A novel medium for the development of in vitro cell culture system from Penaeus monodon

Lack of a valid shrimp cell line has been hampering the progress of research on shrimp viruses. One of the reasons identified was the absence of an appropriate medium which would satisfy the requirements of the cells in vitro. We report the first attempt to formulate an exclusive shrimp cell culture medium (SCCM) based on the haemolymph components of Penaeus monodon prepared in isosmotic seawater having 27 % salinity. The SCCM is composed of 22 amino acids, 4 sugars, 6 vitamins, cholesterol, FBS, phenol red, three antibiotics, potassium dihydrogen phosphate and di-sodium hydrogen phosphate at pH 6.8–7.2. Osmolality was adjusted to 720 ± 10 mOsm kg-1 and temperature of incubation was 25 8C. The most appropriate composition was finally selected based on the extent of attachment of cells and their proliferation by visual observation. Metabolic activity of cultured cells was measured by MTT assay and compared with that in L-15 (29), modified L-15 and Grace’s insect medium, and found better performance in SCCM especially for lymphoid cells with 107 % increase in activity and 85 ± 9 days of longevity. The cells from ovary and lymphoid organs were passaged twice using the newly designed shrimp cell dissociation ‘‘cocktail’’.

“Development of lymphoid cell culture system from Penaeus monodon and molecular approaches for its transformation

Unveiling the molecular and regulatory mechanisms that prevent in vitro transformation in shrimp remains elusive in the development of continuous cell lines, with an arduous history of over 25 years (Jayesh et al., 2012). Despite presenting challenges to researchers in developing a cell line, the billion dollar aquaculture industry is under viral threat. In addition, the regulatory mechanisms that prevent in vitro transformation and carcinoma in shrimps might provide new leads for the development of anti-ageing and anti-cancer interventions in human (Vogt, 2011) and in higher vertebrates. This highlights the importance of developing shrimp cell lines, to bring out effective prophylactics against shrimp viruses and for understanding the mechanism that induce cancer and ageing in human.. Advances in molecular biology and various gene transfer technologies for immortalization of cells have resulted in the development of hundreds of cell lines from insects and mammals, but yet not a single cell line has been developed from shrimp and other marine invertebrates. With this backdrop, the research described in this thesis attempted to develop molecular tools for induced in vitro transformation in lymphoid cells from Penaeus monodon and for the development of continuous cell lines using conventional and novel technologies to address the problems at cellular and molecular level.

INVESTIGATIONS ON THE STRESS EFFECTS OF PETROLEUM HYDROCARBONS ON METAPENAEUS DOBSON (MIERS)

The present investigation has addressed the effects of PHC contaminated culture medium on the morphology, physiology and behaviour of shrimps. The shrimp Metapenaeus dobsoni is an important member of the crustacean animal community abounding the oil contaminated benthic regions of Cochin backwater system. Since it is known that true pollutants can disrupt the sustainability of ecosystems by its effect on species, populations and communities,a representative species was used for the study. The results discussed in this work is bound to help in understanding the ecotoxicant resistance that the animal may display under toxic conditions compared to dynamic steady-state systems in nature.

Modification of Linear Low Density Polyethylene for Improved Photo and Biodegradation

LLDPE was blended with poly (vinyl alcohol) and mechanical, thermal, spectroscopic properties and biodegradability were investigated. The biodegradability of LLDPE/PVA blends has been studied in two environments, viz. (1) a culture medium containing Vibrio sp. and (2) a soil environment over a period of 15 weeks. Nanoanatase having photo catalytic activity was synthesized by hydrothermal method using titanium-iso-propoxide. The synthesized TiO2 was characterized by X-Ray diffraction (XRD), BET studies, FTIR studies and scanning electron microscopy (SEM). The crystallite size of titania was calculated to be ≈ 6nm from the XRD results and the surface area was found to be about 310m2/g by BET method. SEM shows that nanoanatase particles prepared by this method are spherical in shape. Linear low density polyethylene films containing polyvinyl alcohol and a pro-oxidant (TiO2 or cobalt stearate with or without vegetable oil) were prepared. The films were then subjected to natural weathering and UV exposure followed by biodegradation in culture medium as well as in soil environment. The degradation was monitored by mechanical property measurements, thermal studies, rate of weight loss, FTIR and SEM studies. Higher weight loss, texture change and greater increments in carbonyl index values were observed in samples containing cobalt stearate and vegetable oil. The present study demonstrates that the combination of LLDPE/PVA blends with (I) nanoanatase/vegetable oil and (ii) cobalt stearate/vegetable oil leads to extensive photodegradation. These samples show substantial degradation when subsequent exposure to Vibrio sp. is made. Thus a combined photodegradation and biodegradation process is a promising step towards obtaining a biodegradable grade of LLDPE.

Investigations on the stress effects of petroleum hydrocarbons on Metapenaeus dobsoni (Miers)

The present investigation has addressed the eflects of PHC contaminated culture medium on the morphology, physiology and behaviour of shrimps] The shrimp Metapenaeus dobsoni is an important member of the crustacean animal community abounding the oil contaminated benthic regions of Cochin backwater system. Since it is known that true pollutants can disrupt the sustainability of ecosystems by its eflect on species, populations and communities, a representative species was used for the study. The results discussed in this work is bound to help in understanding the ecotoxicant resistance that the animal may display under toxic conditions compared to aynamic steaay-state systems in nature

