Pyocyanin (5-methyl-1-hydroxyphenazine) produced by Pseudomonas aeruginosa as antagonist to vibrios in aquaculture: overexpression, downstream process and toxicity

Pyocyanin is a versatile and multifunctional phenazine, widely used as a bio-control agent. Besides its toxicity in higher concentration, it has been applied as bio-control agents against many pathogens including the Vibrio spp. in aquaculture systems. The exact mechanism of the production of pyocyanin in Pseudomonas aeruginosa is well known, but the genetic modification of pyocyanin biosynthetic pathways in P. aeruginosa is not yet experimented to improve the yield of pyocyanin production. In this context, one of the aims of this work was to improve the yield of pyocyanin production in P. aeruginosa by way of increasing the copy number of pyocyanin pathway genes and their over expression. The specific aims of this work encompasses firstly, the identification of probiotic effect of P. aeruginosa isolated from various ecological niches, the overexpression of pyocyanin biosynthetic genes, development of an appropriate downstream process for large scale production of pyocyanin and its application in aquaculture industries. In addition, this work intends to examine the toxicity of pyocyanin on various developmental stages of tiger shrimp (Penaeus monodon), Artemia nauplii, microbial consortia of nitrifying bioreactors (Packed Bed Bioreactor, PBBR and Stringed Bed Suspended Bioreactor, SBSBR) and in vitro cell culture systems from invertebrates and vertebrates. The present study was undertaken with a vision to manage the pathogenic vibrios in aquaculture through eco-friendly and sustainable management strategies with the following objectives: Identification of Pseudomonas isolated from various ecological niches and its antagonism to pathogenic vibrios in aquaculture.,Saline dependent production of pyocyanin in Pseudomonas aeruginosa originated from different ecological niches and their selective application in aquaculture,Cloning and overexpression of Phz genes encoding phenazine biosynthetic pathway for the enhanced production of pyocyanin in Pseudomonas aeruginosa MCCB117,Development of an appropriate downstream process for large scale production of pyocyanin from PA-pUCP-Phz++; Structural elucidation and functional analysis of the purified compoundToxicity of pyocyanin on various biological systems.

Development of Zero Water Exchange Shrimp Culture System Integrated With Bioremediation of Detritus and Ammonia- Nitrogen

Aquaculture is one of the fastest growing food sectors in the world. Amongst the various branches of aquaculture, shrimp culture has expanded rapidly across the globe because of its faster growth rate, short culture period, high export value and demand in the International market. Indian shrimp farming has experienced phenomenal development over the decades due to its excellent commercial viability. Farmers have adopted a number of innovative technologies to improve the production and to maximize the returns per unit area. The culture methods adopted can be classified in to extensive, modified extensive and semi intensive based on the management strategies adopted in terms of pond size, stocking density, feeding and environmental control. In all these systems water exchanges through the natural tidal effects, or pump fed either from creek or from estuaries is a common practice. In all the cases, the systems are prone to epizootics due to the pathogen introduction through the incoming water, either brought by vectors, reservoir hosts, infected tissue debris and free pathogens themselves. In this scenario, measures to prevent the introduction of pathogen have become a necessity to protect the crop from the onslaught of diseases as well as to prevent the discharge of waste water in to the culture environment.The present thesis deals with Standardization of bioremediation technology for zero water exchange shrimp culture system

Development of a cell culture system from the ovarian tissue of African catfish Clarias gariepinus

A growth medium with Leibovitz-15 L-15.as the base, supplemented with foetal bovine serum 10% vrv., fish muscle extract 10% vrv., prawn muscle extract 10% vrv., lectin concanavalin A. 0.02 mg mly1., lipopolysaccharide 0.02 mg mly1., glucose D 0.2 mg mly1., ovary extract 0.5% vrv.and prawn haemolymph 0.5%. has been formulated with 354"10 mOsm for the development and maintenance of a cell culture system from the ovarian tissue of African catfish, Clarias gariepinus. For its subculturing, a cell dissociationrextracting solution, composed of equal portions of trypsin phosphate versene glucose TPVG. containing 0.0125% wrv.trypsin and 25% vrv.non-enzymatic cell dissociation solution 1 and 2, has also been developed with which the cell culture can be passaged 15 times after which they cease to multiply and consequently perish. The cell cultures can be maintained for 12–15 days without fluid change between the passages. This is the first report of a cell culture system from the ovarian tissues of African catfish

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.

