Extraction of Glucan from Acremonium Diospyri and Its Application in Macrobrachium Rosenbergii Larval Rearing System Along With Bacterians as Microspheres

Present work is aimed at development of an appropriate microbial technology for protection of larvae of macrobrachium rosenbergii from disease and to increase survival rate in hatcheries. Application of immunostimulants to activate the immune system of cultured animals against pathogen is the widely accepted alternative to antibiotics in aquaculture. The most important immunostimulant is glucan. Therefore a research programme entitled as extraction of glucan from Acremonium diospyri and its application in macrobrachium rosenbergii larval rearing system along with bacterians as microspheres. The main objectives of the study are development of aquaculture grade glucan from acremonium diospyri, microencapsulated drug delivery system for the larvae of M. rosenbergii and microencapsulated glucan with bacterian preparation for the enhanced production of M. rosenbergii in larval rearing system. Based on the results of field trials microencapsulated glucan with bacterin preparation, it is concluded that the microencapsulated preparation at a concentration of 25g per million larvae once in seven days will enhance the production and quality seed of M. rosenbergii.

Antibody Based Diagnostic for Detection Of Vibrios and their Biological Control using Antagonistic Bacteria in Macrobrachium Rosenbergii Larval Rearing Systems

The main objective of the work undertaken here was to develop an appropriate microbial technology to protect the larvae of M.rosenbergii in hatchery from vibriosis. This technology precisely is consisted of a rapid detection system of vibrios and effective antagonistic probiotics for the management of vibrios. The present work was undertaken with the realizations that to stabilize the production process of commercial hatcheries an appropriate, comprehensive and fool proof technology is required primarily for the rapid detection of Vibrio and subsequently for its management. Nine species of Vibrio have been found to be associated with larvae of M. rosenbergii in hatchery. Haemolytic assay of the Vibrio and Aeromonas on prawn blood agar showed that all isolates of V. alginolyticus and Aeromonas sp., from moribund, necrotized larve were haemolytic and the isolates of V.cholerae, V.splendidus II, V.proteolyticus and V.fluvialis from the larvae obtained from apparently healthy larval rearing systems were non-haemolytic. Hydrolytic enzymes such as lipase, chitinase and gelatinase were widespread amongst the Vibrio and Aeromonas isolates. Dominance of V.alginolyticus among the isolates from necrotic larvae and the failure in isolating them from rearing water strongly suggest that they infect larvae and multiply in the larval body and cause mortality in the hatchery. The observation suggested that the isolate V. alginolyticus was a pathogen to the larvae of M.rosenbergii. To sum up, through this work, nine species of Vibrio and genus Aeromonas associated with M.rosenbergii larval rearing systems could be isolated and segregated based on the haemolytic activity and the antibodies (PA bs) for use in diagnosis or epidemiological studies could be produced, based on a virulent culture of V.alginolyticus. This could possibly replace the conventional biochemical tests for identification. As prophylaxis to vibriosis, four isolates of Micrococcus spp. and an isolate of Pseudomonas sp. could be obtained which could possibly be used as antagonistic probiotics in the larval rearing system of M.rosenbergii.

Culture and Nutritional Enrichment of the Rotifer Brachionus Rotundiformis ( Tschugunoff ) for the Rearing of Marine Fin Fish and Shrimp Larvae

The present study is an attempt to standardize the environmental condition like pH, salinity and photoperiod, and also the feed for the maximum production of rotifers. Considering the deficiency of essential fatty acids in rotifers, enrichment experiments were carried out and fatty acids profile were analysed. Attempts were made to improve the production of clown fish (Amphiprion sebae) juveniles using enriched rotifers. Attempts were also made to rear various larval stages of Penaeus monodon with enriched rotifers as a substitute for Artemia nauplii.

Biocontrol of Vibrio harveyi in Penaeus monodon larval rearing systems employing probiotics and vibriophages

National Centre for Aquatic Animal Health, School of Environmental Studies, Cochin University of Science and Technology.

