Identification and antifungal sensitivity of Fusarium species isolated from piscine hosts.

Fusarium is a huge genus of filamentous fungi that has the potential to cause emerging diseases. Members of this genus can cause infections in plants, animals and humans. Here, we report the isolation of F. oxysporum and F. equiseti from 2 important fish species, Oncorhynchus mykiss (rainbow trout) and Tor putitora (golden mahseer), respectively. F. oxysporum has emerged as a significant fungal pathogen causing infection in many fish. However, F. equiseti has been isolated mainly from plants. As far as the available literatures are concerned, this is the first report on the isolation of F. oxysporum and F. equiseti from these hosts. The isolates were identified based on growth morphology and microscopic observation. F. oxysporum produced violet pigmentation on potato dextrose agar, while F. equiseti had yellow colouration. F. oxysporum produced 1- to 2-celled microconidia along with straight or curved macroconidia having 3 to 4 septa. F. equiseti produced abundant macroconidia with 4 or more septa. Species were further confirmed based on the nucleotide sequences of the internal transcribed spacer region. In a molecular phylogeny analysis, F. oxysporum and F. equiseti formed 2 different clades. In an antifungal sensitivity assay, F. oxysporum was found to be susceptible to clotrimazole with a minimum inhibitory concentration of 1.0 µg ml-1, whereas F. equiseti was susceptible to clotrimazole, ketoconazole and fluconazole. Overall, the main findings of this study are the infection of new hosts by Fusarium species and the limited activity of many antifungal drugs against these pathogens.

Antibacterial activity of a short de novo designed peptide against fish bacterial pathogens.

In the face of increasing antimicrobial resistance in aquaculture, researchers are exploring novel substitutes to customary antibiotics. One potential solution is the use of antimicrobial peptides (AMPs). We aimed to design and evaluate a novel, short, and compositionally simple AMP with potent activity against various bacterial pathogens in aquaculture. The resulting peptide, KK12YW, has an amphipathic nature and net charge of + 7. Molecular docking experiments disclosed that KK12YW has a strong affinity for aerolysin, a virulence protein produced by the bacterial pathogen Aeromonas sobria. KK12YW was synthesized using Fmoc chemistry and tested against a range of bacterial pathogens, including A. sobria, A. salmonicida, A. hydrophila, Edwardsiella tarda, Vibrio parahaemolyticus, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus epidermidis, and methicillin-resistant S. aureus. The AMP showed promising antibacterial activity, with MIC and MBC values ranging from 0.89 to 917.1 µgmL-1 and 3.67 to 1100.52 µgmL-1, respectively. In addition, KK12YW exhibited resistance to high temperatures and remained effective even in the presence of serum and salt, indicating its stability. The peptide also demonstrated minimal hemolysis toward fish RBCs, even at higher concentrations. Taken together, these findings indicate that KK12YW could be a highly promising and viable substitute for conventional antibiotics to combat microbial infections in aquaculture.

Assessing safety, efficacy and residue depletion in golden mahseer, Tor putitora (Hamilton, 1822): biochemical and physiological responses to graded concentrations of oxytetracycline dietary supplementation.

The safety and effectiveness of oxytetracycline can potentially manage bacterial infections in fish. This, in turn, might reduce the concerns related to its use in aquaculture and human consumption, such as toxicity, antimicrobial resistance, and other associated risks. The primary objective of this study was to assess how adding oxytetracycline dihydrate to the diet affects its effectiveness, safety, and the presence of residues in T. putitora. T. putitora fingerlings, subjected to experimental infection with Aeromonas hydrophila at a concentration of 108 CFU mL- 1, received an oral administration of oxytetracycline dihydrate. The oxytetracycline dihydrate was added to the feed (corresponding to 2% of the fish body weight) at concentrations of 44.1, 88.2, 132.3 and 176.4 mg Kg- 1 fish body weight per day. This treatment was carried out for 10 consecutive days. The biochemical and physiological responses of T. putitora and efficacy of oxytetracycline dihydrate were determined through estimation of microbial load (CFU mL- 1), haematogram, serum biomarkers, behavioral characteristics, non-specific immunity and residue depletion. Experimentally infected fish showed disease progression and induced histopathological conditions with highest microbial load (CFU mL- 1) in the muscle of both control and treated fish. The fish haematogram showed increased leucocyte and haemoglobin content, influenced by dietary oxytetracycline dihydrate. The fish demonstrated adaptive physiological response to oxytetracycline dihydrate at 44.1 to 88.2 mg and resulted in increased albumin and globulin content. The serum-enzyme assay showed significant increase in aspartate aminotransferase (AST), alanine aminotransferase (ALT) and plasma alkaline phosphatase (ALP) activities in the test fish (< 0.05). Oxytetracycline dihydrate at 88.2 to 132.3 mg Kg- 1 fish body weight per day recorded higher feed intake (75%), significant survivability (66-68%) and histopathological recovery. The suppressed immune response was manifested with decreased respiratory burst and lysozyme activity. The palatability, treatment of bacterial infection, histopathological changes and survivability by fingerlings of golden mahseer determined the safety and optimized the therapeutic potential of the oxytetracycline dihydrate at 88.2 mg Kg- 1 fish body weight per day for 10 days to contain the infection by A. hydrophila. A withdrawal period of 8-d was recommended as oxytetracycline dihydrate concentration depleted below the legal maximum residue limit (MRL 2.0 mg g- 1) in the edible muscle of the golden mahseer reared at an average water temperature of 20 °C. This is considered safe for human consumption.

