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.

Conformational analysis of a synthetic fish kisspeptin 1 peptide in membrane mimicking environments.

Kisspeptin 1 is a neuropeptide hormone of the RFamide family, which act as an upstream regulator of brain-pituitary-gonad (BPG) axis in most vertebrates including teleosts. In the present study, a 16 amino acid long putative mature bioactive peptide (kiss 1) from preprokisspeptin 1 of golden mahseer, Tor putitora (Hamilton, 1822), was synthesized and characterized using an integrated (experimental and in silico) approach. The far-UV circular dichroism (CD) spectrum of this peptide was evaluated both in aqueous and membrane mimicking solvents (TFE, HFIP and Dioxane). The results indicate that kiss 1 peptide adopted helical, turn and ß conformations in membrane like environments. The near-UV CD spectroscopy was also carried out to examine the tertiary packing around aromatic residues of kiss 1 peptide and the peptide-membrane complex. The kiss 1 peptide exhibited little signal in water, but a prominent negative band was observed at around 275 nm when membrane mimetic solution was added. The observed ordered conformations of kiss 1 peptide in the different solvents indicated its potential biological activity which could enhance the secretion of gonadotropin-releasing hormone (GnRH) at BPG axis. The conformational information generated from the present study reinforces the application prospects of bioactive synthetic peptide analogs of kisspeptin 1 in improving the reproductive performances of important cultivable fish species.

Buffalo hepcidin: characterization of cDNA and study of antimicrobial property.

Hepcidin, a novel gene encoded, 25 residue, 2-3 KDa cysteine rich cationic peptide synthesized in liver cells play an important role in iron metabolism in addition to its antimicrobial activity. In this study hepcidin cDNA expressed on hepatocytes of Bubalus bubalis has been characterized and the antibacterial activity of buffalo hepcidin analog has been determined. The complete buffalo hepcidin sequence is of 324 bp and have an ORF of 249 nucleotides from 14 to 262. The sequence analysis at nucleotide level showed homology of 98.0% with cattle, 84.3% with pig, whereas with human, chimpanzee and gorilla 80.3%, 80.7% and 81.5% respectively. The percent identity of buffalo hepcidin cDNA to that of japanese macaque, dog, gibbon, mouse and norway rat are81.9%, 79.9%, 79.9%, 67.9% and 65.9% respectively. The hepcidin cDNA of buffalo and cattle showed a total difference of five nucleotides at position 15, 108, 225, 240 and 246 with no difference in deduced amino acid sequence. The base A(15), C(108), A(225), C(240), A(246) in cattle are replaced by G(15), G(108), G(225), T(240) and G(246) respectively in buffalo. The deduced amino acid sequence of buffalo preprohepcidin consists of 82 amino acids containing 10 strongly basic, 3 strongly acidic, 27 hydrophobic and 29 polar amino acids. The predicted molecular weight and isoelectric point (P(I)) for the peptide is 8883.67 Daltons and 8.804 respectively. Phylogenetic analysis both at nucleotide and amino acid level indicate that buffalo and cattle hepcidin sequences comprises one clad which is more closely related to pig compared to human. The chemically synthesized analog of buffalo hepcidin using Fmoc chemistry in solid phase had antibacterial activity.