Insulin-like growth factor II, a marker gene for determining the optimum dietary protein level in clownfish Amphiprion ocellaris.

In vivo study was conducted to determine the role of insulin-like growth factors (IGF-II) related to dietary protein utilization. For this early juvenile stage of marine false clownfish Amphiprion ocellaris, 300 numbers of 20-day-old larvae (initial body weight of 18.2±0.027 mg/fish) were used as an experimental animal. Animals were fed for 12 weeks with different dietary protein levels (35, 40, 45, 50, and 55%), using Spirulina maxima as a major protein source. Proximate compositions and amino acid profile of the formulated diet were analyzed by the standard methods. Eventually, significant (P<0.05) mean, body weight gain, absolute growth rate, specific growth rate, and feed conversion ratio were obtained in the fishes fed with 50% of dietary protein, whereas, poor growth performance was noticed in 35% of the dietary protein level fed juveniles. The growth-responsible gene IGF-II expression studies showed the significant upregulation in the growth of the juveniles at 2.05±0.11 for 40%, 3.13±0.20 for 45%, 4.97±0.13 for 50%, and 4.33± 0.24 for 55%, which were higher than the control group 35%. The study concluded that 50% of dietary protein level is optimal for better growth indices in clownfish; A. ocellaris juveniles and IGF-II can be used as a potential marker gene to assess the growth indices in A. ocellaris.

Study on amyloodiniosis outbreak in captive-bred percula clownfish (Amphiprion percula) and improved control regimens.

The amyloodiniosis outbreak was documented with high mortality of percula clownfish (Amphiprion percula) and subsequently, various therapeutics were evaluated to control the infection. The affected fish exhibited symptoms such as discoloration of the skin and jerky movement with severe respiratory stress. Microscopic examination of the gill and body surface of the infested moribund fish showed the presence of brown coloured spherical shaped trophonts with the size range of 150-300 µM. Copper sulphate (10 ppm) and hydroxychloroquine phosphate (10 ppm) treatment with 14 days of continuous bath showed 76 ± 5.3 and 66 ± 4.3% survival rates. However, formalin (10 ppm) and malachite green (10 ppm) treated groups showed a survival of 41 ± 1.7 and 32.7 ± 1.2% respectively. The present results suggest that, use of copper sulphate to treat amyloodiniosis in clownfish will relatively more effective than other treatment options. The findings will be helpful to mitigate amyloodiniosis in marine finfish aquaculture, particularly marine ornamentals.

Multifunctional murrel caspase 1, 2, 3, 8 and 9: Conservation, uniqueness and their pathogen-induced expression pattern.

Caspases are evolutionarily conserved proteases which play fundamental role in apoptosis. Invasion of pathogen triggers the activation of caspases-mediated pro-inflammatory and pro-apoptotic pathways, where multifunctional caspases are involved. In striped murrel Channa striatus, epizootic ulcerative syndrome (EUS) causes endemics resulting in huge economic loss. Aphanomyces invadans, an oomycete is the primary causative agent of EUS which further induces secondary bacterial infections especially Aeromonas hydrophila. In order to get insights into the caspase gene family in C. striatus during EUS infection, we performed various physicochemical and structural analyses on the cDNA and protein sequences of five different murrel caspases namely CsCasp 1, 2, 3, 8 and 9. Sequence analysis of murrel caspase proteins showed that in spite of the conserved CASC domain, each caspase embraces some unique features which made them functionally different. Tissue distribution analysis showed that all the murrel caspases are highly expressed in one of the immune organs such as liver, kidney, spleen and blood cells. Further, to understand the role of caspase during EUS infection, modulation in expression of each caspase gene was analysed after inducing fungal and bacterial infection in C. striatus. Pathogen-induced gene expression pattern revealed an interesting fact that the expression of all the caspase genes reached a maximum level at 24 h post-infection (p.i) in case of bacteria, whereas it was 48 h in fungus. However, the initiation of elevated expression differed between each caspase based on their role such as pro-inflammatory, initiator and executioner caspase. Overall, the results suggested that the caspases in murrel are diverse in their structure and function. Here, we discuss the similarities and differences of five different murrel caspases.

Fish chemokines 14, 20 and 25: A comparative statement on computational analysis and mRNA regulation upon pathogenic infection.

In this study, we reported a molecular characterization of three CC chemokines namely, CsCC-Chem14, CsCC-Chem20 and CsCC-Chem25 which are were identified from the established cDNA library of striped murrel Channa striatus. Multiple sequence alignment of all the three chemokines revealed the presence of gene specific domains and motifs including small cytokine domain, IL8 like domain, receptor binding site and glycosaminoglycan (GAG) binding sites. Three dimensional structures of the chemokines under study showed an important facet on their anti-microbial property. Tissue specific mRNA expression showed that the CsCC-Chem14 is highly expressed in spleen, CsCC-Chem20 in liver and CsCC-Chem25 in trunk kidney. On challenge C. striatus with oomycete fungus Aphanomyces invadans, both CsCC-Chem20 and CsCC-Chem25 showed significant (P < 0.05) up-regulation compared to CsCC-Chem14. The increase in the expression levels of CsCC-Chem20 and CsCC-Chem25 due to infection showed that they are antimicrobial proteins. But considering the CsCC-Chem14 expression, it is found to be a constitutive chemokine and is involved in homeostatic function in spleen of C. striatus. C. striatus challenged with bacteria Aeromonas hydrophila also exhibited different up-regulation pattern in all the three chemokines at various time points. However, extensive studies are required to determine the functional activities of CsCC-Chem14, CsCC-Chem20 and CsCC-Chem25 in vitro and in vivo to gain more knowledge at the molecular and proteomic levels.

Dietary supplementation of Avicennia marina extract on immune protection and disease resistance in Amphiprion sebae against Vibrio alginolyticus.

The effect of Avicennia marina aqueous leaf extract on innate immune mechanisms such as total white blood cell counts (WBC), serum lysozyme activity, respiratory burst assay, alternative complement (ACH50) assay, phagocytic activity assay, disease resistance, gut bacteria, and survival rate of clownfish (Amphiprion sebae) against Vibrio alginolyticus is reported. Healthy fish challenged with V. alginolyticus (1 × 10(7) cells ml(-1)) were fed with diets supplemented (0, 1, 2, and 4%) with A. marina extract. The survival rate was 85% and 80% in infected fish fed with 4% and 8% supplementation diet; with 1% diet it was 70% while in the infected untreated group it was only 10%. The total gut bacteria flora was high in 8% and 4% supplementation diet groups with 2.8 × 10(5) and 4.7 × 10(4) cfu/g while it was 8.9 × 10(3) cfu/g in 1% diet group. The immunological parameters significantly increased on weeks 6 and 8 when infected fish were fed with 1% or 4% supplementation diet. This study reports that in clownfish challenged with V. alginolyticus, dietary administration of the 1% or 4% of A. marina extract improved the immune status and survival rate.