Pathological analysis and antimicrobial susceptibility of Chryseobacterium balustinum RTFCP 298 isolated from diseased rainbow trout, Oncorhynchus mykiss.

In this study, six isolates of Chryseobacterium balustinum were characterized from diseased rainbow trout fingerlings. The virulence characteristics, pathogenicity, and antimicrobial susceptibility pattern of these isolates were investigated. The bacterium showed positive results for catalase, cytochrome oxidase, and aesculin hydrolysis, while negative results were obtained for DNase, gelatinase, methyl red, Voges-Proskauer's reaction, Simon citrate, Hydrogen sulphide, and starch hydrolysis. Amino acid metabolism analysis revealed the inability to metabolize arginine, lysine, and ornithine decarboxylase. Molecular characterization (16S rRNA) and phylogenetic analysis revealed the test isolates as C. balustinum, closely related to strain WLT (99.85% similarity) and C. balustinum P-27 (99.77%). Virulence assay indicated haemolytic activity and biofilm formation by the test bacterium. The challenge test confirmed moderate pathogenicity in rainbow trout and established Koch's postulates. The clinical manifestations of infection included fin erosion, eye and body surface haemorrhage, exophthalmia, and organ liquefaction. The minimum inhibitory concentrations of various antimicrobials ranged from 1 to > 256 µg mL-1. The novel synthetic antimicrobial peptides exhibited MICs of 8 to > 256 µg mL-1, suggesting a potential control method. These findings suggest that C. balustinum is an opportunistic pathogen with moderate pathogenicity in rainbow trout. Further research on the host-pathogen relationship is necessary to understand virulence characteristics and pathogenicity in aquaculture.

First report of Achlya bisexualis infection in captive-reared Endangered golden mahseer Tor putitora.

Achlya bisexualis is a notorious oomycete pathogen with the potential to cause emerging disease in fish farms. In this study, we report the first isolation of A. bisexualis from captive-reared golden mahseer Tor putitora, an Endangered fish species. The infected fish showed a cotton-like growth of mycelia at the site of infection. The mycelium when cultured on potato dextrose agar produced radially growing white hyphae. The hyphae were non-septate, and some of them carried matured zoosporangium with dense granular cytoplasmic contents. Spherical gemmae with stout stalks were also observed. All the isolates had 100% identity in internal transcribed spacer (ITS)-rDNA sequence and showed highest similarity to that of A. bisexualis. In molecular phylogeny, all the isolates formed a monophyletic group with A. bisexualis which was supported by a bootstrap value of 99%. Based on the molecular and morphological findings, all the isolates were confirmed as A. bisexualis. Further, the anti-oomycete effect of boric acid, a known antifungal agent, against the isolate was evaluated. The minimum inhibitory concentration and minimum fungicidal concentration were found to be 1.25 and >2.5 g l-1, respectively. Isolation of A. bisexualis from a new fish species indicates its possible occurrence in other unreported hosts. Considering its wide infectivity and the potential to cause disease in farmed fishes, its probable prevalence in a new environment and host needs to be closely monitored to prevent the spread of infection, if any, by adopting suitable control measures.

Biosafety, histological alterations and residue depletion of feed administered anti-parasitic drug emamectin benzoate in golden mahseer, Tor putitora (Hamilton, 1822) as a model candidate fish for sport fishery and conservation in temperate waters.

