Genetic dissection of the zebrafish retinal stem-cell compartment

In a large-scale forward-genetic screen, we discovered that a limited number of genes are required for the regulation of retinal stem cells after embryogenesis in zebrafish. In 18 mutants out of almost 2000 F2 families screened, the eye undergoes normal embryonic development, but fails to continue growth from the ciliary marginal zone (CMZ), the post-embryonic stem-cell niche. Class I-A mutants (5 loci) display lower amounts of proliferation in the CMZ, while nearly all cells in the retina appear differentiated. Class I-B mutants (2 loci) have a reduced CMZ with a concomitant expansion in the retinal pigmented epithelium (RPE), suggesting a common post-embryonic stem cell is the source for these neighboring cell types. Class II encompasses three distinct types of mutants (11 loci) with expanded CMZ, in which the progenitor population is arrested in the cell cycle. We also show that in at least one combination, the reduced CMZ phenotype is genetically epistatic to the expanded CMZ phenotype, suggesting that Class I genes are more likely to affect the stem cells and Class II the progenitor cells. Finally, a comparative mapping analysis demonstrates that the new genes isolated do not correspond to genes previously implicated in stem-cell regulation. Our study suggests that embryonic and post-embryonic stem cells utilize separable genetic programs in the zebrafish retina.     

Small scale application and assessment of a Index of Biotic Integrity for a large boreal river

Modifications and use of Karr’s Index of Biotic Integrity (IBI) for assessing the effects of anthropogenic impacts to aquatic ecosystems have typically occurred using data collected at the macro scale. However, some non-point sources of habitat degradation occur at small scales. One possible source of perturbation to fish habitat in boreal rivers is the application of rip rap shoreline armouring in human use areas. In this study we assess the use of IBI in a small scale setting and discuss the potential impact of rip rap shoreline armouring. We captured small and juvenile fishes weekly during 2002–2003 from 12 sample sites within the littoral zone of a human use area using a modified beach seine. Principal component analysis (PCA) was performed on the data to examine the relationship between species composition and IBI scores. We also performed PCA on the IBI metrics to assess our modifications. The IBI method produced higher scores for armoured sites than for unarmoured sites. We found a strong rank order correlation (Spearman’s ρ > 0.93; p < 0.001) between the modified IBI scores and the first principal component, suggesting that Karr’s original empirically-based IBI is strongly linked to species composition. We found a high degree of redundancy between the metrics of the IBI that validate our modifications. These results suggest that IBI can be a suitable method for assessing non-point impacts from within a small study area. Our results also indicate that fish habitat from rip rap armoured sites within the test area had consistently higher IBI scores that unarmoured sites.     

Behavioural Reproductive Isolation in a Rotifer Hybrid Zone

A hybrid zone between two Brachionus plicatilis rotifer mitochondrial DNA (mtDNA) lineages was recently described in the Iberian Peninsula between a pond (Santed 2) and a lake (Gallocanta). The patterns of mitochondrial and nuclear genetic variation observed suggested that gene flow is mainly male-mediated from the lake to the pond. Here we test two hypotheses: (a) that male-mediated gene flow occurs through assortative mating between individuals from these ponds, (b) that behavioural isolation occurs between the two mtDNA lineages. We isolated, reared and genotyped rotifer clones from resting eggs collected in the sediments of these and two other distant ponds. We devised a quick, inexpensive RFLP method to discriminate between B. plicatilis and its sibling species B. ‘Manjavaeas’ and between both mtDNA B. plicatilis lineages. Behavioural no-choice tests using new-born, virgin males and females were performed between five clones. B. ‘Manjavacas’ and B. plicatilis were reproductively isolated. B. plicatilis clones did not show evidence of reproductive isolation, regardless of their mtDNA lineage, except Santed 2 males, which discriminated strongly against Gallocanta females. These results could help to explain the discrepancies between mitochondrial and nuclear genetic variation reported in the two populations.     

