Multiplexed DNA-mediated electrochemistry

The aromatic core of double helical DNA possesses the unique and remarkable ability to form a conduit for electrons to travel over exceptionally long molecular distances. This core of π-stacked nucleobases creates an efficient pathway for charge transfer to proceed that is exquisitely sensitive to even subtle perturbations. Ground state electrochemistry of DNA-modified electrodes has been one of the major techniques used both to investigate and to harness the property of DNA-mediated charge transfer. DNA-modified electrodes have been an essential tool for both gaining insights into the fundamental properties of DNA and, due to the exquisite specificity of DNA-mediated charge transfer for the integrity of the π-stack, for use in next generation diagnostic sensing. Here, multiplexed DNA-modified electrodes are used to (i) gain new insights on the electrochemical coupling of metalloproteins to the DNA π-stack with relevance to the fundaments of in vivo DNA-mediated charge transfer and (ii) enhance the overall sensitivity of DNA-mediated reduction for use in the detection of low abundance diagnostic targets.

First, Methylene Blue (MB′) was covalently attached to DNA through a flexible C12 alkyl linker to yield a new redox reporter for DNA electrochemistry measurements with enhanced sensitivity. Tethered, intercalated MB′ was reduced through DNA-mediated charge transport. The redox signal intensity for MB′-dT-C12-DNA was found to be at least 3 fold larger than that of previously used Nile Blue (NB)-dT-DNA, which is coupled to the base stack via direct conjugation. The signal attenuation, due to an intervening mismatch, and therefore the degree of DNA-mediated reduction, does, however, depend on the DNA film morphology and the backfilling agent used to passivate the surface. These results highlight two possible mechanisms for the reduction of MB′ on the DNA-modified electrode that are distinguishable by their kinetics: reduction mediated by the DNA base pair stack and direct surface reduction of MB′ at the electrode. The extent of direct reduction at the surface can be minimized by overall DNA assembly conditions.

Next, a series of intercalation-based DNA-mediated electrochemical reporters were developed, using a flexible alkane linkage to validate and explore their DNA-mediated reduction. The general mechanism for the reduction of distally bound redox active species, covalently tethered to DNA through flexible alkyl linkages, was established to be an intraduplex DNA-mediated pathway. MB, NB, and anthraquinone were covalently tethered to DNA with three different covalent linkages. The extent of electronic coupling of the reporter was shown to correlate with the DNA binding affinity of the redox active species, supporting an intercalative mechanism. These electrochemical signals were shown to be exceptionally sensitive to a single intervening π-stack perturbation, an AC mismatch, in a densely packed DNA monolayer, which further supports that the reduction is DNA-mediated. Finally, this DNA-mediated reduction of MB occurs primarily via intra- rather than inter duplex intercalation, as probed through varying the proximity and integrity of the neighboring duplex DNA. Further gains to electrochemical sensitivity of our DNA-modified devices were then achieved through the application of electrocatalytic signal amplification using these solvent accessible intercalative reporters, MB-dT-C8, and hemoglobin as a novel electron sink. Electrocatalysis offers an excellent means of electrochemical signal amplification, yet in DNA based sensors, its application has been limited due to strict assembly conditions. We describe the use of hemoglobin as a robust and effective electron sink for electrocatalysis in DNA sensing on low density DNA films. Protein shielding of the heme redox center minimizes direct reduction at the electrode surface and permits assays on low density DNA films. Electrocatalysis of MB that is covalently tethered to the DNA by a flexible alkyl linkage allows for efficient interactions with both the base stack and hemoglobin. Consistent suppression of the redox signal upon incorporation of single CA mismatch in the DNA oligomer demonstrates that both the unamplified and the electrocatalytically amplified redox signals are generated through DNA-mediated charge transport. Electrocatalysis with hemoglobin is robust: it is stable to pH and temperature variations. The utility and applicability of electrocatalysis with hemoglobin is demonstrated through restriction enzyme detection, and an enhancement in sensitivity permits femtomole DNA sampling.

Finally, we expanded the application of our multiplexed DNA-modified electrodes to the electrochemical characterization of DNA-bound proteins containing [4Fe-4S] clusters. DNA-modified electrodes have become an essential tool for the characterization of the redox chemistry of DNA repair proteins that contain redox cofactors. Multiplexed analysis of EndonucleaseIII (EndoIII), a DNA repair protein containing a [4Fe-4S] cluster known to be accessible via DNA-mediated charge transport, elucidated subtle differences in the electrochemical behavior as a function of DNA morphology. DNA-bound EndoIII is seen to have two different electron transfer pathways for reduction, either through the DNA base stack or through direct surface reduction. Closely packed DNA films, where the protein has limited surface accessibility, produce electrochemical signals reflecting electron transfer that is DNA-mediated. The electrochemical comparison of EndoIII mutants, including a new family of mutations altering the electrostatics surrounding the [4Fe-4S] cluster, was able to be quantitatively performed. While little change in the midpoint potential was found for this family of mutants, significant variations in the efficiency of DNA-mediated electron transfer were apparent. Based on the stability of these proteins, examined by circular dichroism, we propose that the electron transfer pathway can be perturbed not only by the removal of aromatic residues, but also through changes in solvation near the cluster.

