Studies on some aspects of the reproductive physiology of the female grey mullet mugil cephalus l.

Aquaculture is the dynamic pursuit of production of organisms from water a process analogous to agriculture on land. The field of aquaculture is an emerging bioindustry, based upon the culture and husbandry of economically utilizable aquatic organisms. Of late, there has been a global upsurge for aquaculture, the main reasons for which include the requirement of protein source for the increasing world population, the decision by various world nations to increase the fish yield by developing unutilized or partially utilized water bodies and depletion of natural stock which is evident in recent years due to excessive exploitation .The present study has been taken up on the reproductive physiology of the female grey mullet, M. cephalus. The thesis is presented in seven chapters. In the present study, variations in the major biochemical parameters namely, moisture, proteins, lipids, carbohydrates cholesterol, carotenoid, ash, calcium and iron in four tissues E. muscle, liver, ovary and bloodserum of cephalus have been analysed at different maturity stages.

Virulence Potential And Antibiotic Susceptibility Pattern Of Motile Aeromonads Associated With Freshwater Ornamental Fish Culture Systems: A Possible Threat To public Health

Aeromonas spp. are ubiquitous aquatic organisms, associated with multitude of diseases in several species of animals, including fishes and humans. In the present study, water samples from two ornamental fish culture systems were analyzed for the presence of Aeromonas. Nutrient agar was used for Aeromonas isolation, and colonies (60 No) were identified through biochemical characterization. Seven clusters could be generated based on phenotypic characters, analyzed by the programme NTSYSpc, Version 2.02i, and identified as: Aeromonas caviae (33.3%), A. jandaei (38.3%) and A. veronii biovar sobria (28.3%). The strains isolated produced highly active hydrolytic enzymes, haemolytic activity and slime formation in varying proportions. The isolates were also tested for the enterotoxin genes (act, alt and ast), haemolytic toxins (hlyA and aerA), involved in type 3 secretion system (TTSS: ascV, aexT, aopP, aopO, ascF–ascG, and aopH), and glycerophospholipid-cholesterol acyltransferase (gcat). All isolates were found to be associated with at least one virulent gene. Moreover, they were resistant to frequently used antibiotics for human infections. The study demonstrates the pathogenic potential of Aeromonas, associated with ornamental fish culture systems suggesting the emerging threat to public health

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’’.

Characterization and virulence potential of phenotypically diverse Aeromonas veronii isolates recovered from moribund freshwater ornamental fishes of Kerala, India

In the present study, we investigated the involvement of Aeromonas spp. in eliciting disease outbreaks in freshwater ornamental fishes across the state of Kerala, India. We investigated three incidences of disease, in which the moribund fishes exhibited clinical signs such as haemorrhagic septicemia (in gouramy, Trichogaster sp.), dropsy (in Oscar, Astronotus ocellatus) and tail rot/fin rot (in gold fish, Carassius carassius). Pure cultures (n = 20 from each fish; 60 in total) of Aeromonas spp. were recovered from the abdominal fluid as well as from internal organs of affected fishes, although they could not be identified to species level because of the variations in their phenotypic characters. The molecular fingerprinting of the isolates using Enterobacterial Repetitive Intergenic Consensus PCR proved the genetic diversity of the isolates from the three sites. The phylogenetic trees constructed using concatenated sequences (using 16S rRNA, gyrA, gyrB and rpoD genes) indicated that they were related to Aeromonas veronii. They exhibited marked cytotoxic and haemolytic activity, which were responsible for the pathogenic potential of the isolates. The isolates possessed multiple virulence genes such as enterotoxins (act and alt), haemolytic toxins (aerA and hlyA), genes involved in type III secretion system (ascV, aexT and ascF–ascG), glycerophospholipid-cholesterol acyltransferase (gcat) and a type IV pilus (tapA) gene, as determined by PCR. Virulence of representative isolates to goldfish was also tested, and we found LD50 values of 104.07–105.35 cfu/fish. Furthermore, the organisms could be recovered as pure cultures from the lesions as well as from the internal organs.

