FLUCTUATING ALGAL FOOD POPULATIONS AND THE OCCURRENCE OF LESSER FLAMINGOS (PHOENICONAIAS MINOR) IN THREE KENYAN RIFT VALLEY LAKES1

The last two decades have witnessed increasing episodes of lesser flamingo die‐offs in East Africa. Based on data on phytoplankton composition, biomass, and flamingo population density in three alkaline‐saline lakes of Kenya (Bogoria, Nakuru, and Oloidien) in 2001–2010, this study explored the link between sudden flamingo deaths and fluctuations in algal food quantity and quality. The phytoplankton biomass ranged from 13 to 768 mg · L^?1. Similarly, flamingo numbers varied widely from <1,000 to >500,000 individuals in the study lakes. The dominance of the cyanobacterium Arthrospira fusiformis (Woron.) Komárek et J. W. G. Lund was interrupted at irregular intervals in each lake and replaced partly by populations of different species of the nostocalean Anabaenopsis or by the picoplanktonic chlorophyte Picocystis salinarum Lewin. The populations of Anabaenopsis have the potential of blocking the flamingo food filtration system with their large and slimy colonies; moreover, they are able to produce cyanotoxins. Estimates of flamingo populations suggest that low flamingo numbers coincided with periods of low algal food quantity and/or poor quality. A food deficit can be theorized to have two effects on the flamingos: (i) it weakens them to the point of being susceptible to attacks of infective diseases, such as the ones caused by Mycobacterium avium and Pseudomonas aeruginosa, and (ii) it predisposes them to poisoning by cyanotoxins and pollutants, by reducing their capacity to handle toxic substances. This study therefore concludes that the challenges facing the flamingos are associated with changes in their environment, which affect food and water supply.     

Solute accumulation and decreased photosynthesis in leaves of potato plants expressing yeast-derived invertase either in the apoplast, vacuole or cytosol

Potato (Solanum tuberosum cv. Désirée) plants expressing yeast invertase directed either to the apoplast, vacuole or cytosol were biochemically and physiologically characterised. All lines of transgenic plants showed similarities to plants growing under water stress. Transformants were retarded in growth, and accumulated hexoses and amino acids, especially proline, to levels up to 40-fold higher than those of the wild types. In all transformants rates of CO₂ assimilation and leaf conductance were reduced. From the unchanged intercellular partial pressure of CO₂ and apoplastic cis-abscisic acid (ABA) content of transformed leaves it was concluded that the reduced rate of CO₂ assimilation was not caused by a limitation in the availability of CO₂ for␣the ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco). In the transformants the amount of Rubisco protein was not reduced, but both activation state and carboxylation efficiency of photosynthesis were lowered. In vacuolar and cytosolic transformants this inhibition of Rubisco might be caused by a changed ratio of organic bound and inorganic phosphate, as indicated by a doubling of phosphorylated intermediates. But in apoplastic transformants the pattern of phosphorylated intermediates resembled that of leaves of water-stressed potato plants, although the cause of inhibition of photosynthesis was not identical. Whereas in water-stressed plants increased contents of the phytohormone ABA are supposed to mediate the adaptation to water stress, no contribution of ABA to reduction of photosynthesis could be detected in invertase transformants. Received: 29 May 1996 / Accepted: 30 December 1996     

The maize autonomous element Activator (Ac) shows a minimal germinal excision frequency of 0.2%–0.5% in transgenic Arabidopsis thaliana plants

Summary The autonomous mobile element Activator from Zea mays was introduced into Arabidopsis thaliana via Agrobacterium-mediated gene transfer. The use of a chimaeric construct, where the Ac element is located in the leader of the neomycin phosphotransferase (NPT II) gene, enabled the excision of Ac to be monitored by assaying for the reconstitution of NPT II gene activity. Using this approach, the transpositional activity of AC was initially studied in primary transformants. About 50% of the regenerating Ac transformants showed evidence for excision of the element. Reintegration of Ac was confirmed by Southern blot analysis. Transposition events are transmitted to the F1 generation with a minimal frequency of 0.3%. In a few exceptional cases they are detected in a high proportion of the F1 generation. Seedlings from the F2 and F3 generations were assayed for the rate of germinal excisions by scoring for kanamycin resistance. The minimal frequency of germinal excision events amounts to 0.2%–0.5% and hence allows the use of the Ac element for gene tagging purposes in A. thaliana.     