National report of IFMP catch assessment survey (CAS) for August 2005

The first comprehensive CAS was carried out during the month of July 2005 This is the second report of CAS for the month of August 2005 following the July report. The design and methodology followed was the same as in July. This report highlights the results obtained in August catch assessment survey. The report gives estimates of mean catch rates in Kgs./boat/day, total catches in M.tons and values of the catch by species. The total catch for August was 31,633.0 M. tons. This is lower when compared with the July catch which was 39,745.1 M. tons. In August the catch composed of Dagaa (45%), Nile perch (33%), Haplochromines (16%), Tilapiines (5%) and all other species combined (1%). (PDF contains 14 pages)

The changing ecosystem of Lake Victoria, East Africa

Dramatic changes are occurring in the Lake Victoria ecosystem. Two-thirds of the endemic haplochromine cichlid species, of international interest for studies of evolution, have disappeared, an event associated with the sudden population explosion of piscivorous Nile perch (Lates: order Perciformes, family Centropomidae) introduced to the lake some thirty years ago. The total fish yield has, however, increased 5-fold from 1970 to 1990, but this yield is now dominated by just three fish species: the introduced Nile perch (Lates niloticus), Nile tilapia (Oreochromis niloticus), and a small endemic pelagic cyprinid (Rastrineobola argentea); these three have replaced a multispecies fishery. Contemporaneously the lake is becoming increasingly eutrophic with associated deoxygenation of the bottom waters, thereby reducing fish habitats. Conditions appear to be unstable.

Estimation of growth and financial analysis through the application of Ipil ipil (Leucaena leucocephala) leaf meal as supplements to soybean and fish meal in the diet of juvenile monosex tilapia (Oreochromis niloticus)

Among plant protein ingredients,ipil ipil (Leucaena leucocephala) leafmeal (ILLM) is considered the most nutritive plant protein source after soybean meal in aquatic feeds. That was proven in a 21-day experiment conducted to assess the response of juvenile Monosex Nile tilapia Oreochromis niloticus with four iso-nitrogenous formulated diets: One control diet was formulated based on fishmeal, one on soybean meal and one on rice bran, ipil ipil leafmeal was also included in experimental diets.

The design of multimesh, multidepth gillnet fleets for use in the Lake Victoria Fisheries Research Project

Multimesh, multidepth gillnet fleets are useful in assessing fish stock abundance, size distribution and depth distribution. Using data collected on net mesh selectivity for Nile perch, Lates niloticus (L.), in the Kenyan waters of Lake Victoria, suitable mesh sizes for gillnet fleets for use in the Lake Victoria Fisheries Research Project were determined. The modal selection length for Nile perch in the mesh sized used in the earlier experiment were determined, as was the size range vulnerable to capture. Initial trials suggest 60% of the Nile perch swim within 5 m of the bottom. Setting and hauling of the nets is simple and quick, allowing the nets to be used at the same time as other sampling programmes.

Current status of the fish stocks of Lake Victoria (Uganda)

A total of 457 hauls were taken during experimental bottom trawl surveys in the Uganda sector of Lake Victoria between November 1997 and June 1999 to estimate composition, distribution and abundance of the major fish species in waters 4-60 m deep. Fifteen fish groups were caught with Nile perch, Lates niloticus (L.), constituting 94% by weight. Haplochromines and L. niloticus occurred in all areas sampled, while Nile tilapia, Oreochromis niloticus (L.) and other tilapiines were restricted to waters 30 m deep or less. The mean trawl catch rate in the zone where artisanal fishermen operate (i.e. in waters less deep than 30 m was 165 kg hr, of which 93.6% comprised L. niloticus. Species diversity and relative abundance decreased with increasing water depth.

The status of the artisanal fishery of Lake Victoria, Kenya, with notes on improvements to the catch data collection system

Catch and effort assessment surveys have been used to assess trends in fish landings in Kenyan waters of Lake Victoria since 1976. Landings reached a maximum of 200000 t annually in 1989-1991 as Nile perch, Lates niloticus (L.), catches increased due to an expansion in stock size and increased fishing effort. CPUE peaked at 180 kg boat day-1 in 1989 and decreased thereafter with increasing effort. By 1998 total Nile perch catches were half those at the beginning of the decade despite increased effort. Catches of Rastrineobola argentea (Pellegrin) have levelled off despite increased effort.

