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Aim: This study aimed to investigate the effectiveness of ethanolic extract of three Himalayan plants Myrica esculenta, Thymus linearis and Butea monosperma on hyphal germination, colonisation and sporulation of two species of Saprolegnia (Saprolegnia parasitica and S. australis) isolated from rainbow trout, Oncorhynchus mykiss. Molecular docking of active ingredients of M. esculenta, Myricetin with effector proteins of S. parasitica was also performed to investigate the target binding sites for drug development. Methodology: Minimum inhibitory concentration (MIC), mycelium growth inhibition, spore germination, and inhibition was performed with the most effective concentrations. Molecular docking was carried out with AutoDock Vina software to investigate target binding sites with S. parasitica. Results: Extracts from Myrica esculenta, Thymus linearis and Butea monosperma showed MIC values of the 25, 100, 50 mg ml-1 against S. parasitica and 25, 50, 25 mg ml-1 against S. australis hyphal growth, respectively. Nevertheless, malachite green as reference control was effective with a MIC value of 2.5 mg l-1. The concentration required to inhibit S. parasitica and S. australis spores were (50) Myrica esculenta, (25) Thymus linearis, (100) Butea monosperma in mg ml-1 and (50) Myrica esculenta, (50) Thymus linearis, (100) Butea monosperma in mg ml-1, respectively. Interpretation: The study concludes that M. esculenta and B. monosperma are effective against Saprolegniasis and could be used as phyto additives.

The present investigation was conducted to estimate the acute toxicity of Thymus linearis plant extract, its effect on hemato-biochemical parameters and behavioural response in the golden mahseer (Tor putitora). The phytochemical composition present in T. linearis plant extrat were Alkaloids, Flavonoids, Phenols and Tannin. The fishes were subjected to eight different concentrations of T. linearis leaves extract (8.25, 8.50, 8.75, 9.00, 9.25, 9.50 and 9.75 mg/kg) and the control group without plant extract for 96-h LD50 study. The mortality was recorded every 24 h post-treatment. Minimum mortality was recorded in the 8.25 mg/kg, whereas 100% mortality was recorded in the 9.75 mg/kg T. linearis extract after 96-h periods. The LD50 was estimated by probit analysis, and the value of T. linearis at 96 h was found to be 8.71 mg/kg for golden mahseer. A non-lethal dose of 1/10th of 96-h LD50 value (0.87 mg/kg) was taken for the sublethal study. After 96 h, the red blood cell (RBC), white blood cell (WBC), haemoglobin (Hb), packed cell volume (PCV) and blood glucose were measured. RBC (×106/mm3), Hb (%) and PCV (%) significantly decreased at 8.25, 8.50, 8.75, 9.00 mg/kg, but WBC and blood glucose significantly increase at 8.25, 8.50, 8.75, 9.00 mg/kg of T. linearis plant extract. The observations on behaviour response of golden mahseer were also recorded. In the present study, the acute toxicity of wild ajwain was more significant than short-term toxicity. The mortality rate was very high during the study period of T. linearis exposure.