Acute salinity stress alters the haemolymph metabolic profile of Penaeus monodon and reduces immunocompetence to white spot syndrome virus infection

Influence of acute salinity stress on the immunological and physiological response of Penaeus monodon to white spot syndrome virus (WSSV) infection was analysed. P. monodon maintained at 15‰ were subjected to acute salinity changes to 0‰ and 35‰ in 7 h and then challenged orally with WSSV. Immune variables viz., total haemocyte count, phenol oxidase activity (PO), nitroblue tetrazolium salt (NBT) reduction, alkaline phosphatase activity (ALP), acid phosphatase activity (ACP) and metabolic variables viz., total protein, total carbohydrates, total free amino acids (TFAA), total lipids, glucose and cholesterol were determined soon after salinity change and on post challenge days 2 (PCD2) and 5 (PCD5). Acute salinity change induced an increase in metabolic variables in shrimps at 35‰ except TFAA. Immune variables reduced significantly (Pb0.05) in shrimps subjected to salinity stress with the exception of ALP and PO at 35‰ and the reduction was found to be more at 0‰. Better performance of metabolic and immune variables in general could be observed in shrimps maintained at 15‰ that showed significantly higher post challenge survival following infection compared to those under salinity stress. Stress was found to be higher in shrimps subjected to salinity change to lower level (0‰) than to higher level (35‰) as being evidenced by the better immune response and survival at 35‰. THC (Pb0.001), ALP (Pb0.01) and PO (Pb0.05) that together explained a greater percentage of variability in survival rate, could be proposed as the most potential health indicators in shrimp haemolymph. It can be concluded from the study that acute salinity stress induces alterations in the haemolymph metabolic and immune variables of P. monodon affecting the immunocompetence and increasing susceptibility to WSSV, particularly at low salinity stress conditions

Soft Computing Approach for Optimization of siRNA Efficiency Prediction for Post-transcriptional Gene Silencing

Post-transcriptional gene silencing by RNA interference is mediated by small interfering RNA called siRNA. This gene silencing mechanism can be exploited therapeutically to a wide variety of disease-associated targets, especially in AIDS, neurodegenerative diseases, cholesterol and cancer on mice with the hope of extending these approaches to treat humans. Over the recent past, a significant amount of work has been undertaken to understand the gene silencing mediated by exogenous siRNA. The design of efficient exogenous siRNA sequences is challenging because of many issues related to siRNA. While designing efficient siRNA, target mRNAs must be selected such that their corresponding siRNAs are likely to be efficient against that target and unlikely to accidentally silence other transcripts due to sequence similarity. So before doing gene silencing by siRNAs, it is essential to analyze their off-target effects in addition to their inhibition efficiency against a particular target. Hence designing exogenous siRNA with good knock-down efficiency and target specificity is an area of concern to be addressed. Some methods have been developed already by considering both inhibition efficiency and off-target possibility of siRNA against agene. Out of these methods, only a few have achieved good inhibition efficiency, specificity and sensitivity. The main focus of this thesis is to develop computational methods to optimize the efficiency of siRNA in terms of “inhibition capacity and off-target possibility” against target mRNAs with improved efficacy, which may be useful in the area of gene silencing and drug design for tumor development. This study aims to investigate the currently available siRNA prediction approaches and to devise a better computational approach to tackle the problem of siRNA efficacy by inhibition capacity and off-target possibility. The strength and limitations of the available approaches are investigated and taken into consideration for making improved solution. Thus the approaches proposed in this study extend some of the good scoring previous state of the art techniques by incorporating machine learning and statistical approaches and thermodynamic features like whole stacking energy to improve the prediction accuracy, inhibition efficiency, sensitivity and specificity. Here, we propose one Support Vector Machine (SVM) model, and two Artificial Neural Network (ANN) models for siRNA efficiency prediction. In SVM model, the classification property is used to classify whether the siRNA is efficient or inefficient in silencing a target gene. The first ANNmodel, named siRNA Designer, is used for optimizing the inhibition efficiency of siRNA against target genes. The second ANN model, named Optimized siRNA Designer, OpsiD, produces efficient siRNAs with high inhibition efficiency to degrade target genes with improved sensitivity-specificity, and identifies the off-target knockdown possibility of siRNA against non-target genes. The models are trained and tested against a large data set of siRNA sequences. The validations are conducted using Pearson Correlation Coefficient, Mathews Correlation Coefficient, Receiver Operating Characteristic analysis, Accuracy of prediction, Sensitivity and Specificity. It is found that the approach, OpsiD, is capable of predicting the inhibition capacity of siRNA against a target mRNA with improved results over the state of the art techniques. Also we are able to understand the influence of whole stacking energy on efficiency of siRNA. The model is further improved by including the ability to identify the “off-target possibility” of predicted siRNA on non-target genes. Thus the proposed model, OpsiD, can predict optimized siRNA by considering both “inhibition efficiency on target genes and off-target possibility on non-target genes”, with improved inhibition efficiency, specificity and sensitivity. Since we have taken efforts to optimize the siRNA efficacy in terms of “inhibition efficiency and offtarget possibility”, we hope that the risk of “off-target effect” while doing gene silencing in various bioinformatics fields can be overcome to a great extent. These findings may provide new insights into cancer diagnosis, prognosis and therapy by gene silencing. The approach may be found useful for designing exogenous siRNA for therapeutic applications and gene silencing techniques in different areas of bioinformatics.