David M. Rawson

Genome 10K: A Proposal to Obtain Whole-Genome Sequence for 10 000 Vertebrate Species

The human genome project has been recently complemented by whole-genome assessment sequence of 32 mammals and 24 nonmammalian vertebrate species suitable for comparative genomic analyses. Here we anticipate a precipitous drop in costs and increase in sequencing efficiency, with concomitant development of improved annotation technology and, therefore, propose to create a collection of tissue and DNA specimens for 10,000 vertebrate species specifically designated for whole-genome sequencing in the very near future. For this purpose, we, the Genome 10K Community of Scientists (G10KCOS), will assemble and allocate a biospecimen collection of some 16,203 representative vertebrate species spanning evolutionary diversity across living mammals, birds, nonavian reptiles, amphibians, and fishes (ca. 60,000 living species). In this proposal, we present precise counts for these 16,203 individual species with specimens presently tagged and stipulated for DNA sequencing by the G10KCOS. DNA sequencing has ushered in a new era of investigation in the biological sciences, allowing us to embark for the first time on a truly comprehensive study of vertebrate evolution, the results of which will touch nearly every aspect of vertebrate biological enquiry.

Whole-cell biosensors for environmental monitoring

Concern over the pollution risk to drinking water from industry and agriculture is growing, and the need for continuous on-line monitoring recognised. There is increasing use of living organisms as the sensitive agent to detect the presence of pollutants, and whole-cell biosensors are seen to have particular advantages in such environmental monitoring. The development of a mediated amperometric biosensor, incorporating the cyanobacterium Synechococcus as the biocatalyst, for on-line herbicide monitoring is described. The biosensor is able to detect a wide range of herbicides with sites of action on the photosynthetic electron transport chain, at concentrations down to 20 micrograms litre-1 and possesses a working life of up to 7 days. The use of alginate immobilisation of the biocatalyst to overcome the problems associated with obtaining a realistic shelf life for the biosensor is discussed.

Whole cell biosensors — electrochemical and optical approaches to ecotoxicity testing

Two different approaches to the interrogation of prokaryotic and eukaryotic cells have been explored, with the objective of developing biosensor based rapid ecotoxicity test protocols for use in a wide range of applications. Prokaryotic cells and some eukaryotic cell types lend themselves to interrogation by mediated amperometry, a technique that allows metabolic activity of the cell to be monitored by accessing cellular redox events with a chemical mediator. Reduction of the mediator by the cell is followed by re-oxidation at an electrode surface poised at a fixed potential. The resulting current flow is proportional to the metabolic status of the cell. A commercial ecotoxicity test, CellSense, employing such whole cell biosensors has now been developed. An alternative approach to interrogation of vertebrate cells has been the use of luminescent reporter genes to determine changes in the metabolic status of the cells following environmental challenge. Several clones have been established of epithelial cells from the bluegill sunfish (BF-2), transfected with the pCIneoLuc plasmid, that express luciferase constitutively. Protocols using these transformed cells are being developed to provide an alternative to the standard neutral red retention test.

Comparison of two types of sensors using eukaryotic algae to monitor pollution of aquatic systems

Abstract Two types of amperometric environmental sensor incorporating eukaryotic algae were investigated for use in monitoring pollution of aquatic systems. Both sensors allowed the monitoring of photosynthetic events, one by measuring the rate of reduction of a redox mediator by the illuminated biocatalyst, the other by monitoring its photosynthetic oxygen production using a semi-protected oxygen electrode. Comparison of sensor response obtained prior to challenge with pollutants with that following challenge allows rapid detection of disturbance in photosynthetic activity. The oxygen electrode-based biosensor gave good sensitivity with long operational life, and proved to be a better approach than mediator system to monitor algal biocatalysts for detection of aquatic pollutants displaying toxicity on photosynthetic organisms.

Effect of cooling rate and partial removal of yolk on the chilling injury in zebrafish (Danio rerio) embryos.