Apptication of Biofloc Technology (BFI) in Ihe nunery rearing and farming of giant freshwater prawn, Macrobrachillm rosenbergii (de Man)

This thesis Entitled Application of Biofloc technology (BFT) In the Nursery Rearing and Farming of Giant Freshwater Prawn,Macrobrachium Rosenbergii(De Man). Aquaculture, rearing plants and animals under controlled conditions is growing with an annual growth rate of 8.3% in the period 1970-2008 (FAO, 2010). This trend of growth is essential for the supply of protein-rich food for ever increasing world population. But growth and development of aquaculture should be in sustainable manner, preferably without jeopardizing the aquatic environment.In the present study, the application of BFT in the nursery rearing and farming ofgiant freshwater prawn, M. rosenbergii, is attempted. The result of the study is organised into eight chapters. In the first chapter, the subject is adequately introduced. Various types of aquaculture practices followed, development and status of Indian aquaculture, present status of freshwater pravm culture, BF T and its use for the sustainable aquaculture systems, theory of BFT based aquaculture practices, hypothesis, objective and outline of the thesis are described. An extensive review of literature on studies carried out so far on biofloc based aquaculture are given in chapter 2. The third chapter deals with the application of BFT in the primary nursery phase of freshwater prawn. Several workers suggested the need for an intermediate nursery phase in the culture system of freshwater prawn for the successful production. Thirty day experiment was conducted to study the effect of BFT on the water quality, and animal welfare under the various stocking densities. The study concluded that stocking finfishes in biofloc-based monoculture system of freshwater prawns has the potential of increasing total yield. Prawns having a higher commercial value than finfishes besides ensuring economic sustainability. Results showed that prawn yield and survival was better in catla dominated tanks. Based on the results of the study, it is recommended to incorporate 25% rohu and 75% catla in the biofloc-based culture system of giant freshwater prawns. The results of the present study also recommend to stock relatively larger catla for biofloc-based culture system. Fish production was also higher in the 100% catla tank. When catla was added in higher percentages it should ensured that the hiding objects in the culture ponds shall be used in order to reduce the chance of cannibalism among prawns. rohu and catla equally have the ability to harvest the biofloc, catla consumes the planktonic contributes in the floc whereas rohu grazed on the bacterial consortium suspended in the water column. In Chapter 8, recommendations and future research perspectives in the field of biofloc based aquaculture is presented.

Studies on Reproductive Biology euui Mass Larval Rearing of Selected Species of Macrobrach

In India scientific commercial farming of Macrobrachium is yet to get a start, the major constraint being the lack of seed. Seed collection from the natural sources alone may not be sufficient, and in such cases seed production in hatcheries is inevitable. This necessitates knowledge of the reproductive biology of the species technology development for brood stock and larval rearing, and the optimum conditions required for larval development to undertake large scale seed production.The present study was taken up with the major objectives of developing a proper, simple and feasible technology for the larval rearing of Macrobrachium, with special emphasis on. mass larval rearing suitable for commercial level operations.

Application of Biofloc Technology (BFT) in the nursery rearing and farming of giant freshwater prawn, Macrobrachium rosenbergii (deMan)

In India a study conducted by CIFE and CIBA (1997), concluded that shrimp farming does more good than harm and it is not eco-unfriendly (Krishnan and Birthal, 2002). Upsurge in coastal aquaculture activity induced by high profitability is reported to have caused adverse impacts on coastal ecosystems and social environments (Parthasarathy and Nirmala, 2000). The crustacean farming sector has received criticism for excessive use of formulated feed containing high protein, of which around 50% gets accumulated at the pond bottom as unconsumed (Avnimelech, 1999; Hari et al., 2004, 2006). The wasted feeds undergo the process of degradation and results in the release of toxic metabolites to the culture system. Reduction of protein in the feed, manipulation and utilisation of natural food in the culture system are the remedy for the above problems. But before reducing the feed protein, it should be confirmed that the feed with reduced protein is not affecting the growth and health of the cultured animal. In the present study, biofloc technology is identified as one of the innovative technologies for ensuring the ecological and environmental Sustainability and examines the compatibility of BFT for the sustainable aquaculture of giant prawn, M. rosenbergii. This thesis starts with a general introduction (Chapter-1), a brief review of the most relevant literature (Chapter-2), results of various experiments (Chapter-3-6), summary (Chapter-7) and recommendations and future research perspectives in the field of biofloc based aquaculture (Chapter – 8). The major objectives of this thesis are, to improve the ecological and economical sustainability of prawn farming by the applicationof BFT and to improve the nutrient utilisation in aquaculture systems.