Coastal regulation zone rules in coastal panchayats (villages) of Kerala, India vis-a` -vis socio-economic impacts from the recently introduced peoples’ participatory program for local self-governance and sustainable development

Coastal Regulation Zone (CRZ) notification was issued by the Ministry of Environment and Forest of Government of India in February 1991 as a part of the Environmental Protection Act of 1986 to protect the coast from eroding and to preserve its natural resources. The initial notification did not distinguish the variability and diversity of various coastal states before enforcing it on the various states and Union Territories. Impact assessments were not carried out to assess its impact on socio-economic life of the coastal population. For the very same reason, it was unnoticed or rather ignored till 1994 when the Supreme Court of India made a land mark judgment on the fate of the coastal aquaculture which by then had established as an economically successful industry in many South Indian States. Coastal aquaculture in its modern form was a prohibited activity within CRZ. Lately, only various stakeholders of the coast realized the real impact of the CRZ rules on their property rights andbusiness. To overcome the initial drawbacks several amendments were made in the regulation to suit regional needs. In 1995, another great transformation took place in the State of Kerala as a part of the reorganization of the local self government institutions into a decentralized three tier system called ‘‘Panchayathi Raj System’’. In 1997, the state government also decided to transfer the power with the required budget outlay to the grass root level panchayats (villages) and municipalities to plan and implement the various projects in their localities with the full participation of the local people by constituting Grama Sabhas (Peoples’ Forum). It is called the ‘‘Peoples’ Planning Campaign’’(Peoples’ Participatory Programme—PPP for Local Level Self-Governance). The management of all the resources including the local natural resources was largely decentralized to the level of local communities and villages. Integrated, sustainable coastal zone management has become the concern of the local population. The paper assesses the socio-economic impact of the centrally enforced CRZ and the state sponsored PPP on the coastal community in Kerala and suggests measures to improve the system and living standards of the coastal people within the framework of CRZ.

Design and Development of a Remotely Operated Underwater Multi-Robot Manipulator Controller

For the scientific and commercial utilization of Ocean resources, the role of intelligent underwater robotic systems are of great importance. Scientific activities like Marine Bio-technology, Hydrographic mapping, and commercial applications like Marine mining, Ocean energy, fishing, aquaculture, cable laying and pipe lining are a few utilization of ocean resources. As most of the deep undersea exploration are beyond the reachability of divers and also as the use of operator controlled and teleoperated Remotely Operated Vehicles (ROVs) and Diver Transport Vehicles (DTVs) turn out to be highly inefficient, it is essential to have a fully automated system capable providing stable control and communication links for the unstructured undersea environment.

Ideotyping integrated aquaculture systems to balance soil nutrients

Due to growing land scarcity and lack of nutrient inputs, African farmers switched from shifting cultivation to continuous cropping and extended crop area by bringing fragile lands such as river banks and hill slopes into production. This accelerated soil fertility decline caused by erosion, harvesting and insufficient nutrient replenishment. We explored the feasibility to reduce nutrient depletion by increasing nutrient utilization efficiencies, while diversifying and increasing food production through the development of integrated aquaculture – agriculture (IAA). Considering the climatic conditions prevailing in Kenyan highlands, aquaculture production scenarios were ideotyped per agro-ecological zone. These aquaculture production scenarios were integrated into existing NUTrient MONitoring (NUTMON) farm survey data for the area. The nutrient balances and flows of the resulting IAA-systems were compared to present land use. The effects of IAA development on nutrient depletion and total food production were evaluated. With the development of IAA systems, nutrient depletion rates dropped by 23–35%, agricultural production increased by 2–26% and overall farm food production increased by 22–70%. The study demonstrates that from a bio-physical point of view, the development of IAA-systems in Africa is technically possible and could raise soil fertility and total farm production. Further studies that evaluate the economic feasibility and impacts on the livelihood of farming households are recommended.

The Predation of Artemia Nauplii by the Larvae of the Amazon River Prawn, Macrobrachium amazonicum (Heller, 1862), is Affected by Prey Density, Time of Day, and Ontogenetic Development

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Food intake of Macrobrachium rosenbergii during larval development