Setting Fish Quotas Based on Holistic Ecosystem Modelling Including Environmental Factors and Foodweb Interactions – A New Approach

We present a new approach to set fish quotas from holistic aquatic foodweb modelling (the LakeWeb-approach). This modelling includes changes in environmental conditions (nutrients, salinity, temperature, oxygen), process-based mass-balance calculations of nutrient concentrations from inflow, internal processes and outflow, calculations of how changes in nutrient concentrations affect primary production, how such changes influence secondary production and how this influence fish production and biomass. This approach gives dynamic, quantitative responses to alterations in driving variables and abiotic/biotic feedbacks. We have applied this approach for preliminary simulations of the cod biomass in the Baltic. We also show that this approach adds a new dimension in setting fish quotas, which in the future could complement, rather than compete with, the more established methods used today based on fish catch statistics and models based on other presuppositions. Our preliminary results indicate that under present environmental conditions (2003), the cod is likely to be extinct if the annual catch is between 95 and 100 kt. The present fish quota is 75 kt/yr in the Baltic, but the overfishing may be 35 kt/yr. We discuss cause–effect relationships regulating fish production, key factors influencing thresholds and points of no return connected to overfishing and changes in environmental conditions, factors regulating recovery and methods for setting optimal fish quotas using this modelling approach.     

Increase of histidine content in Brassica rapa subsp. oleifera by over-expression of histidine-rich fusion proteins

An approach that enables the increase of the quantity of a specific amino acid in crop plants is reported. Oleosin gene from Arabidopsis thaliana or 30K movement protein gene of Tobacco mosaic virus (TMV; genus Tobamovirus) were cloned under the control of napin or hybrid promoters, and in fusion to synthetic poly-histidine (poly-His) sequences for transformation into spring turnip rape (Brassica rapa subsp. oleifera; synonym to B. campestris). The most stable expression cassettes for the poly-His production prior to the plant transformation were selected by analyzing the protein expression in in vitro translation and in transient plant expression systems using GFP as marker. Expression of the poly-His-constructs in transgenic Brassica rapa plants was analyzed using dot and western blotting and PCR. The constructs were stably expressed in the third generation of the transgenic plant lines. Histidine content was measured from the seeds of the transgenic plants, and some plant lines had more than 20% increase in histidine content compared to wild type. The methodology may be widely applicable to increase the content of any amino acid in crop plants including those encoded by rare codons.     

Multi-scale impacts of crested wheatgrass invasion in mixed-grass prairie

Most of North America’s northern Great Plains have been cultivated for crop production, leaving remnants of natural mixed-grass prairie fragmented and threatened by alien plant invasions. The region’s most widespread alien perennial forage crop, crested wheatgrass (Agropyron cristatum sensu amplo), has invaded native grassland and raised concerns regarding its ecological impact. To evaluate impacts at multiple scales of organization, adjacent invaded and uninvaded mixed-grass prairie were sampled at eight widely separated locations. At the population level, native C₃ mid-grasses and forbs were less abundant in invaded grasslands, while native C₃ and C₄ short-grass abundance was not different. At community and landscape levels, diversity was lower in invaded grasslands largely because of lower forb species richness and cover, and crested wheatgrass dominance of both cover (14% basal cover) and seedbank (404 seeds m⁻²). At the ecosystem level, both vegetation and litter biomass were greater in invaded grasslands, however, below ground organic matter (roots and litter), soil organic carbon, total nitrogen and phosphorus were not different. Crested wheatgrass invasion of mixed-grass prairie was associated with lower diversity within and among plant communities, and appears to simplify the composition of mixed-grass prairie landscapes. Hypotheses for crested wheatgrass dominance and persistence following invasion are suggested.     