A brackishwater isolate of Pseudomonas PS-102, a potential antagonistic bacterium against pathogenic vibrios in penaeid and non-penaeid rearing systems

A Pseudomonas sp PS-102 recovered from Muttukkadu brackish water lagoon, situated south of Chennai, showed significant activity against a number of shrimp pathogenic vibrios. Out of the 112 isolates of bacterial pathogens comprising Vibrio harveyi, V. vulnificus, V. parahaemolyticus, V. alginolyticus, V. fluvialis, and Aeromonas spp, 73% were inhibited in vitro by the cell-free culture supernatant of Pseudomonas sp PS-102 isolate. The organism produced yellowish fluorescent pigment on King's B medium, hydrolysed starch and protein, and produced 36.4% siderophore units by CAS assay and 32 μM of catechol siderophores as estimated by Arnow's assay. The PS-102 isolate showed wide ranging environmental tolerance with, temperatures from 25 to 40 °C, pH from 6 to 8, salinity from 0 to 36 ppt, while the antagonistic activity peaked in cultures grown at 30 °C, pH 8.0 and at 5 ppt saline conditions. The antagonistic activity of the culture supernatant was evident even at 30% v / v dilution against V. harveyi. The preliminary studies on the nature of the antibacterial action indicated that the antagonistic principle as heat stable and resistant to proteolytic, lipolytic and amylolytic enzymes. Pseudomonas sp PS 102 was found to be safe to shrimp when PL-9 stage were challenged at 107 CFU ml−1 and by intramuscular injection into of ∼5 g sub-adults shrimp at 105 to 108 CFU. Further, its safety in a mammalian system, tested by its pathogenicity to mice, was also determined and its LD50 to BALB/c mice was found to be 109 CFU. The results of this study indicated that the organism Pseudomonas sp PS 102 could be employed as a potential probiont in shrimp and prawn aquaculture systems for management and control of bacterial infections

A marine bacterium, Micrococcus MCCB 104, antagonistic to vibrios in prawn larval rearing systems

marine bacterium, Micrococcus MCCB 104, isolated from hatchery water, demonstrated extracellular antagonistic properties against Vibrio alginolyticus, V. parahaemolyticus, V. vulnificus, V. fluviallis, V. nereis, V. proteolyticus, V. mediterranei, V. cholerae and Aeromonas sp., bacteria associated with Macrobrachium rosenbergii larval rearing systems. The isolate inhibited the growth of V. alginolyticus during co-culture. The antagonistic component of the extracellular product was heat-stable and insensitive to proteases, lipase, catalase and α-amylase. Micrococcus MCCB 104 was demonstrated to be non-pathogenic to M. rosenbergii larvae

Penaeus monodon larvae can be protected from Vibrio harveyi infection by pre-emptive treatment of a rearing system with antagonistic or non-antagonistic bacterial probiotics

This study shows that the disease resistance and survival rate of Penaeus monodon in a larval rearing systems can be enhanced by supplementing with antagonistic or non-antagonistic probiotics. The antagonistic mode of action of Pseudomonas MCCB 102 and MCCB 103 against vibrios was demonstrated in larval mesocosm with cultures having su⁄cient concentration of antagonistic compounds in their culture supernatant. Investigations on the antagonistic properties of Bacillus MCCB 101, Pseudomonas MCCB 102 and MCCB 103 and Arthrobacter MCCB 104 against Vibrio harveyi MCCB111under in vitro conditions revealed that Pseudomonas MCCB 102 and MCCB 103 were inhibitory to the pathogen.These inhibitory propertieswere further con¢rmed in the larval rearing systems of P. monodon. All these four probionts signi¢cantly improved larval survival in long-term treatments as well as when challengedwith a pathogenic strain ofV. harveyiMCCB111. We could demonstrate that Pseudomonas MCCB 102 andMCCB103 accorded disease resistance and a higher survival rate in P. monodon larval rearing systems throughactive antagonism of vibrios,whereas Bacillus MCCB 101 and Arthrobacter MCCB 104 functioned as probiotics through immunostimulatory and digestive enzyme-supporting modes of action.