In the present experiment, the attempt has been made to study the biosafety, toxicity, residue depletion and drug tolerance of graded doses of emamectin benzoate (EB) in juveniles of golden mahseer, Tor putitora as a model candidate fish for sport fishery and conservation in temperate waters through an extended medicated feeding. The graded doses of EB viz., 1× (50 µg/kg fish/day), 2 × (100 µg/kg fish/day), 5 × (250 µg/kg fish/day) and 10 × (500 µg/kg fish/day) were administered to golden mahseer juveniles through medicated diet for 21 days at water temperature of 18.6°C. The higher doses of EB did not cause any mortality during and 30 days after the end of medication period, but considerable variations in feeding and behavior were observed. Severe histological alterations observed after EB-diets (5 × and 10×) were vacuolation, pyknotic nuclei, melanomacrophage centre and necrosis in liver; Bowman's capsule dilation, degenerated renal tubules in kidney; myofibril disintegration, muscle oedema, splitting of muscle fibres, migration of inflammatory cells in muscle; and abundant goblet cells, dilated lamina propria and disarrangement of mucosa in intestine tissues. The residual concentrations of EB metabolites Emamectin B1a and B1b were analyzed using muscle extracts and were found to be peaked during medication period followed by gradual depletion in post-medication period. The outcome of this study showed that the Emamectin B1a residual concentration in fish muscle in 1×, 2×, 5×, and 10× EB treatment groups were 1.41 ± 0.49, 1.2 ± 0.7, 9.7 ± 3.3, and 37.4 ± 8.2 µg/kg at 30 days of post-medication period, respectively, which falls under the maximum residue limits (MRLs) of 100 µg/kg. The results support the biosafety of EB at recommended dose of 50 µg/kg fish/day for 7 days. As residue of EB is recorded falling within the MRL, no withdrawal period is recommended for golden mahseer.

Temperature alters the oxidative and metabolic biomarkers and expression of environmental stress-related genes in chocolate mahseer (Neolissochilus hexagonolepis).

Long-term acclimation temperature effects on biomarkers of oxidative stress, metabolic stress, expression of heat shock proteins (Hsps), and warm-temperature acclimation related 65-kDa protein (Wap65) were evaluated in the threatened chocolate mahseer (Neolissochilus hexagonolepis). Fifteen-day-old larvae were acclimated to different water temperatures (15, 19, 23-control group, 27, and 31 °C) for 60 days prior to the sampling for quantification of mRNA, enzyme, nitric oxide, and malondialdehyde (MDA) content. Acclimation to 31 °C increased the basal mRNA level of glutathione S-transferase alpha 1 (GSTa1), and activities of catalase (CAT), glutathione reductase (GR), and GST enzymes and but downregulated the expression of superoxide dismutase 1 (SOD1) in the whole-body homogenate. Other antioxidant genes, i.e., CAT and GPx1a, were unaffected at 31 °C, and nitric oxide (NO) concentration was significantly lower. In contrast, fish acclimated to 15 °C showed an upregulated transcript level of all the antioxidant genes and no significant difference in the CAT, GR, and GST enzymes. Activities of the metabolic enzymes, aspartate transaminase (AST) and alanine transaminase (ALT), were significantly lower at 15 °C. The expression of Hsp47 was upregulated at both 15 and 31 °C groups, whereas Hsp70 was elevated at 27 and 31 °C groups. Wap65-1 transcription did not show significant variation in treatment groups compared to control. Fish in the high (31 °C) and low-temperature (15 °C) acclimation groups were capable of maintaining oxidative stress by modulating their antioxidant transcripts, enzymes, and Hsps.

Computational analysis and functional characterisation of Tor putitora toll-like receptor 4 with the elucidation of its binding sites for microbial mimicking ligands.

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.

Development of multiplex PCR assay for species-specific detection and identification of Saprolegnia parasitica.

Saprolegnia parasitica is the most important pathogen under the genus, Saprolegnia which causes devastating oomycete diseases in freshwater fish. At present, the most common molecular method for identification of Saprolegnia species is sequencing of ribosomal DNA internal transcribed spacer (rDNA-ITS) region. In this study, a highly sensitive multiplex PCR targeting rDNA-ITS region and a hypothetical protein gene was developed using two sets of primer pair. In this PCR, two amplicons of different size of 750 bp and 365 bp are produced only in case of S. parasitica while other Saprolegnia species had single amplicon. This protocol could also differentiate Saprolegnia species from other fungus based on the size of rDNA-ITS region. The protocol does not require sequencing and can identify S. parasitica in a single reaction. Therefore, the multiplex PCR developed in this study may prove to be an easier, faster and cheaper molecular method for identification of S. parasitica.

