoxic activity even at higher concentrations. In peptide-DNA binding assay, KK16 showed its binding potential with bacterial genomic DNA and thus, may interfere with replication. Fluorescent microscopy revealed the uptake of propidium iodide by peptide treated bacterial cells, indicating its membrane disruption activity. In in vivo experiment, KK16 peptide completely inhibited the growth of Saprolegnia parasitica fungus at ≥ 30 µM peptide concentrations in embryonated fish eggs. The results indicate that KK16 peptide is stable, possess potent antibacterial and antifungal activity, less cytotoxic to host cells, and hence may prove to be a promising anti-infective agent for combating common bacterial and fungal infections.

Taurine and/or inorganic potassium as dietary osmolyte counter the stress and enhance the growth of GIFT reared in ion imbalanced low saline water.

The effects of dietary osmolytes for alleviating osmotic stress and enhancing growth are not well elucidated in fish reared in inland saline water. The present study evaluated the effects of dietary taurine or potassium (K+) individually or in combination on growth, ionic homeostasis, and stress response of GIFT tilapia reared in potassium deficient low saline water (PDLSW, 10 ppt salinity) mimicking inland saline water. Isonitrogenous and isoenergetic diets supplemented with five potassium concentrations (0, 0.3, 0.45, 0.6 and 0.75 %), two taurine (T) concentrations (0.5 and 1.0 %) and two combinations of both (K+ 0.1 % + T 0.5 % and K+ 0.2 % + T 0.5 %) were fed to GIFT juveniles (4.4 ± 0.02 g body weight) and reared in PDLSW for 45 days. The fish fed on the diet fortifying with K+ 0.2 % + T 0.5 % showed the highest growth performance among the controls and other treatment groups. Dietary supplementation had no effects on PDLSW induced increase in osmoregulatory endpoints. The optimum dietary potassium requirement of GIFT reared in PDLSW was 0.57 and 0.599 g/100 g diet. Dietary K+ down-regulated the PDLSW induced expression of NKAa1, AQP1, and ClC2, whereas inhibited taurine-induced up-regulation of AQP1 and CLC2, which is the first report in tilapia. In addition, dietary K+ and taurine modulated antioxidant and metabolic enzyme activities for easing stress and balancing energy requirements. Thus, blending of potassium (0.2 %) and taurine (0.5 %) in the diet appears best to mitigate stress and enhance GIFT growth reared in inland saline water.

Bicistronic DNA vaccine macromolecule complexed with poly lactic-co-glycolic acid-chitosan nanoparticles enhanced the mucosal immunity of Labeo rohita against Edwardsiella tarda infection.

DNA vaccine is an important tool to elicit both humoral and cellular immunity. Present study investigates mucosal immune response of Labeo rohita (15 ± 04 g) to plasmid DNA (pDNA) vaccine macromolecule complexed with nanoparticles (NPs). Poly lactic-co-glycolic acid (PLGA), Chitosan (Chit) and PLGA-Chit-NPs were synthesized by double emulsion solvent evaporation method. Synthesized NPs were complexed with pDNA (pGPD + IFN) vaccine construct. Size and zeta potential of PLGA-NPs, Chit-NPs and PLGA-Chit-NPs-pDNA complex were recorded to be 120 nm and +0.5 mV, 117 nm and +32 mV, 189 nm and +11 mV, respectively. Immunization by immersion was carried out in two groups receiving PLGA-Chit-NPs-pDNA (T1) and PLGA-NPs-pDNA (T2) respectively. After immersion, samples were collected on 0, 2, 4, 7, 15 and 30 days from mucosa-associated lymphoid tissues (MALT) for mRNA expression studies of IgM, IgD and IgZ using qRT-PCR. Significant up-regulation of the mRNA expression of IgM, IgD, and IgT were observed in MALT in immunized fish compared to control. After 30 days post-immunization fish were infected with a virulent strain of Edwardsiella tarda. The highest relative percentage survival was observed in T1 (64.7%) compared to T2. The study showed better efficiency of pDNA-PLGA-Chit-NPs compared to pDNA-PLGA-NPs for inducing adaptive mucosal immunity in fish.