Detection of Trichinella spiralis, T. britovi and T. pseudospiralis in muscle tissue with real-time PCR

Infections caused by Trichinella species occur throughout the world in many wild and domestic animals resulting in trichinellosis in men. In Europe, domestic pigs are predominantly infected by three Trichinella species: T. spiralis, T. britovi and T. pseudospiralis. Present methods for detection of Trichinella spp. (compressorium method, artificial digestion) do not always sufficiently recognize Trichinella larvae and these techniques are labor-intensive, time consuming and do not differentiate isolates on the species level since there are no distinguishing morphological features. Additionally, conventional PCRs cannot quantify numbers of larvae in infectious material. In order to better meet these requirements, we developed a real-time PCR assay for the accurate, rapid and specific identification of the three common European species of the genus Trichinella. The assay targets the large subunit of the mitochondrial rRNA (rrnL) and enables sensitive determination and discrimination of larvae in muscle tissue samples. The real-time PCR assay was developed and validated using reference and field strains from T. spiralis, T. britovi and T. pseudospiralis. In the described real-time PCR assay, the melting points of specific amplificates were always discernable via the melting curve from melting points of unspecific amplificates. This is important for the methods workflow because only C(T) values connected with the additional melting curve analysis allow a distinction of the individual species with confidence. The sensitivity of the technique enabled detection down to 0.1 Trichinella larva per gram meat sample. High disruption levels of tissues by mincing generally resulted in higher sensitivities than protocols without mincing. With its short completion time as well as accurate and specific detection of selected species this assay could become a convenient tool for the fast detection of Trichinella larvae in meat.     

Identification of metabolic and biomass QTL in Arabidopsis thaliana in a parallel analysis of RIL and IL populations

Plant growth and development are tightly linked to primary metabolism and are subject to natural variation. In order to obtain an insight into the genetic factors controlling biomass and primary metabolism and to determine their relationships, two Arabidopsis thaliana populations [429 recombinant inbred lines (RIL) and 97 introgression lines (IL), derived from accessions Col-0 and C24] were analyzed with respect to biomass and metabolic composition using a mass spectrometry-based metabolic profiling approach. Six and 157 quantitative trait loci (QTL) were identified for biomass and metabolic content, respectively. Two biomass QTL coincide with significantly more metabolic QTL (mQTL) than statistically expected, supporting the notion that the metabolic profile and biomass accumulation of a plant are linked. On the same basis, three out the six biomass QTL can be simulated purely on the basis of metabolic composition. QTL based on analysis of the introgression lines were in substantial agreement with the RIL-based results: five of six biomass QTL and 55% of the mQTL found in the RIL population were also found in the IL population at a significance level of P  ≤ 0.05, with >80% agreement on the allele effects. Some of the differences could be attributed to epistatic interactions. Depending on the search conditions, metabolic pathway-derived candidate genes were found for 24–67% of all tested mQTL in the database AraCyc 3.5. This dataset thus provides a comprehensive basis for the detection of functionally relevant variation in known genes with metabolic function and for identification of genes with hitherto unknown roles in the control of metabolism.     

A detailed study of the regulation and evolution of the two classes of patatin genes in Solanum tuberosum L.

The class-specific expression of patatin genes was investigated by analysing four new patatin genes. A class I patatin gene from cv. Berolina as well as a class I and two class II patatin genes from the monohaploid cultivar AM 80/5793 were isolated and partially sequenced. Sequence comparison indicates rearrangements as the major source for the generation of diversity between the different members of the classes. The expression of single genes was studied in potato plants transformed with chimaeric genes where the putative patatin promoters were fused to the GUS reporter gene. A detailed histochemical analysis reveals that both class I genes are expressed as the previously described class I patatin gene B33 from cv. Berolina [1], i.e. in the starch-containing cells of potato tubers and in sucrose-induced leaves. The class II gene pgT12 shows the same pattern as the previously described class II gene pgT2 [2], i.e. expression in root tips and in the vascular tissue of tubers, whereas no activity was detectable for pgT4. Thus the expression pattern of both classes of genes seems to be stable at least within or even between different cultivars.     