In the current study, full-length Toll-like receptor 4 (TLR4) cDNA was cloned and characterised in Tor putitora, an important fish inhibiting Himalayan rivers. The complete coding sequence of TpTLR4 is 2457 bp with nine key structural domains, including six leucine-rich repeats (LRRs). The phylogenetic tree revealed that TpTLR4 showed the closest relationship with TLR4 of Cyprinus carpio (96%), Labeo rohita (91%) and Megalobrama amblycephala (88%), all belonging to the Cyprinidae family. CELLO2GO tool revealed that TpTLR4 protein is highly localised in the plasma (67.7%), and the protein has a strong association with myeloid differentiation primary response 88 (MYD88) followed by Tumor necrosis factor receptor-associated factor (TRAF) family. In the toll-interleukin-1 receptor (TIR) domain of TpTLR4, the proline is replaced by the alanine amino acid, thus may give plasticity to the receptor to recognise both bacterial and viral ligands. Molecular docking has revealed that TpTLR4 showed the strongest affinity towards poly (I:C) with the binding energy of -6.1 kcal/mol and five hydrogen bonds among all ligands. Based on our molecular docking results, it can be presumed that TpTLR4 can sense bacterial, fungal and viral molecular patterns with binding sites mainly present in the TpTLR4 LRR9 motif, which spans between 515 and 602 amino acids. Tor putiora TLR4 transcript was ubiquitously expressed in all the tested fish tissues. Although, transcript level was found to be highest in blood and spleen followed by the kidney. The TpTLR4 transcripts showed peak expression in spleen and kidney at 12 h post-injection (hpi) (p 0.05) of poly (I:C). The constitutive expression of TpTLR4 in various tissues, up-regulation in different tissues and strong binding affinities with poly (I:C) indicate that TpTLR4 may play an essential role in sensing pathogen-associated molecular patterns (PAMPs), particularly of viral origin.

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.

An experiment was conducted to study the lethal dose (LD50-96h) and histopathological changes occurring in several organs of grass carp challenged with different concentrations of Edwardsiella tarda. The healthy grass carps were challenged with the bacterial suspension of 106,107, 108, 109 and 1010 CFU ml−1. The study demonstrated that the lethal dose (LD50-96h) of E. tarda for grass carp is 1.3 × 109 CFU ml−1. The infected fish showed abnormal swimming behavior, slower movements, skin necrosis, hemorrhages, and open lesion on the fontanelle of the frontal bone of the skull during the initial phase of infection. About 60% of the fish which received the bacterial suspension of 1010 CFU ml−1 died within 24 h of infection. The histopathological examination of the infected tissue section demonstrated the severe damages in the internal organs. In gills, oedema, secondary lamellae fusion, and hyperplasia of basal epithelial lining between secondary lamellae were reported. The microscopic observation showed the disruption of submucosa to the mucosa, which finally led to muscularis in the intestine, necrosis of hepatocytes and infiltration of red blood cells in the liver. The tubular disintegration in kidney and loss of capsular boundary of red pulp in spleen were also reported. In conclusion, the result indicates that the infection caused by E. tarda can cause severe damages and alterations in grass carp tissues and potential mass mortality. Moreover, The bacteria isolated from the mobribund fish was characterized by biochemical tests and expression of five critical virulence genes like citC, fimA, gadB, mukF and gyrB were detected from the microorganism. The study aims to provide a research foundation for further studies on the susceptibility and pathological changes of grass carp induced by E. tarda infection.

Eurycomanone (EN), a quassinoid present in E. longifolia plants acts as a testosterone booster in male animals. The present study was aimed to explore the role of EN in females using fish as model organism. Chitosan nano-conjugated EN nanoparticles were synthesised and tested for their role in reproductive response in female catfish. Three injections of the formulation were given for a period of 21 days and the mRNA expression profile of important genes as well as serum hormones involved in steroid hormone synthesis were evaluated. In gonads, the mRNA expression of genes was upregulated except CYP19a1 (aromatase), that significantly decreased in the treatment groups. The hormone genes, FSH-β and LH-β mRNA levels in brain were upregulated whereas CYP19a2 was sharply downregulated following treatments. The testosterone varied non-significantly while the estradiol-17β serum hormone was significantly diminished. The effect was sustained in chitosan conjugated treatments compared to EN alone. The Gonadosomatic index (GSI) varied non-significantly in different treatments and control. In treatment groups, histological analysis showed the ovarian follicles in the regressed phase. Analysis of the results of the undertaken research leads to conclusion that that EN may not be beneficial for female reproduction due to its obstructive role in estrogenic production.