Lipase Production by Candida rugosa

Strain improvement is one of the major objectives for maximizing the microbial production of industrially significant primary and secondary metabolites. This goal can be achieved by judicious tuning of the organisms by monitoring its growth parameters and optimizing adequate supply of micro and macro nutrients, inducers, pH, temperature and other factors which control fermentation. Though C. rugosa has been under extensive studies for lipases, maximum world production is only 36 units. In fact, in India, enhanced production conditions for lipases have not yet been initiated. C. rugosa has been cultivated in diverse environments like liquid, semi-solid, solid—state and immobilized conditions, though major emphasis is on SmF or suspension culture. Hence the present investigations mainly focused on increasing the yield by adjusting the physico-chemical growth parameters and to characterize the lipase isoforms secreted by C. rugosa in the culture medium. Maximum possible improved methods were investigated to achieve these objectives. Within this under-optimised background, enhancement of lipase production and its characterization were investigated, employing modified liquid, semi-solid, solid—state and immobilized fermentation strategies

Biodegradability Studies on LDPE-Starch Blends using Amylase-Producing Vibrios

Low-density polyethylene, (LDPE) was mixed with two grades of tapioca starch–lowgrade and high-grade. Various compositions were prepared and mechanical and thermal studies performed. The biodegradability of these samples was checked using a culture medium containing Vibrios (an amylase-producing bacteria), which was isolated from a marine benthic environment. The soil burial test and reprocessability of these samples were checked. The studies on biodegradability show that these blends are partially biodegradable. These low-density polyethylene-starch blends are reprocessable without sacrificing much of their mechanical properties

Effect of Amylase Producing Vibrios From the Benthic Environment on the Biodegradation of Low Density Polyethylene-Dextrin Blends

Low-density polyethylene was mixed with dextrin having different particle sizes (100, 200 and 300 mesh). Various compositions were prepared and their mechanical properties were evaluated and thermal studies have been carried out. Biodegradability of these samples has been checked using liquid culture medium containing Vibrios (an amylase producing bacteria), which were isolated from marine benthic environment. Soil burial test was done and reprocessability of these samples was evaluated. The results indicate that the newly prepared blends are reprocessable without sacrificing much of their mechanical properties. The biodegradability tests on these blends indicate that these are partially biodegradable

Establishment of Shrimp Cell Lines: Perception and Orientation

Development of continuous shrimp cell lines for effective investigation on shrimp viruses remains elusive with an arduous history of over 25 years. Despite presenting challenges to researchers in developing a cell line, the billion dollar aquaculture industry is under viral threat. Advances in molecular biology and various gene transfer technologies for immortalization of cells have resulted in the development of hundreds of cell lines from insects and mammals, but yet not a single cell line has been developed from shrimp and other marine invertebrates. Though improved growth and longevity of shrimp cells in vitro could be achieved by using modified growth media this did not make any leap to spontaneous transformation; probably due to the fact that shrimp cells inhibited neoplastic transformations. Oncogenic induction and immortalization are considered as the possible ways, and an exclusive medium for shrimp cell culture and an appropriate mode of transformation are crucial. In this review status of shrimp cell line development and its future orientation are discussed

Multifactorial interaction of growth factors on Penaeus monodon lymphoid cells and the impact of IGFs in DNA synthesis and metabolic activity in vitro

Development of continuous cell lines from shrimp is essential to investigate viral pathogens. Unfortunately, there is no valid cell line developed from crustaceans in general and shrimps in particular to address this issue. Lack of information on the requirements of cells in vitro limits the success of developing a cell line, where the microenvironment of a cell culture, provided by the growthmedium, is of prime importance. Screening and optimization of growth medium components based on statistical experimental designs have been widely used for improving the efficacy of cell culture media. Accordingly, we applied Plackett–Burman design and response surface methodology to study multifactorial interactions between the growth factors in shrimp cell culture medium and to identify the most important ones for growth of lymphoid cell culture from Penaeus monodon. The statistical screening and optimization indicated that insulin like growth factor-I (IGF-I) and insulin like growth factor-II (IGF-II) at concentrations of 100 and 150 ng ml-1, respectively, could significantly influence the metabolic activity and DNA synthesis of the lymphoid cells. An increase of 53 % metabolic activity and 24.8 % DNA synthesis could be obtained, which suggested that IGF-I and IGFII had critical roles in metabolic activity and DNA synthesis of shrimp lymphoid cells

Characterization of Linear Low-Density Polyethylene/ Poly(vinyl alcohol) Blends and Their Biodegradability by Vibrio sp. Isolated from Marine Benthic Environment

Increasing amounts of plastic waste in the environment have become a problem of gigantic proportions. The case of linear low-density polyethylene (LLDPE) is especially significant as it is widely used for packaging and other applications. This synthetic polymer is normally not biodegradable until it is degraded into low molecular mass fragments that can be assimilated by microorganisms. Blends of nonbiodegradable polymers and biodegradable commercial polymers such as poly (vinyl alcohol) (PVA) can facilitate a reduction in the volume of plastic waste when they undergo partial degradation. Further, the remaining fragments stand a greater chance of undergoing biodegradation in a much shorter span of time. In this investigation, LLDPE was blended with different proportions of PVA (5–30%) in a torque rheometer. Mechanical, thermal, and biodegradation studies were carried out on the blends. The biodegradability of LLDPE/PVA blends has been studied in two environments: (1) in a culture medium containing Vibrio sp. and (2) soil environment, both over a period of 15 weeks. Blends exposed to culture medium degraded more than that exposed to soil environment. Changes in various properties of LLDPE/PVA blends before and after degradation were monitored using Fourier transform infrared spectroscopy, a differential scanning calorimeter (DSC) for crystallinity, and scanning electron microscope (SEM) for surface morphology among other things. Percentage crystallinity decreased as the PVA content increased and biodegradation resulted in an increase of crystallinity in LLDPE/PVA blends. The results prove that partial biodegradation of the blends has occurred holding promise for an eventual biodegradable product