Consequences of genetic erosion on fitness and phenotypic plasticity in European tree frog populations (Hyla arborea)

The detrimental effects of genetic erosion on small isolated populations are widely recognized contrary to their interactions with environmental changes. The ability of genotypes to plastically respond to variability is probably essential for the persistence of these populations. Genetic erosion impact may be exacerbated if inbreeding affects plastic responses or if their maintenance were at higher phenotypic costs. To understand the interplay 'genetic erosion-fitness-phenotypic plasticity', we experimentally compared, in different environments, the larval performances and plastic responses to predation of European tree frogs (Hyla arborea) from isolated and connected populations. Tadpoles from isolated populations were less performant, but the traits affected were environmental dependant. Heterosis observed in crosses between isolated populations allowed attributing their low fitness to inbreeding. Phenotypic plasticity can be maintained in the face of genetic erosion as inducible defences in response to predator were identical in all populations. However, the higher survival and developmental costs for isolated populations in harsh conditions may lead to an additional fitness loss for isolated populations.     

Improving gene replacement by intracellular formation of linear homologous DNA

BACKGROUND: Gene targeting is a potential tool for gene therapy but is limited by the low rate of homologous recombination. Using highly homologous linear DNA improves gene targeting frequency but requires microinjection into nuclear cells to be effective. Because transfection of circular DNA is more efficient than transfection of linear DNA and adaptable to viral vectors, we developed a system for the intracellular release of linear fragments from circular plasmids. METHODS: Only one cutting site inside the "donor" DNA was not convenient because it led to integration of exogenous sequences into the target. So we constructed several "donor" plasmids containing the homologous sequences flanked by two I-Sce I recognition sites. Expression of I-Sce I allowed intracellular delivery of "ends-out" (replacement) vectors. We compared the efficiency of different constructions to correct a mutated gfp target. RESULTS: Co-transfection of "donor" plasmids and an I-Sce I expression vector into CHO cells enhanced the correction of an extrachromosomal mutated gfp target by at least 10 times. Maximum correction was observed with the greatest homology size and maximum effect of I-Sce I was obtained when the long hemi-sites of the duplicated I-Sce I sites were contiguous to the homologous sequence. Unexpectedly, the reverse orientation of I-Sce I sites provided little or no effect, probably due to the asymmetrical activity of the I-Sce I meganuclease. CONCLUSIONS: Releasing homologous DNA fragments with I-Sce I enhances gene replacement. This work provides the basis for the future design of viral vectors for gene replacement.     

Impairing the bioenergetic status and the biogenesis of mitochondria triggers mitophagy in yeast

Autophagy, a highly regulated programme found in almost all eukaryotes, is mainly viewed as a catabolic process that degrades nonessential cellular components into molecular building blocks, subsequently available for biosynthesis at a lesser expense than de novo synthesis. Autophagy is largely known to be regulated by nutritional conditions. Here we show that, in yeast cells grown under nonstarving conditions, autophagy can be induced by mitochondrial dysfunction. Electron micrographs and biochemical studies show that an autophagic activity can result from impairing the mitochondrial electrochemical transmembrane potential. Furthermore, mitochondrial damage-induced autophagy results in the preferential degradation of impaired mitochondria (mitophagy), before leading to cell death. Mitophagy appears to rely on classical macroautophagy machinery while being independent of cellular ATP collapse. These results suggest that in this case, autophagy can be envisioned either as a process of mitochondrial quality control, or as an ultimate cellular response triggered when cells are overwhelmed with damaged mitochondria.     

Aphid parasitoid complex in potato in the context of IPM in Belgium

Parasitic hymenoptera of potato aphids were collected and identified in 2000 and 2001 in 11 potato fields in Belgium. Nine Aphidiidae species, parasiting seven aphid species, were found: Aphidius ervi, Aphidius matricariae, Aphidius picipes, Binodoxys angelicae, Diaeretiella rapae, Praon abjectum, Praon gallicium, Praon volucre and Toxares deltiger. A. ervi and A. picipes were the dominant and sub-dominant species, with 54% and 28% of the primary parasite collected, respectively. Both species and T. deltiger were found on Aphis nasturti, Aulacorthum solani, Macrosiphum euphorbiae and Myzus persicae, the four most important aphid potato in Belgium. Parasitism rate of A. nasturtii and, to a lesser extent, M. euphorbiae was low compared to A. solani and M. persicae. Parasitism of A. solani was particularly high, with 63.5% in 2000 and 89.2% in 2001, and this species was the preferred host of several Aphidiid species. The abundance of alternative hosts as other crops pest aphids or non-pest aphids on wild plants in agroecosystems could explains the efficacy of these species. The biological control of A. nasturtii and M. euphorbiae by parasitic hymenoptera was poorer, and several studies need to be undertaken to find suitable parasitic hymenoptera species effective on these aphids and agro-environmental measures able to promote them.     