Catch trends from Lake Victoria - Tanzanian waters

Catch trends from Fisheries Department reports from the last eleven years (1985-1995) were analyzed. These showed a shift in the fishery from a cichlid-based system to one dominated by Nile perch and tilapias. In recent years, catches have declined from a peak in the early 1990s. Catch per unit effort appears to have remained stable except for a drop in 1995, however, this is considered dubious because the effort showed a doubling in that year. Limitations of the fisheries data collection system for the Tanzanian sector of Lake Victoria are highlighted and discussed.

Improved fisheries productivity and management in tropical reservoirs

The main objective of this 3.5 year project was to increase the productivity of reservoir fisheries and provide sustainable livelihoods to the rural poor through a series of interventions in tropical reservoirs. The main project activities focused on two reservoirs in the Indo-Gangetic basin (Dahob in Madhya Pradesh, and Pahuj in Uttar Pradesh, both in India), the Lake Nasser in Egypt as part as the Nile basin, and the Volta Lake in Ghana as part as the Volta basin. The project also encompassed an initial data inventory from a wide variety of tropical reservoirs within the three basins, and the detailed assessment of the selected reservoirs, including market evaluation and post-capture improvements. (PDF contains 117 pages)

Reproductive biology of Oreochromis niloticus (L.) in the Nyanza Gulf of Lake Victoria

The reproduction of Nile tilapia, Oreochromis niloticus (L.), in the Nyanza Gulf of Lake Victoria was studied from June 1998 to May 1999. Length at maturity ranged from 28-30 cm TL for females and from 32-34 cm TL for males. Males were more abundant in all length classes longer than 36 cm TL. Relative condition factor was above unity, except in August, October and May for males, and October for females. Gonadosomatic index (GSI) was low during the post spawning period (July to October) and high during the protracted breeding period (December-June).

The regulators and the regulated: fisheries management, options and dynamics in Kenya's Lake Victoria fishery

The study divides the history of the fishery into five 'regulatory periods': the pre-colonial fishery (pre-1901), the colonial fishery (1901-1963), the post indipendence fishery (1963-1980), the Nile perch 'boom' years (1980-1989), and finally the fishery in the 1990's. Within each of these periods, the nature of and the relationship between, formal and informal regulations differs and changes with time. In the pre-colonial period, the outcome of formal and informal regulations largely sustained the fishery in a productive and species diverse state. However, at no time since then have formal regulations worked, with the result that the nature of production from the fishery changes over time and is dependent on a number of factors, amongst which the most important are effort level increases, technological introductions, species introductions, changes in regional and national job markets, the change from community-based controls to state-based controls within the fishery, and finally, considerable changes to the fish markets.

Africa: the FBA connection

Since its inception in the 1930's the Freshwater Biological Association at Windermere, England has been involved in research on African lakes and rivers. Research has included general and multidisciplinary surveys of many lakes including Lake Victoria, Lake Tanganyika, Lake Nyasa (Lake Malawi) and Lake George. The hydrobiology of the River Nile has also been studied. Research into physical and chemical limnology, phytoplankton ecology and primary productivity, invertebrate biology, freshwater fish and fisheries.

Approaches to national fish breeding programs: pointers from a tilapia pilot study

Details are given of a framework for developing breeding programmes using experience from the Genetic Improvement of Farmed Tilapias project which focussed on Nile tilapias (Oreochromis niloticus ). The following aspects are outlined: Analysis of targeted production and marketing systems; Breeding goals; Systematic documentation and evaluation of available genetic resources and choice and genetic base; Number of strains; Breeding strategy; Selection criteria and evaluation; Production and dissemination of improved strains; and, social, economic and environmental impacts.

Breeding and propagation of tilapia (Oreochromis niloticus) in a floating hatchery, Gabon

Based on the encouraging results obtained by earlier workers, the concept for a floating hatchery was developed for producing tilapia for both farming and enhanced fisheries in the freshwater lakes and coastal lagoons of Gabon. The research and development work to test this concept was undertaken with Nile tilapia (Oreochromis niloticus). Two places in Gabon were selected, representing climatic and environmental condition similar to other parts of the country. The study aims to improve fish production in the freshwater lakes of the Lambarene area, and in the numerous coastal lagoons of Gabon, to compensate for the current overexploitation of natural stocks, or the alternative of introducing other species, and to reduce the drift of fishing population away from lakes.

From drawing board to dining table: the success story of the GIFT project

As compared to crops and livestock, the genetic enhancement of fish is in its infancy. While significant progress has been achieved in the genetic improvement of temperate fish such as salmonids, no efforts were made until the late 1980s for the genetic improvement of tropical finfish, which account for about 90 percent of global aquaculture production. This paper traces the history of the Genetic Improvement of Farmed Tilapia (GIFT) project initiated in 1988 by the WorldFish Center and its partners for the development of methods for genetic enhancement of tropical finfish using Nile tilapia (Oreochromis niloticus) as a test species. It also describes the impacts of the project on the adoption of these methods for other species and the dissemination of improved breeds in several countries in Asia and the Pacific.