The study was undertaken to explore the molecular mechanism of eurycomanone, a major compound of Eurycoma longifolia plant in increasing the reproductive processes in the male fish model. Chitosan-nanoconjugated eurycomanone nanoparticles with a significant particle size [130 nm (CED1); 144.1 nm (CED2)] and stable zeta potentials (+49.1 mV and +30 mV) were synthesized and evaluated against naked eurycomanone (ED1 and ED2). In present study, short-term and long-term experiments were conducted to evaluate the effect of nano-formulation on expression of endocrine-related genes, circulating hormone concentrations (Follicle stimulating hormone, FSH; luteinizing hormone, LH; progesterone, testosterone and 17-β estradiol) and reproductive capacity of male Clarias magur. In short-term experiment, the sampling of tissues was done on hourly basis after injection of eurycomanone either alone or with chitosan and long-term experiment was carried for 21 days and in this the injection was repeated after 7 days and 14 days. Treatments CED1 and CED2 showed controlled and sustained surge of the transcript level of selected genes (except aromatase) and serum hormones (except 17β-estradiol) compared to ED1 and ED2 groups. The transcript levels of aromatase and serum 17β-estradiol hormone showed the declining trend in the chitosan conjugated groups. The gonadosomatic index (GSI), reproductive capacity, intracellular calcium and selenium and cellular structure of testes were improved in CED1 and CED2 groups compared to other treatments. Furthermore, the effect of chitosan conjugated eurycomanone was evaluated in primary testicular cells and an increase in the mRNA expression level of endocrine-related genes was detected. This is the first report of the use of chitosan conjugated eurycomanone and present study elucidates the molecular mechanism of eurycomanone in increasing the reproductive output in animals.

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.

The pathogenic oomycetes Saprolegnia parasitica, causative agent of saprolegniosis in salmonid fish, and Aphanomyces astaci, causative agent of crayfish plague, have a negative impact on freshwater aquaculture. As they are controlled worldwide with chemicals that are harmful to humans and the environment, there is a growing need to replace them with ecologically acceptable alternatives. Propolis has documented antimicrobial activity as well as positive effects on the immune response, growth performance and/or reproductive capacity of fish and crayfish. Therefore, we aimed to investigate the possible inhibition of S. parasitica and A. astaci by fluid propolis formulations. Chemical analysis showed that the propolis formulations were rich in volatile and non-volatile phenolic bioactive components (chrysin was the dominant flavone and pinocembrin the dominant flavanone). Overall, the fluid propolis formulations showed good in vitro inhibition of mycelial growth and zoospores of both pathogens, but we found differences in sensitivity depending on species and life cycle stage. Mycelial growth was more sensitive for A. astaci, with EC50 values (the samples concentrations causing 50% inhibition) up to 8.59 μg/mL, compared to 206.60 μg/mL for S. parasitica. Zoospore motility was more affected in S. parasitica, where MIC (minimum inhibitory concentration) values were up to 61.88 μg/mL, compared to 154.68 μg/mL for A. astaci. Zoospore germination of both pathogens was similarly sensitive to the fluid propolis preparations, with EC50 values of up to 19.52 μg/mL for A. astaci and up to 23.62 μg/mL for S. parasitica. In addition, molecular docking was used to analyze the binding of selected propolis components to oomycete proteins suggested to play a role in pathogenesis. Apigenin, chrysin and pinocembrin were predicted to bind strongly to the endochitinase of A. astaci, which is mainly expressed in the mycelium, and to the thrombospondin of S. parasitica, mainly expressed in the cysts, which is consistent with the results of the in vitro inhibition experiments. Overall, our results suggest that propolis could be used in salmonid and crayfish aquaculture not only as an immunostimulant but also as an antioomycetic agent.