High chilling sensitivity is one of the main obstacles to successful cryopreservation of zebrafish embryos. So far the nature of the chilling injury in fish embryos has not been clear. The aim of this study is to investigate the effect of cooling rate and partial removal of yolk on chilling injury in zebrafish embryos. Zebrafish embryos at 64-cell, 50%-epiboly, 6-somite and prim-6 stages were cooled to either 0 degrees C or -5 degrees C at three different cooling rates: slow (0.3 degrees C/min or 1 degree C/min), moderate (30 degrees C/min), and rapid (approximately 300 degrees C/min). After chilling, embryos were warmed in a 26 degrees C water bath, followed by 3-day culturing in EM at 26 +/- 1 degrees C for survival assessment. When embryos were cooled to 0 degrees C for up to 30 min, 64-cell embryos had higher survival after rapid cooling than when they were cooled at a slower rate. When 64-cell embryos were held at -5 degrees C for 1 min, their survival decreased greatly after both slow and rapid cooling. The effect of cooling rate on the survival of 50%-epiboly and 6-somite embryos was not significant after 1 h exposure at 0 degrees C and 1 min exposure at -5 degrees C. However, rapid cooling resulted in significantly lower embryo survival than a cooling rate of 30 degrees C/min or 1 degree C/min after 1 h exposure to 0 degrees C for prim-6 stage or 1 h exposure to -5 degrees C for all stages. Chilling injury in 64-cell embryos appears to be a consequence of exposure time at low temperatures rather than a consequence of rapid cooling. Results also indicate that chilling injury in later stage embryos (50%-epiboly, 6-somite and prim-6) is a consequence of the combination of rapid cooling and exposure time at low temperatures. Dechorionated prim-6 embryos were punctured and about half of yolk was removed. After 24 h culture at 26 +/- 1 degrees C after removal of yolk, the yolk-reduced embryos showed higher embryo survival than did control embryos after rapid cooling to -5 degrees C for 10 to 60 min. Results suggest that cold shock injury after rapid cooling can be mitigated after partial removal of yolk at the prim-6 stage. These findings help us to understand the nature of chilling sensitivity of fish embryos and to develop protocols for their cryopreservation.

Cryobanking of viable biomaterials: implementation of new strategies for conservation purposes

Cryobanking, the freezing of biological specimens to maintain their integrity for a variety of anticipated and unanticipated uses, offers unique opportunities to advance the basic knowledge of biological systems and their evolution. Notably, cryobanking provides a crucial opportunity to support conservation efforts for endangered species. Historically, cryobanking has been developed mostly in response to human economic and medical needs — these needs must now be extended to biodiversity conservation. Reproduction technologies utilizing cryobanked gametes, embryos and somatic cells are already vital components of endangered species recovery efforts. Advances in modern biological research (e.g. stem cell research, genomics and proteomics) are already drawing heavily on cryobanked specimens, and future needs are anticipated to be immense. The challenges of developing and applying cryobanking for a broader diversity of species were addressed at an international conference held at Trier University (Germany) in June 2008. However, the magnitude of the potential benefits of cryobanking stood in stark contrast to the lack of substantial resources available for this area of strategic interest for biological science — and society at large. The meeting at Trier established a foundation for a strong global incentive to cryobank threatened species. The establishment of an Amphibian Ark cryobanking programme offers the first opportunity for global cooperation to achieve the cryobanking of the threatened species from an entire vertebrate class.

Detrimental effects of cryopreservation of loach (Misgurnus fossilis) sperm on subsequent embryo development are reversed by incubating fertilised eggs in caffeine

Cryopreservation can cause changes to the genetic material of cells, but the mechanism and significance of these changes are still unknown. It has been suggested that some damage to the sperm genome could be repaired by the DNA repair system of the oocyte after fertilisation. Caffeine has been reported to be an inhibitor of such repair processes. In this study the effect of caffeine on the repair system of Loach (Misgurnus fossilis) oocytes was investigated. Loach eggs were fertilised using cryopreserved sperm. Embryos derived from cryopreserved sperm were exposed to 2.6mM caffeine for 1h after fertilisation. The experiments were carried out using 32313 embryos from four females and eight males. Embryo survival was evaluated for 46 h until the hatching stage. Reduction in embryo survival after 20th stage is generally believed to result from the failure in the genome function of embryos. Cryopreservation of sperm significantly decreased embryo survival (53.4+/-2.8% compared to 68.4+/-2.8% of control) after the 20th stage. However, the addition of caffeine to the embryos derived from cryopreserved sperm, in contrast to our expectation, significantly increased survival of loach embryos (70.9+/-2.8% compared to 53.4+/-2.8% of embryos derived from cryopreserved sperm in the absence of caffeine). The effect of individual donors of sperm and eggs on overall embryo survival was also studied. Whilst no significant differences were observed between males, the effect of individual females on embryo survival was significant. The analysis of embryo survival at different developmental stages showed that embryo survival both before and after 20th stage decreased with embryo development. When fresh sperm were used the decline of embryo survival with development was more pronounced compared with those embryos derived from cryopreserved sperm. Possible explanations of these effects are presented.

