Cloning, characterization and ontogenetic expression profile of RAG-2 and IgM in angelfish (Pterophyllum scalare).

Early larval developmental stages of fish are highly susceptible to opportunistic pathogens until the complete maturation of the lymphoid organs. Knowledge of the expression pattern of important markers of adaptive immune system during the ontogenetic development is essential before vaccinating the fish. In the present study, Pterophyllum scalare (angelfish) was taken to explore the relative expression profile of developmental markers of adaptive immunity, recombination activating gene-2 (RAG-2) and immunoglobulin M (IgM). The fishes were bred and early developmental stages (0-45 days post-hatched) were used to assess the expression profile. The genes, RAG-2 and IgM were cloned and sequenced with the base pair lengths of 1958 bp and 225 bp respectively. The mRNA expression of RAG-2 appeared at insignificant level at the first day of hatching, but the expression was significantly increased from 24 dph (days post-hatching) onwards and reached its peak at 27 dph. The results proved that the maturation of lymphoid organs was completed at 27 dph as the respective protein is involved in the V(D)J recombination, important for the maturation of lymphoid organs. A similar trend was also observed in the mRNA transcript levels of IgM gene and a significantly high expression was detected from 27 dph onwards. The present study suggested that the suitable time for vaccination in P. scalare could be taken at 27 dph, as the maturation and development of lymphoid organs is completed thus helps in stimulating the adaptive response of immunity against any pathogen.

Expression and activity of key lipases during the larval development of walking catfish (Clarias magur).

The study was conducted to investigate the expression and activity of key lipolytic enzymes during the ontogenetic development of Clarias magur. After partial characterization, the messenger RNA (mRNA) expression analysis of lipoprotein lipase (LPL), pancreatic triacylglycerol lipase (PL), and bile salt-activated lipase (BAL) genes along with the specific lipase activity were performed in larvae from Day 1 after hatching till 34-day posthatch (dph). Heterogeneous patterns of mRNA expression were shown by the important lipolytic enzymes and were detected before first exogenous feeding during the yolk-sac stage. LPL started increasing from 13 dph and peaked at 16 dph followed by a declining trend till 34 dph. However, the PL observed to be peaking at 9, 22, and 30 dph. Similarly, BAL showed an increasing trend from 11 to 22 dph with a significantly high level of mRNA expression at 16 dph. Later, the specific lipase activity was evaluated which appears at Day 1 after hatching with a progressive increase from 7 to 16 dph and a further declining trend afterwards with a peak at 22 dph. The results indicated the development of exocrine pancreas at 16 dph. Furthermore, the transcript levels and the activity of lipases were regulated with the age. Hence, the present study can be helpful in devising different strategies containing optimum lipid levels at a suitable stage of development for improving the survival during larval rearing. Furthermore, the study could be a baseline for elucidating the optimized dietary lipid levels of this catfish during its larval rearing.

In vitro screening of selected antiviral drugs against betanodavirus.

The inhibitory effects of ammonium chloride (NH4Cl) and chlorpromazine hydrochloride on betanodavirus were evaluated on Sahul Indian sea bass kidney (SISK) cell line. The cytotoxicity of different concentrations of NH4Cl (0.1 mM, 1 mM, 10 mM, 100 mM and 500 mM) and chlorpromazine hydrochloride (1 µM, 10 µM, 100 µM, 200 µM and 500 µM) were assessed in SISK cells using different cytotoxic assays. Among the selected concentrations, 0.1 mM, 1 mM and 10 mM of NH4Cl and chlorpromazine hydrochloride at the dose of 1 µM, 10 µM and 100 µM were found to be non-toxic to the SISK cell line and same were chosen for the trials against nodavirus. The presence of nodavirus in the infected cells was confirmed by cytopathic effect (CPE) and RT-PCR (Reverse transcriptase PCR). NH4Cl of 1 mM and 10 mM, and chlorpromazine hydrochloride of 10 µM and 100 µM could successfully inhibit betanodavirus infection in SISK cells, which was confirmed by indirect ELISA and real-time PCR analysis. The result further suggested that the chlorpromazine hydrochloride drug could be more effective in inhibiting the betanodavirus with much lower dose than NH4Cl which was more effective at a higher dose. The present study thus suggested that NH4Cl and chlorpromazine hydrochloride drugs could be successfully used for controlling the nodavirus infection in aquaculture.