This study aimed to investigate the effectiveness of five natural plant extract compounds Curcumin (CUR); Eugenol (EUG), Cinnamaldehyde (CIN), Stigmasterol (ST) and Morin (MOR), on two species of Saprolegnia; Saprolegnia parasitica and S. australis. Selective compounds were screened for the minimum inhibitory concentration, first for anti-oomycetes activity and then mycelium growth inhibition, spore germination inhibition and colonisation test. Nitric oxide production and myeloperoxidase activity of the compounds were tested in head kidney leukocytes of rainbow trout, Oncorhynchus mykiss to assess the immunostimulatory potential. Molecular docking of effective compounds was carried out with effector proteins of S. parasitica to investigate the target binding sites. Among all, CUR could completely inhibit zoospore production and significantly (p ≤ .05) inhibit hyphal growth at 16 mg l−1 against S. parasitica and S. australis. CIN at the concentration of 50 mg l−1 completely inhibited hyphal growth of both Saprolegnia spp., although the zoospore production of S. parasitica and S. australis was reduced at 25 mg l−1 and 10 mg l−1. In the case of EUG, significant inhibition of the hyphal growth and germination of S. parasitica zoospores was observed at 50 mg l−1. ST and MOR did not show antioomycetes activity. The molecular docking results were consistent with in vitro studies, possibly due to the binding with the vital proteins (Plasma membrane ATPase, V-type proton ATPase, TKL protein kinase, Host targeting protein 1) of S. parasitica and ultimately inhibiting their activity. CUR and CIN showed increased nitric oxide production at the highest concentration of 250 and 256 mg l−1 but the value was not significant (p ≤ .05) with control. CUR showed significantly higher peroxidase activity (p ≤ .05) at a concentration of 256 mg l−1 though values were significantly similar with concentration from 16 to 128 mg l−1. The nitric oxide and total peroxidase activity of rainbow trout leukocytes in the case of CIN showed a significant difference only at 250 mg l−1 against the control. The results conclude that CUR, CIN showed the better anti-Saprolegnia activity and could be used as phyto-additives in aquaculture. Among all, the inclusion of CUR as phyto-additives will provide additional immunostimulatory activity.

Genome editing, a technique of introducing sequence-specific modifications by means of engineered nucleases, has opened new vistas in genome manipulation. It allows molecular biologists to precisely edit, delete, or alter the genomic sequences of an organism using a specific set of engineered nucleases. This technology can enable to introduce precise, specific, and accurate changes in the genetic material of an organism that can alter the phenotypic characteristics and genotypic composition. Such changes in the nucleotide sequences of a gene or its disruption due to imperfect repair of a genomic segment can have a tremendous impact on development of novel gene-based therapies, besides acceleration of discoveries in both basic and translational scientific fields. The technology of gene editing can be very well applied in the treatment of genetic diseases, cancer, elimination of pests as well as diseases.

The present investigation estimated the potential anti-oomycetes activity of Thymus linearis leaf extract and identified the phytochemical compounds by Gas chromatography-Mass spectrometry (GC-MS). Saprolegnia parasitica isolated from the golden mahseer, Tor putitora was used for efficacy study. The effect of T. linearis plant extract on hyphal growth, zoospore production was employed by a agar diffusion method and microwell plate, respectively. The ethanolic extract of T. linearis exhibited hyphal growth inhibition to 54.45 ± 0.9% at 0.32 mg ml−1 and complete inhibition (100%) at 5.12 mg ml−1. The zoospore production was ultimately arrested at 0.32 mg ml−1 against S. parasitica. A total of 18 volatile constituents were identified from the GC–MS of the whole plant extract. The major volatile constituents in the extracts were ethyl (9z, 12z)-9, 12-octadecadienoate (22.58%), palmitic acid (11.95%), ethyl palmitate (9.89%), phytol (5.03%), stigmast-5-En-3-Ol, (3.Beta.)- (4.54%), (Z, Z)-6, 9-Cis-3, 4-epoxy-nonadecadiene (3.60%), carvacrol (3.59%), cryptomeridiol (3.22%), Heptadecanoic acid, ethyl ester (2.03%) and naphthalene, decahydro-(1.28%). Molecular docking was performed by Auto Dock Vina 4.0 of the constituents for obtaining the binding mode predictions with target proteins of S. parasitica. Phytol and carvacrol were identified to interact with plasma membrane ATPase, host target protein-1 and TKL protein kinase and V-type protein ATPase. Molecular interaction of phytol was stronger to V-type protein ATPase. From the results, it is evident that T. linearis contain various natural compounds and is recommended plant of phytopharmaceutical importance. T. linearis extract could be explored as antioomycetes compounds for treating oomycetes infections, saprolegniasis in aquaculture.