Peroxisome proliferator-activated receptor gamma overexpression inhibits pro-fibrogenic activities of immortalised rat pancreatic stellate cells

Pancreatic stellate cells (PSCs) play a key role in the development of pancreatic fibrosis, a constant feature of chronic pancreatitis and pancreatic cancer. In response to pro-fibrogenic mediators, PSCs undergo an activation process that involves proliferation, enhanced production of extracellular matrix proteins and a phenotypic transition towards myofibroblasts. Ligands of the peroxisome proliferator-activated receptor gamma (PPARgamma), such as thiazolidinediones, are potent inhibitors of stellate cell activation and fibrogenesis in pancreas and liver. The effects of PPARgamma ligands, however, are at least in part mediated through PPARgamma-independent pathways. Here, we have chosen a different approach to study regulatory functions of PPARgamma in PSCs. Using immortalised rat PSCs, we have established a model of tetracycline (tet)-regulated PPARgamma overexpression. Induction of PPARgamma expression strongly inhibited proliferation and enhanced the rate of apoptotic cell death. Furthermore, PPARgamma-overexpressing cells synthesised less collagen than controls. To monitor effects of PPARgamma on PSC gene expression, we employed Affymetrix microarray technology. Using stringent selection criteria, we identified 21 up- and 19 down-regulated genes in PPARgamma-overexpressing cells. Most of the corresponding gene products are either involved in lipid metabolism, play a role in signal transduction, or are secreted molecules that regulate cell growth and differentiation. In conclusion, our data suggest an active role of PPARgamma in the induction of a quiescent PSC phenotype. PPARgamma-regulated genes in PSCs may serve as novel targets for the development of antifibrotic therapies.     

Hybridisation between oilseed rape (Brassica napus) and tetraploid Brassica rapa under field conditions

Cross-compatible relatives of crop species contribute to the uncertainty regarding the potential risk of transgene escape from genetically modified varieties. The most successful crossing partner of oilseed rape (Brassica napus L.) is diploid Brassica rapa L. Variation of ploidy level among B. rapa cultivars has, until recently, been neglected in the context of gene flow and hybridisation with oilseed rape. We estimated the extent of hybridisation between autotetraploid B. rapa varieties (female) and B. napus (pollen donor) under experimental field conditions. Morphology, variation of relative DNA amount, and microsatellite markers were used to distinguish between intraspecific offspring of tetraploid B. rapa and interspecific hybrids with B. napus. Of 517 seed progenies of tetraploid B. rapa, 45 juvenile plants showed species specific morphological traits of oilseed rape. The detection of putative hybrids based on variation in relative DNA amounts was problematic due to the occurrence of aneuploidy. In total, 84 offspring showed relative DNA amounts deviating from tetraploid B. rapa, four of which were hexaploids. Of the 205 offspring analysed at three microsatellite loci, 67 had oilseed rape alleles. Based on molecular evidence a minimum hybridisation rate of 13.0% was estimated. A few mother plants accounted for the majority of hybrids. The mean pollen viability of hybrids between B. napus and tetraploid B. rapa (80.6%) was high in comparison with mean pollen viability of triploid hybrids between B. napus and diploid B. rapa. Therefore, the occurrence of tetraploid B. rapa should be taken into consideration when estimating the likelihood of gene flow from oilseed rape to close relatives at the landscape level. Tetraploid B. rapa is a common component of several seed mixtures and establishes feral populations in northwest Germany. Assuming a similar abundance of diploid and tetraploid B. rapa, gene flow from B. napus to tetraploid may be more likely than gene flow to diploid B. rapa.     

Adjusting for Ordinal Covariates by Inducing a Partial Ordering

In the context of randomized clinical trials, multiplicity arises in many forms. One prominent example is when a key endpoint is measured and analyzed both at baseline and after treatment. It is common to analyze each separately, but more efficient to adjust the post‐treatment comparisons for the baseline values. Adjustment techniques generally treat the covariate (baseline value, in this case) as either nominal or continuous. Either is problematic when applied to an ordinal covariate, the former because it fails to exploit the natural ordering and the latter because it relies on an artifical notion of linear prediction and differences between values. We propose new methods for adjusting for ordinal covariates without having to treat them as nominal or continuous. Specifically, the information‐preserving composite endpoint consists of the pair of values for each patient, one at baseline and one after treatment. Some of these patterns will indicate more improvement than others, yet some pairs of patterns are not comparable. Hence, the ordering is only partial. We develop an approach to testing and deriving estimators of magnitudes of the treatment effect based on comparing each observation in one group to each observation in the other group to which it is comparable. (© 2004 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)