This work investigates the acceptance of different food types and sizes by Macrobrachium rosenbergii during each larval stage. Food intake of dry and wet formulated diets of four different size classes (250-425, 425-710, 710-1000 and 1000-1190 mum), as well as Artemia nauplii, was determined. Larvae of each zoeal stage were stocked in beakers and fed ad libitum. After 30-45 min, the digestive tract of each larva was observed under a stereomicroscope. Acceptance was evaluated by food intake frequency (FFI). There was no significant interaction (P<0.05) between inert diet size and FFI for each larval stage. Therefore, food intake during larval development is independent of food particle size. The ingestion of Artemia nauplii, was significantly higher by larvae between stages II and VI. Between stages VII and XI, FFI for Artemia nauplii and wet diet was similar, while the FFI of the dry diet was similar to live food between stages IX and XI. The wet diet was ingested by more than 50% of the larvae only from stage VII onwards, while the dry diet from stage VIII onwards. These results indicate that larvae could be fed Artemia nauplii only until stage VI. Diet supplementation should start from stage VII onwards, using food particles varying from 250 to 1190 mum. (C) 2003 Elsevier B.V. B.V. All rights reserved.

Effect of nitrite on larval development of giant river prawn Macrobrachium rosenbergii

The effects of ambient nitrite concentrations on larval development of giant river prawn Macrobrachium rosenbergii were evaluated. The trials were conducted in two phases: phase 1, larvae from stages I through VIII and phase 2, larvae from stage VIII until post-larvae. In both phases larvae were kept in water with nitrite (NO2-N) concentrations of 0, 2, 4, 8 and 16 mg/L. Oxygen consumption was analyzed for larvae in stage II at nitrite concentrations of 0, 4, and 8 mg/L. Survival, weight gain, larval stage index and metamorphosis rate decreased linearly with increasing ambient nitrite concentration. However, there was no significant difference between larvae subjected to 0 and 2 mg/L NO2-N. In phase 1, there was total mortality at 16 mg/L NO2-N, while in phase 2 larval development stopped at stage X in this treatment. The oxygen consumption in stage II increased significantly at NO2-N concentration from 0 to 4 mg/L, but there was no difference between 4 and 8 mg/L NO2-N. In conclusion, increasing ambient nitrite up to 16 mg/L NO2-N delays larval development, reduces larval growth rate and causes mortality, whereas no significant effect occurs for levels below 2 mg/L NO2-N. However, the establishment of a general safe level of nitrite to M rosenbergii hatchery may be difficult due to the great variability in larvae individual sensitivity. (c) 2006 Elsevier B.V. All rights reserved.

The influence of initial feeding on muscle development and growth in pacu Piaractus mesopotamicus larvae

The effects of different feeding schemes on pacu Piaractus mesopotamicus early development were evaluated with respect to growth, survival, muscle development, and differential gene expression of MyoD and myogenin. The pacu larvae (4 days post hatch-dph, 0.77 mg wet weight) were given six feeding treatments intentionally designed to cause variations in the larvae growth rate: (A) only artemia nauplii; (CD) only a commercial diet; (ED) only a semi-purified experimental diet; (ACD) and (AED) two treatments that involved weaning; and (S) starvation. Early weaning from artemia nauplii to the formulated diets (ACD and AED) affected growth and survival of the pacu larvae compared with the exclusive use of artemia (A). Starvation (S) and the commercial diet (CD) caused total mortality in pacu larvae at 18 dph. The experimental diet (ED) assured low fish survival and growth. The skeletal muscle morphology was not affected by the delay in somatic growth from early weaning onto the formulated diets. Three distinct muscle compartments were observed throughout the larval development in treatments A, ACD and AED: superficial, deep and intermediate, accompanied by muscle thickening. Severe undernourishment caused drastic differences in growth and in the morphology of the muscle fibers. Pacu larvae fed only formulated diets (CD and ED) showed muscle characteristics similar to the larvae in starvation (S) during the first 15 dph. At 27 and 35 dph, a slight increase in epaxial muscle mass was noted in larvae fed only the experimental diet (ED). At 35 dph, we observed a high frequency of fibers >= 40 mu m in the larvae that were weaned onto the formulated diets (ACD and AED), indicative of hypertrophy. In contrast, the larvae fed only artemia nauplii (A) displayed a larger number of fibers with diameters <= 20 mu m, which is indicative of hyperplasia. The expression of the MyoD and myogenin genes in pacu larvae at 35 dph was not affected by initial feeding (p>0.05). In conclusion, the formulated diets used impaired pacu larvae growth and survival; therefore, they were inadequate for pacu, at least at the times they were introduced. Artemia nauplii were the most adequate food source during first feeding of the pacu, and they produced bigger fish upon completion of the experiment. Moreover, the contribution of hyperplasia to the skeletal muscle growth appeared higher in fast- than in slow-growing pacu larvae. (C) 2011 Elsevier By. All rights reserved.