Captive breeding programs for Neolissochilus hexagonolepis are essential for population restoration. To develop an efficacious method for enhancing N. hexagonolepis spawning in captivity, there was examination of: (1) different types of spawning substrate and (2) area of spawning in the substrate. The study was conducted to describe spawning behavior of males and females. There was a choice of three substrates in which to spawn: gravel, small cobble, and coarse sand. There was preferential choosing of gravel followed by cobble with there being no use of sand for spawning. Behavior of N. hexagonolepis included preparation of a spawning pit by females, a behavior that has not been previously ascertained for cyprinids. Males expressed courting behaviors, including chasing, nudging, and quivering. Courting males expressed aggressive behaviors towards other males. Results from the present study are the first on the volitional spawning of N. hexagonolepis in captivity using spawning substrate. It was further revealed that using a gravel substrate tray would also be a feasible approach for egg production. Mean total eggs per female and mean fertilized eggs collected were less when there was siphoning used for egg collections in the preference study. Hence, stripping was implemented to increase the egg collection when spawning behaviors were observed. Total eggs collected were 40,540 with 3685 eggs per female, 90.3% fertilization rate, 82.8% hatching rate, and 97.4% free-swimming larvae survival rate. The implications of this study could be beneficial for enhancing the natural population through environmental management and developing a viable egg production technique in captivity.

The present study was carried out to investigate the antimicrobial and immunomodulatory activity of Myrica esculenta ethanolic leaf extract (MeALE) in rainbow trout. The juvenile fish were immersed in 20 mg/l (T1) and 40 mg/l (T2) MeALE for 2 and 6 h and then challenged with pathogenic bacteria, A. hydrophila (ATCC 7966, USA) after 6 h post immersion in MeALE. The immuno-haematological parameters and relative percentage survival were recorded over 7 days post-exposure. The amino acid interaction of functional proteins of bacteria with myricetin, the active ingredient of M. esculenta was performed by AutoDock Vina software. FTIR detected the characteristic peak values and revealed the presence of various functional groups, viz. alkenes, amines, alcohols, phenols and aromatic compounds. The MIC of MeALE was found to be 2 mg/ml while MBC was recorded to be 25 mg/ml. Molecular docking revealed a strong affinity between myricetin and functional proteins of the pathogen. The fish immersed in 40 mg/l MeALE showed a significant increase in haematological indices and non-specific immunological parameters. The current study reports the successful use of MeALE as an antimicrobial and immunostimulatory agent in rainbow trout and recommends the dosage of 40 mg/l MeALE for preventing Aeromonas infection.

Snow trout, Schizothorax richardsonii, in early as well as in advance life stages are highly susceptible to Saprolegnia spp. infections. In the present study, we aimed to identify the Saprolegnia spp. from different life stages of infected snow trout eggs, juveniles, and adults from two geographic regions, Bhimtal and Champawat of Central Himalaya, India. Morphological identification was carried out by microscopic observation of life stages of Saprolegnia. For the molecular characterization, the internal transcribed spacer region (ITS) was amplified using universal primers ITS 1 and ITS 4. On the basis of the morphological and molecular findings, we have identified two Saprolegnia species from snow trout, i.e., Saprolegnia parasitica from eggs and adults; and S. australis from eggs and juveniles. In particular, S. parasitica was isolated from both geographical regions and S. australis only from Bhimtal, where a comparatively higher temperature regime prevails. An in-vitro experiment was conducted to study the growth of Saprolegnia spp. in varying incubation temperatures. The results confirmed that S. australis could not thrive at or below 7 °C, whereas S. parasitica could grow within the temperature range of 4–20 °C.

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.