Biosensors for the measurement of toxicity of wastewaters to activated sludge

When wastewaters from chemical manufacturing are discharged for biological treatment it is important to ensure that the presence of potentially toxic chemicals is below concentrations that may impair the performance of the treatment plant. This report describes the use of mediated amperometric biosensors incorporating activated sludge to provide rapid determination of toxicity in wastewaters to activated-sludge-based water-treatment plants. Redox-mediated interrogation of the microbial consortium comprising the biosensor biocatalyst allowed real-time monitoring of metabolic activity and the detection of perturbation due to toxic challenge. A time-independent inhibition value was determined within 30 min of exposure, using a mathematical model based on a hyperbolic relationship between exposure time and biosensor inhibition. Comparative testing of the biosensors and an activated sludge respiration inhibition test (ASRIT) against 35 wastewater samples, from a wide variety of industrial discharges, showed the close correlation coefficient of 0·924 between the two test methods.

Cryopreservation of zebrafish (Danio rerio) oocytes using improved controlled slow cooling protocols.

Cryopreservation of gametes provides a promising method to preserve fish genetic material. Previously we reported some preliminary results on cryopreservation of zebrafish (Danio rerio) oocytes using controlled slow cooling and determined the optimum cryoprotective medium and cooling rate for stage III zebrafish oocytes. In the present study, the effects of two different cryopreservation media, cryoprotectant removal method, final sample freezing temperature before LN(2) plunge, warming rate, and the post-thaw incubation time on oocyte viability were investigated. Commonly used cryoprotectant methanol and glucose were used in this study. Stage III zebrafish oocytes were frozen in standard culture medium 50% L-15 or in a sodium-free KCl buffer medium. Oocyte viability was assessed using trypan blue staining and ATP assay. The viability of oocytes frozen in KCl buffer was significantly higher than oocytes frozen in L-15 medium. The results also showed that fast thawing and stepwise removal of cryoprotectant improved oocyte survival significantly, with highest viability of 88.0+/-1.7% being obtained immediately after rapid thawing when assessed by trypan blue staining. However, after 2h incubation at 22 degrees C the viability of freeze-thawed oocytes decreased to 29.5+/-5.1%. Results also showed that the ATP level in oocytes decreased significantly immediately after thawing. All oocytes became translucent after freezing which complicated the use of GVBD test (in vitro maturation of oocytes followed by observation of germinal vesicle breakdown which results in oocytes becoming translucent). New oocyte viability assessment methods are urgently needed.

Effects of methanol and developmental arrest on chilling injury in zebrafish (Danio rerio) embryos

Stage-dependent chilling sensitivity has been reported for many species of fish embryos. Most of these studies reveal that developmental stages beyond 50% epiboly are less sensitive to chilling, but the chilling sensitivity accelerates rapidly at subzero temperatures. In this study, the effects of methanol and developmental arrest on chilling injury were studied using zebrafish (Danio rerio) embryos at 64-cell, 50% epiboly, 6-somite, prim-6 and long-bud stages. Embryos were exposed to methanol or anoxic conditions before they were cooled to 0 or -5 degrees C with slow (1 degrees C/min), medium (30 degrees C/min) or fast ( approximately 300 degrees C/min) cooling rates and were held at these temperatures for different time periods. Embryo survival was evaluated in terms of the percentage of treated embryos with normal developmental appearance after 3-day culture. Experiments on the effect of methanol on chilling sensitivity of the embryos showed that the addition of methanol to embryo medium increased embryo survival significantly at all developmental stages and under all cooling conditions. Higher concentration of methanol treatment generally improved embryo survival when embryos were cooled at a fast cooling rate of 300 degrees C/min. Experiments on the effect of developmental arrest on chilling sensitivity of embryos showed that embryos at 50% epiboly and prim-6 stages underwent developmental arrest almost immediately after 15 min oxygen deprivation. After 4h in anoxia, the survival rates of the embryos were not significantly different from their respective aerobic controls. Anoxia and developmental arrest had no effect on the chilling sensitivity of zebrafish embryos.