Development of nitrifying bacterial consortia for immobilizing in nitrifying bioreactors designed for penaeid and non-penaeid larval rearing systems in the tropics

Two ammonia oxidizing (AMOPCU-1 and AMONPCU-1) and two nitrite oxidizing (NIOPCU-1 and NIONPCU-1) consortia for activating nitrifying bioreactors and thereby establishing nitrification in penaeid and non-penaeid hatchery systems were developed by enrichment. For further amplification of the consortia a simple medium having seawater (either salinity 30 ‰ or 15 ‰) as base, supplemented with NH4+-N/NO2--N and PO4- and pH adjusted to 8 was identified. During the amplification in a fermentor the consortia exhibited excessive wall growth and diminished their yield coefficient posing difficulty in harvesting the cells completely. The consortia consisted of both Gram negative and Gram-positive bacterial cells embedded in a mucilaginous matrix of glycocalyx - like material presumably composed of polysaccharides. The consortia besides being useful in activating nitrifying bioreactors developed for shrimp/prawn hatchery systems can also be used as bioaugmentors in the bioremediation of ammonia and nitrite toxicity in aquaculture systems.

Antimicrobial activity of chitosan against vibrios from freshwater prawn Macrobrachium rosenbergii larval rearing systems

Chitosan is a biocompatible and biodegradable natural polymer with established antimicrobial properties against specific microorganisms. The present study demonstrates its antibacterial activity against 48 isolates of Vibrio species from prawn larval rearing systems. The antibacterial activity had a positive correlation with the concentration of chitosan. This work opens up avenues for using chitosan as a prophylactic biopolymer for protecting prawn larvae from vibriosis.

Physiological analysis of central and peripheral insect circadian pacemaker neurons