Development and validation of a mastication simulator

More and more research are being done on food bolus formation during mastication. However, the process of bolus formation in the mouth is difficult to observe. A mastication simulator, the Artificial Masticatory Advanced Machine (AM2) was developed to overcome this difficulty and is described here. Different variables can be set such as the number of masticatory cycles, the amplitude of the mechanical movements simulating the vertical and lateral movements of the human lower jaw, the masticatory force, the temperature of the mastication chamber and the injection and the composition of saliva. The median sizes of the particles collected from the food boluses made by the AM2 were compared with those of human boluses obtained with peanuts and carrots as test foods. Our results showed that AM2 mimicked human masticatory behavior, producing a food bolus with similar granulometric characteristics.     

Optimizing rumen functions in the close-up transition period and early lactation to drive dry matter intake and energy balance in cows

The large deficit in energy intake in relation to energy requirements during the transition and early lactation periods means that high-producing cows need energy-dense rations. High-starch diets are intensively fermented by the microbial ecosystem in the rumen, giving rise to a high production of VFAs and resulting in a drop in pH and the accumulation of lactic acid, which exacerbates the decline in pH and is considered as the major cause of rumen acidosis. This rumen dysfunction affects rumen microbes and results in less efficient digestion, thereby decreasing feed intake and exacerbating the energy deficit in the cows. The main way to limit the risk of acidosis is by diet management. Thus, animals must be progressively adapted to grain ingredients during the 3-week period before calving. Furthermore, the diet management strategy will be to privilege low-degradable starch and include enough fiber in the diet to stimulate rumination and salivation. Chemical additives can be used to prevent rumen acidosis. Rumen pH can be controlled by direct addition of chemical buffers at doses of 1-2% of DM intake. Metabolic hydrogen can be mobilized through specific metabolic pathways such as propionogenesis to compete with the synthesis of lactic acid. This can be achieved by adding propionate precursors such as aspartate, malate or fumarate. However, these additives are not economically viable, and will not easily be accepted by consumers. Probiotics, which are mainly supplied as live yeasts, have to be regarded as a solution for preventing subacute acidosis but they are not considered as an efficient cure for acute acidosis. Although the mechanisms of action of probiotics are not fully understood, this paper proposes a plausible model of their mode of action. Recently, additives based on plant extracts have been proposed as a solution for optimizing rumen functions, but further efficacy studies and safety trials are required before these additives can be marketed. The addition of fibrolytic enzymes has been suggested to improve the digestion of the dietary fiber fraction during acidosis. Although the results obtained so far are encouraging, more ruminant-specific research needs to be carried out on these enzymes. Some antibiotics are potential inhibitors of Gram-positive bacteria, which are involved in rumen acidosis, but their use as feed additives has been banned from 01/01/2006 in the EU.     

Survival of Listeria monocytogenes and Enterococcus faecium in sludge evaluated by real-time PCR and culture methods

AIMS: This study evaluates the behaviour in spiked sludge of a pathogenic bacteria, Listeria monocytogenes, by cultural and molecular techniques, and compares its survival with the one of a faecal indicator, Enterococcus faecium. METHODS AND RESULTS: Listeria monocytogenes strain Scott A and E. faecium(T) were followed for 17 days after inoculation in sludge. Kinetics of survival depended on the bacteria and on the technique used [most probable number method, direct plate count or real-time quantitative PCR (qPCR)]. The concentration of L. monocytogenes decreased rapidly regardless of the technique, but the decrease was much more dramatic with culture techniques than with qPCR. On the contrary, the concentrations of culturable E. faecium(T) were stable. CONCLUSIONS: The results suggest that the cells of L. monocytogenes strain Scott A might have entered a viable, but nonculturable (VBNC) status, whereas cells of the indicator bacteria, E. faecium(T), maintained themselves better and stayed culturable. SIGNIFICANCE AND IMPACT OF THE STUDY: The difference of survival kinetics in the sludge of a faecal indicator (E. faecium) and a pathogenic bacterium (L. monocytogenes) may be linked to the fact that they either enter or do not enter into a VBNC status.