Quantification of intracellular polyhydroxyalkanoates by virtue of personalized flow cytometry protocol.

Polyhydroxyalkanoates (PHAs) are natural polyesters produced by microbes, a potential alternative to synthetic plastics. Various methods ranging from gravimetry to spectrophotometry are routinely used for qualitative analysis of extracted PHA. There is a great need for accurate quantification of intracellular PHA during bioprocess. Hence, the present study aims to improvise the existing Nile red-based flow cytometry protocol. It was achieved using respective cells in a non-PHA accumulating state as gating control to minimize non-specific staining. The optimal Nile red concentration required for PHA staining is 5 × 10(3) pg mL(-1), which is ~10(3)-fold less than that of earlier reports. Further, it was inferred that flow-based quantification was more accurate than the gravimetric method. The intracellular PHA content was highest in Pseudomonas sp. MNNG-S (52.06 %) among the Pseudomonas strains tested by the flow-based method. Both gravimetric and flow-based cell cycle analyses revealed that DNA synthesis (S phase) and PHA production (log phase) are synchronous at 24-48 h of culture. This study supports flow-based PHA quantification for real time online measurement of intracellular PHA for bioreactor monitoring, control and optimization enduing industrial applications.

Diel variations in the selected serum immune parameters in Oreochromis mossambicus.

Almost all metabolic processes in an organism alternate through high and low activity phases with a regular periodicity of nearly 24h. These daily/diel variations are governed by factors such as light, weather conditions, availability of food or predator activity. The immune system in fish is expected to follow the same routine based on external cues from the environment which it lives. The present study was carried out to investigate such daily/diel variations in selected immune parameters such as serum lysozyme and peroxidases activity, total serum globulin level and peripheral blood leukocyte count in Oreochromis mossambicus. The fish were maintained in semi natural condition (i.e.12L:12D). The results showed significant rise in serum peroxidases and lysozyme between 0200 h and 0600 h of the day and serum cortisol exhibited elevated level between 2200 h and 0600 h. Total serum globulin exhibited peak concentration from 1400 h to 1800 h. Thus suggesting the possibility of rhythmic functioning of immune system in O. mossambicus.

Immune response in the tilapia, Oreochromis mossambicus on exposure to tannery effluent.

The objective of the study was to investigate the effect of chronic exposure to sublethal concentrations of tannery effluent (TE) on the specific immune response and nonspecific immunity in tilapia, Oreochromis mossambicus. The effluent from the tannery was collected directly from a chrome-tanning factory situated in Dindigul district, Tamil Nadu, India. Apart from chromium (88.2 ppm), the effluent contained appreciable amount of calcium carbonate and sodium sulphate. Groups of fish (45-50 g) were exposed to 0.0053, 0.053 or 0.53% [0.1%, 1% or 10% LC50] of TE for 28 days. The specific immune response of fish was assessed by antibody response to heat-killed Aeromonas hydrophila by ELISA and bacterial agglutination assay. Nonspecific immune mechanisms were assessed in terms of serum lysozyme activity, production of intracellular reactive oxygen species (ROS) and reactive nitrogen intermediates (RNI) by peripheral blood leucocytes (PBL). The results indicate that chronic exposure of fish to 0.53% of TE, significantly suppressed antibody response, nonspecific serum lysozyme activity, and ROS and RNI production. Exposure to 0.053% (1% LC50) of TE also caused a similar suppressive effect though at a lesser degree. In conclusion, the study shows, that exposure to sublethal concentrations of TE, can lead to adverse effects on selected immune reactions in tilapia. Further, these findings may be important in terms of monitoring fish health and risk assessment during periods of fluctuating levels of pollutants in the natural and farm environments.

Immune response and disease resistance of Oreochromis mossambicus to Aeromonas hydrophila after exposure to hexavalent chromium.

The objective of this study was to investigate the effect of chronic exposure to sublethal concentrations of hexavalent chromium (K2Cr2O7) on the immune response and disease resistance of Oreochromis mossambicus (Peters) to bacterial Aeromonas hydrophila infection. Fish (45 to 50 g) were exposed to 0.005, 0.05, 0.5, and 5 mg l(-1) [0.01, 0.1, 1, and 10% LC50, respectively] of hexavalent chromium Cr (VI) for 28 d. The specific immune response was assessed by antibody response to A. hydrophila by bacterial agglutination assay, and to sheep red blood cells (SRBC) by plaque forming cell (PFC) assay. In addition, nonspecific immune mechanisms were assessed by serum lysozyme activity and reactive nitrogen intermediates, the latter in terms of nitric oxide (NO) production by peripheral blood leucocytes. Overall immunity was assessed by disease resistance against live virulent A. hydrophila. The study clearly indicated that chronic exposure of fish to 0.5 and 5 mg l(-1) of chromium (VI) decreased both nonspecific and specific parameters of the immune system, which resulted in a lower disease resistance to A. hydrophila. Interestingly, 0.05 mg l(-1) of Cr (VI) enhanced disease resistance and both nonspecific and specific immune responses to A. hydrophila. Our study revealed a concentration-dependent modulation of the immune system by chromium (VI), as demonstrated by suppressive or stimulatory effects on lymphocytes, lysozyme, phagocytic killing mechanisms, and disease resistance in O. mossambicus.

Effect of sex ratio on the immune system of Oreochromis mossambicus (Peters).

The social environment of fish has a crucial role to play on the immune system and hence on the overall health status. Stressors of social origin such as dominance, subordination, and fight for mate have a major impact on the immune system of fish. The present study was designed with the objective of finding the effect of sex ratio of the population on the immune system of Oreochromis mossambicus. Groups of fish were maintained for 28 days in three different sex ratios i.e., (i) all-male (ii) all-female (iii) equal male and female (mixed). The specific immune response of fish was assessed by antibody response to Aeromonas hydrophila by ELISA and bacterial agglutination assay, and to SRBC by plaque forming cell assay. Nonspecific immune mechanisms were assessed in terms of serum lysozyme activity, production of intracellular reactive oxygen species (ROS) and reactive nitrogen species (RNS) by peripheral blood leukocytes. Disease resistance against live, virulent A. hydrophila was performed to assess the overall functional immunity. The results showed that antibody responses and numbers of antibody producing cells were increased in fish in the equal male and female sex ratio group compared to fish in monosex ratio groups. Similar enhancement was also observed in nonspecific serum lysozyme level and the ROS and RNS production. The host resistance test revealed that enhanced immunity in equal male and female sex ratio group was protective against A. hydrophila infection. The study clearly reveals positive and negative effect of sex ratio on the immune system of O. mossambicus.