Temporally regulated expression of Cre recombinase in neural stem cells

The use of mouse gene targeting to study molecules important in neural development is oftentimes impaired by early embryonic lethality. In order to address later roles for such molecules, specifically in neural stem cells, we generated transgenic mice that express both the tetracycline-inducible molecule rtTA-M2 and GFP under the control of the neural precursor specific form of nestin. Developmental analysis of these mice demonstrates that GFP expression is exclusive to the neural tube. Adult expression of GFP is seen only in known areas of adult neurogenesis, namely, the subventricular zone and the dentate gyrus. When crossed with a second transgenic mouse (TetOp-Cre) that expresses the Cre recombinase under the control of the tetracycline responsive promotor, we demonstrate temporal induction of Cre in bigenic animals exposed to doxycycline. We further demonstrate the feasibility of this approach by using the ROSA-26 reporter mouse to mediate recombination in neural precursor cells.     

Separation and detection of vitellogenin in fish plasma by capillary zone electrophoresis

A method for coupling capillary zone electrophoresis (CZE) with rapid membrane chromatography purification (RMCP) was established for the analysis of vitellogenin (VTG) in male fish plasma induced with 17ss-estrodiol. CZE analyses of purified VTG were performed in a buffer containing 25 mM sodium borate (pH 8.4). A 50 microm i.d. fused-silica capillary was used for separation and the detection was carried out by UV-diode array at 214 nm. Inter- and intra-assay variabilities of the proposed method were less than 10.06 and 1.95%, respectively. The method has good linear relationship over the scope of 15-2250 microg/ml with a correlation coefficient of R2 = 0.9965 and a detection limit of 7.0 microg/ml. The established CZE method was also applied to directly separate and identify VTG from fish plasma. The results indicated this method could minimize interferences from plasma proteins, allowing the detection of at least 62.5 microg/ml of VTG proteins in total proteins. This is a rapid and easy method to determine the quantity and purity of VTG compared to Bradford method and SDS-PAGE.     

Utilization and management of organic wastes in Chinese agriculture: Past, present and perspectives

Recycling and composting of organic materials such as animal waste, crop residues and green manures has a long tradition in China. In the past, the application of organic manures guaranteed a high return of organic materials and plant mineral nutrients and thus maintained soil fertility and crop yield. As a result of rapid economic development coupled with the increasing urbanization and labour costs, the recycling rate of organic materials in Chinese agriculture has dramatically declined during the last two decades, in particular in the more developed eastern and southeastern provinces of China. Improper handling and storage of the organic wastes is causing severe air and water pollution. Because farmers are using increasing amounts of mineral fertilizer, only 47% of the cropland is still receiving organic manure, which accounted for 18% of N, 28% of P and 75% of K in the total nutrient input in 2000. Nowadays, the average proportion of nutrients (N+P+K) supplemented by organic manure in Chinese cropland is only 35% of the total amount of nutrients from both inorganic and organic sources. In China, one of the major causes is the increasing de-coupling of animal and plant production. This is occurring at a time when "re-coupling" is partly being considered in Western countries as a means to improve soil fertility and reduce pollution from animal husbandry. Re-coupling of modern animal and plant production is urgently needed in China. A comprehensive plan to develop intensive animal husbandry while taking into account the environmental impact of liquid and gaseous emissions and the nutrient requirements of the crops as well as the organic carbon requirements of the soil are absolutely necessary. As a consequence of a stronger consideration of ecological aspects in agriculture, a range of environmental standards has been issued and various legal initiatives are being taken in China. Their enforcement should be strictly monitored.     

Production of fuculose-1-phosphate aldolase using operator-repressor titration for plasmid maintenance in high cell density Escherichia coli fermentations

We report a novel application for the operator-repressor titration (ORT) plasmid maintenance system. The ability of ORT to maintain a plasmid during production of DNA has been demonstrated previously. In this study, we have used the ORT system to maintain a plasmid during high cell density cultivation and expression of a recombinant protein. No evidence of plasmid loss was seen during protein expression at high cell densities. In addition, the quantity of protein produced using this system was similar to traditional plasmid maintenance systems.