Anti-oomycete Activity of Chlorhexidine Gluconate: Molecular Docking and in vitro Studies.

Saprolegniosis is one of the most catastrophic oomycete diseases of freshwater fish caused by the members of the genus Saprolegnia. The disease is responsible for huge economic losses in the aquaculture industry worldwide. Until 2002, Saprolegnia infections were effectively controlled by using malachite green. However, the drug has been banned for use in aquaculture due to its harmful effect. Therefore, it has become important to find an alternate and safe anti-oomycete agent that is effective against Saprolegnia. In this study, we investigated the anti-oomycete activity of chlorhexidine gluconate (CHG) against Saprolegnia. Before in vitro evaluation, molecular docking was carried out to explore the binding of CHG with vital proteins of Saprolegnia, such as S. parasitica host-targeting protein 1 (SpHtp1), plasma membrane ATPase, and TKL protein kinase. In silico studies revealed that CHG binds with these proteins via hydrogen bonds and hydrophobic interactions. In an in vitro study, the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of CHG against S. parasitica were found to be 50 mg/L. Further, it was tested against S. australis, another species of Saprolegnia, and the MIC and MFC were found to be 100 and 200 mg/L, respectively. At 500 mg/L of CHG, there was complete inhibition of the radial growth of Saprolegnia hyphae. In propidium iodide (PI) uptake assay, CHG treated hyphae had bright red fluorescence of PI indicating the disruption of the cell membrane. The results of the present study indicated that CHG could effectively inhibit Saprolegnia and hence can be used for controlling Saprolegniasis in cultured fish.

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.

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.

Clinical signs, lethal dose and histopathological lesions in grass carp, Ctenopharyngodon idella experimentally infected with Edwardsiella tarda.

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 degenerative changes 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.

Spawning substrate preference and spawning behavior of chocolate mahseer, Neolissochilus hexagonolepis.

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.

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.

Effects of rearing temperature on egg incubation, growth, standard metabolic rate, and thermal tolerance of chocolate mahseer, Neolissochilus hexagonolepis.

The present study was aimed to assess the effect of temperatures on egg incubation, growth, standard metabolic rate (SMR), and thermal tolerance of a near threatened Himalayan hill stream chocolate mahseer (Neolissochilus hexagonolepis). For the hatching study, eggs were incubated in four temperatures (17, 20, 23, and 26 °C). The total hatching and free-swimming larvae percentage were higher at 23 °C (p < 0.05). Experiment I (for validation of the CTmax method) was carried out by incubating eggs at 17 °C and 23 °C. The CTmax was estimated in response to different warming rates (1-18°C h-1), acclimation temperatures (17 and 23°C), and the age of fishes (8, 15, 35 dph). The results suggested that a warming rate of 18°C h-1 could be used for the thermal tolerance study of yolk-sac larvae (8 dph) and 35 dph larvae, but for free-swimming larvae (15 dph) up to 3°C h-1 is suitable. Experiment II (for growth, SMR and thermal tolerance) was carried by acclimatizing 15 dph larvae in five temperatures (15, 19, 23, 27, and 31 °C) for 60 days. The mean growth rate increased with the increase in temperature from 15°C to 27°C (1.30-3.58% day-1) and decreased at 31°C. The mean SMR of the chocolate mahseer in the above acclimation temperatures was ranged from 1.14 ± 0.36 to 2.81 ± 0.15 µgO2h-1mg-1 and were significantly different (p < 0.01). The Q10 with the SMR of the fish suggested the preferred temperature ranged between 23 and 27 °C, and the optimum temperature for growth (ToptG) was estimated to be 25 °C. Chocolate mahseer is an eurythermal species which is advantageous for aquaculture practices due to its wide thermal tolerance zone (411.68°C2 in 15 to 31 °C acclimation temperature range) and high ARR values (0.49 - 0.54).

Evaluation of the acute toxicity of Thymus linearis ethanol extract and its effect on the hemato-biochemical and behavioural response of the Golden mahseer, Tor putitora (Hamilton, 1923).

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.

Anti-oomycetes and immunostimulatory activity of natural plant extract compounds against Saprolegnia spp.: Molecular docking and in-vitro studies.

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.

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.