Alle bisher untersuchten Lebewesen besitzen (circadiane) innere Uhren, die eine endogene Perioden-länge von ungefähr 24 Stunden generieren. Eine innere Uhr kann über Zeitgeber mit der Umwelt synchronisiert werden und ermöglicht dem Organismus, rhythmische Umweltveränderungen vorweg zu nehmen. Neben einem zentralen Schrittmacher, der Physiologie und Verhalten des Organismus steuert, gibt es in unterschiedlichen Organen auch periphere Uhren, die die zeitlichen Abläufe in der spezifischen Funktion dieser Organe steuern. In dieser Arbeit sollten zentrale und periphere Schrittmacherneurone von Insekten physiologisch untersucht und verglichen werden. Die Neurone der akzessorischen Medulla (AME) von Rhyparobia maderae dienten als Modellsystem für zentrale Schrittmacher, während olfaktorische Rezeptorneurone (ORNs) von Manduca sexta als Modellsystem für periphere Schrittmacher dienten. Die zentralen Schrittmacherneurone wurden in extrazellulären Ableitungen an der isolierten AME (Netzwerkebene) und in Patch-Clamp Experimenten an primären AME Zellkulturen (Einzelzellebene) untersucht. Auf Netzwerkebene zeigten sich zwei charakteristische Aktivitätsmuster: regelmäßige Aktivität und Wechsel zwischen hoher und niedriger Aktivität (Oszillationen). Es wurde gezeigt, dass Glutamat ein Neurotransmitter der weitverbreiteten inhibitorischen Synapsen der AME ist, und dass in geringem Maße auch exzitatorische Synapsen vorkommen. Das Neuropeptid pigment-dispersing factor (PDF), das von nur wenigen AME Neuronen exprimiert wird und ein wichtiger Kopplungsfaktor im circadianen System ist, führte zu Hemmungen, Aktivierungen oder Oszillationen. Die Effekte waren transient oder langanhaltend und wurden wahrscheinlich durch den sekundären Botenstoff cAMP vermittelt. Ein Zielmolekül von cAMP war vermutlich exchange protein directly activated by cAMP (EPAC). Auf Einzelzellebene wurde gezeigt, dass die meisten AME Neurone depolarisiert waren und deshalb nicht feuerten. Die Analyse von Strom-Spannungs-Kennlinien und pharmakologische Experimente ergaben, dass unterschiedliche Ionenkanäle vorhanden waren (Ca2+, Cl-, K+, Na+ Kanäle sowie nicht-spezifische Kationenkanäle). Starke, bei hohen Spannungen aktivierende Ca2+ Ströme (ICa) könnten eine wichtige Rolle bei Ca2+-abhängiger Neurotransmitter-Ausschüttung, Oszillationen, und Aktionspotentialen spielen. PDF hemmte unterschiedliche Ströme (ICa, IK und INa) und aktivierte nicht-spezifische Kationenströme (Ih). Es wurde angenommen, dass simultane PDF-abhängige Hyper- und Depolarisationen rhythmische Membranpotential-Oszillationen verursachen. Dieser Mechanismus könnte eine Rolle bei PDF-abhängigen Synchronisationen spielen. Die Analyse peripherer Schrittmacherneurone konzentrierte sich auf die Charakterisierung des olfaktorischen Corezeptors von M. sexta (MsexORCO). In anderen Insekten ist ORCO für die Membran-Insertion von olfaktorischen Rezeptoren (ORs) erforderlich. ORCO bildet Komplexe mit den ORs, die in heterologen Expressionssystemen als Ionenkanäle fungieren und Duft-Antworten vermitteln. Es wurde die Hypothese aufgestellt, dass MsexORCO in pheromonsensitiven ORNs in vivo nicht als Teil eines ionotropen Rezeptors sondern als Schrittmacherkanal fungiert, der unterschwellige Membranpotential-Oszillationen generiert. MsexORCO wurde mit vermeintlichen Pheromonrezeptoren in human embryonic kidney (HEK 293) Zellen coexprimiert. Immuncytochemie und Ca2+ Imaging Experimente zeigten sehr schwache Expressionsraten. Trotzdem war es möglich zu zeigen, dass MsexORCO wahrscheinlich ein spontan-aktiver, Ca2+-permeabler Ionenkanal ist, der durch den ORCO-Agonisten VUAA1 und cyclische Nucleotide aktiviert wird. Außerdem wiesen die Experimente darauf hin, dass MsexOR-1 offensichtlich der Bombykal-Rezeptor ist. Eine weitere Charakterisierung von MsexORCO in primären M. sexta ORN Zellkulturen konnte nicht vollendet werden, weil die ORNs nicht signifikant auf ORCO-Agonisten oder -Antagonisten reagierten.

The use of insect cells to identify potent and selective inhibitors of the replication of the Dengue and Chikungunya viruses and unravel their molecular mechanism of action

Dengue and Chikungunya viruses cause the most important arthropod-borne viral infections for humans. These viruses are predominant in tropical and subtropical regions. In addition, these viruses are predominant in tropical and subtropical regions. Dengue mortality rate is around 1.2 to 3.5% and deaths due to chikungunya fever are around 1 in 1000; however, half of chikungunya-infected patients evolve into a chronic state that can persist for months up to years. There are no antiviral drugs available for DENV and CHIKV treatment and prevention. Moreover, vector control strategies have failed so far. Thus, the development of potent inhibitors for a broad spectrum of RNA viruses is urgently needed. We established and characterized a new embryonic insect cell line from Culex quinquefasciatus mosquito. Also we established the flaviviruses and alphavirus replication, both in C6/36 and Lulo insect cell lines, as well as in Vero cell line. In addition we carried out a reference compound library and reference panel of assays and data for DENV, which provides a benchmark for further studies. During this study, a panel of 9 antiviral molecules, with proven in vitro anti-dengue virus activity and that act at different stages of the DENV life cycle, was selected. Finally, Favipiravir or T-705, was identified as inhibitor in vitro and in vivo of alphaviruses and the mutation K291R in nsP4, which is responsible of the polymerase activity, was found as the mode of action in CHIKV. Interestingly, lysine in motif F1 is also highly conserved in positive-stranded RNA viruses and this might explain the broad spectrum of T-705 antiviral activity.