Reproductive cycle and ovarian development of the marine ornamental shrimp Stenopus hispidus in captivity

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Effects of nitrate concentration on larval development of the giant river prawn, Macrobrachium rosenbergii

The effect of nitrate concentration on giant river prawn, Macrobrachium rosenbergii, larvae was investigated. Survival rate, weight gain, and larval development were evaluated for different concentrations of nitrate in three experiments. The experiments were divided i n to two phases. In the first phase, larvae from stages I through VIII were analysed, while in the second phase larvae from stage VIII through post-larvae metamorphosis were analysed. Oxygen consumption was also determined for zoea I, II, and VIII exposed to 0, 700, and 1,000 mg/L of nitrate-N. No effect was observed for concentrations up to 180 mg/L NO3-N (experiments I and II), and nitrate levels as 1,000 mg/L NO3-N did not affect survival in the first phase of the third experiment. On the other hand, larval stage index (LSI) and weight gain decreased as nitrate-N concentration increased from 0 to 1,000 mg/L. In the second phase, survival and metamorphosis rate decreased as nitrate concentration increased, according to a linear model. The effect of nitrate levels on weight gain followed a curvilinear pattern. Larval respiration decreased in the water where nitrate was added, but only during stage II. The results demonstrated that nitrate presents extremely low toxicity for giant river prawn larvae, and data were related to the levels of nitrate that usually occur in larviculture systems also discussed. Therefore, nitrate is not a limiting factor for giant river prawn larviculture. © 2003 by The Haworth Press, Inc. All rights reserved.

Larval development of the giant river prawn Macrobrachium rosenbergii at different ammonia concentrations and pH values

The effect of ammonia and pH levels on giant river prawn Macrobrachium rosenbergii larvae were evaluated to provide science-based information on safe levels of ammonia and pH for larviculture. Survival rate, developmental stage, and larval weight gain were determined for larvae kept in water with total ammonia (NH4-N) concentrations of 0, 1, 2, 4, and 8 mg\L and pH 7, 8, and 9. The trials were conducted in two phases: phase 1, larvae from stages I through VIII and phase 2, larvae from stage VIII until metamorphose. Oxygen consumption was determined for larvae in stages I and VIII at total ammonia concentrations of 0, 4, and 8 mg/L and pH 8. Survival rate up to stage VIII varied from 86 to 98% and did not differ for total ammonia concentrations in pH 7 and 8 and for 0 mg/L NH4-N in pH 9. Survival rate was significantly lower (0-20%) for total ammonia concentrations from 1 to 8 mg/L (0.43-3.41 mg/L of unionized ammonia) in pH 9. Larval stage indexes (7.9-8.0 range) and weight gain (1.572-2.931 mg range) of larvae at the end of phase 1 of the experiment did not differ for the different ammonia concentration solutions, but were significantly lower in pH 9. In phase 2, no parameter differed among treatments for pH 7 and 8; however there was total mortality at pH 9 until 96h. Respiration rates diminished when larvae were exposed to total ammonia concentrations of 4 and 8 mg/L (0.28 and 0.55 mg/L of unionized ammonia), but development remained unaltered. Therefore, M. rosenbergii larvae tolerate high levels of total ammonia, while toxicity depends primarily on unionized ammonia concentrations. In addition, alkaline pH (9) acted directly on the larvae, curbing development and causing severe mortality. Larval tolerance to high ammonia and pH levels decreases for the last zoeal stages. © Copyright by the World Aquaculture Society 2005.

Detection of post-F1 fish hybrids in broodstock using molecular markers: Approaches for genetic management in aquaculture

Herein, we have developed molecular markers for nuclear genes to use in multiplex-PCR and PCR-RFLP, with the goal of characterising hybrid lines derived from crosses between pintado Pseudoplatystoma corruscans and cachara P. reticulatum. These markers, together with others described previously, were used to perform molecular identification analyses as genetic subsidies for Brazilian aquaculture. These analyses were performed due to the problems of high mortality in the offspring reported by the aquaculturist. From a total of 16 broodstock samples, 13 were genetically identified as hybrids; surprisingly, nine of these hybrids were found to be post-F1 lineages. These data show that the fertility of these animals can seriously affect the cultivated stocks, thus causing financial damage in this aquaculture system. The establishment of PCR-RFLP and multiplex-PCR as molecular techniques allows for both the correct management of these animals and the routine monitoring of production and trade of fish hybrids in aquaculture. Consequently, such tools will enable a sustainable development in the aquaculture industry. © 2012 Blackwell Publishing Ltd.