Exogenous DNA Uptake of Boar Spermatozoa by a Magnetic Nanoparticle Vector System

The sperm‐mediated gene transfer method is applicable to transgenesis in many species that use spermatozoa for reproduction recently, which has been shown various results. In the current study, we show that transgenic porcine embryos can be efficiently produced by employing a simple transfection method that uses magnetic nanoparticles (MNPs). The complexes formed between plasmid DNA and MNPs were bounded on ejaculated boar spermatozoa at a higher efficiency compared to methods using DNA alone or lipofection. Using confocal microscopy, rhodamine fluorophore‐labelled MNPs were detected on external surfaces of the spermatozoa membrane, which were bounded on zona pellucida of in vitro maturated oocyte during in vitro fertilization. Electron microscopy revealed that clusters of MNPs were detected in inside of plasma membrane and nucleus of the spermatozoa head. Additionally, we found that magnetofected boar spermatozoa could be fertilized with oocytes in vitro and that the resulting gene of green fluorescent protein was detected in fertilized eggs by genomic PCR analysis. Taken together, these results suggest that MNPs can be used to efficiently introduce a transgene into embryo via spermatozoa.     

Impact of the potato inoculation date on potato virus Y load and viral distribution in daughter tubers at harvest

Aged plants are more difficult to infect than young plantlets. This modification of susceptibility is described as mature plant resistance (MPR). For potato virus Y (PVY), MPR is known to lead to low infection rates of plants inoculated at the postflowering stage and a decrease in the number of infected daughter tubers. However, the impact of inoculation date on the capacity of PVY to accumulate in daughter tubers has not been studied so far. Field and greenhouse experiments were carried out to better understand PVY epidemiology and to help potato growers to evaluate consequences of early/late infections on the quality of their crops. In field trials, potato plants (cv. Bintje) were covered by insectproof nets from planting to harvest except for a 14-day period to expose plants to natural PVY infections. Under controlled conditions, potato plants were mechanically inoculated with PVY at different dates from preflowering stages (early inoculations) to postflowering stage (late inoculations). At harvest, daughter tubers were individually collected and analysed to define proportions and viral load of infected tubers according to the time between virus inoculation and harvest. Our results showed that although the age of plants at the time of inoculation can modify their susceptibility to PVY infection, in return, early and late PVY inoculations lead to similar rates of infected tubers at the plant scale and equivalent viral accumulation in infected tubers. All together, these data revealed that both early/late infections are high risks for the sanitary quality of potato tubers.     

Enhancing Embryo Yield in Superovulated Holstein Heifers by Immunization Against Inhibin

Eight heifers, aged 16–17 months and showing normal oestrous cycles, were immunized against a recombinant porcine inhibin α subunit immunogen, together with another 10 heifers of the same age as controls and treated with placebo immunogen. Primary (1 mg immunogen) and two booster (0.5 mg immunogen each) immunizations were administered at 28‐day intervals. Ten days after the second booster immunization, both groups of heifers underwent a superovulation treatment. Each animal was given an intravaginal progesterone releasing sponge, which was withdrawn 7 days following an i.m. injection of 0.5 mg cloprostenol. Heifers were treated with FSH for 4 days and artificially inseminated after oestrus occurred. The embryos were flushed and evaluated 7 days after insemination. Immunization significantly (p < 0.01) increased blood antibody titres against recombinant porcine inhibin α subunit, from pre‐immunizaion and control values of approximately 0.06 of ELISA 450 nm reading to 0.6 to 0.7 after two or three immunizations. The immunized heifers produced on average 15.8 ± 2.8 embryos, significantly (p < 0.05) higher than the yield of 8.3 ± 1.5 in the controls. The number of transferable embryos were non‐significantly higher in immunized than in control heifers (9.6 ± 3.1 vs 5.8 ± 1.6, p > 0.05). The peak plasma oestradiol concentrations were significantly higher in immunized than in control heifers, both immediately after FSH treatment and 20 days thereafter. Plasma P4 concentrations after superovulation were in the range of 20 ng / ml in the immunized heifers, significantly (p < 0.05) higher than the values approximately 15 ng / ml in control heifers. These results indicated that prior immunization against inhibin α subunit stimulated production of antibodies against inhibin, which enhanced follicular developmental response to superovulation and lead to higher yield of total and transferable embryos. Therefore immunization combined with the conventional superovulatory gonadotrophin treatment, can be a simple and efficient method to produce low cost bovine embryos.     

融合表达淋巴细胞脉络丛脑膜炎病毒和卵清白蛋白CD8+T细胞表位的减毒鼠伤寒沙门氏菌的构建与鉴定

依据淋巴细胞脉络丛脑膜炎病毒(LCMV)主要保护性抗原CD8 T细胞表位VRRPQASGVYMGNLTAQ和卵清白蛋白(OVA)CD8 T细胞表位SIINFEKL,设计、合成两条编码LCMV和OVA CD8 T细胞表位的寡核苷酸片段,经退火后,克隆入绿色荧光蛋白表达质粒pYAGFP,经PCR扩增和序列测定分析,证实成功构建T细胞表位与绿色荧光蛋白融合表达的重组质粒pYAGFPL-O,将此重组质粒pYAGFPL-O转化减毒鼠伤寒沙门氏菌X4550,获得重组沙门氏菌X4550(pYAGFPL-O).用SDS-PAGE电泳测得重组菌表达的融合蛋白约为30kD.同时,荧光显微镜下观察到X4550(pYAGFPL-O)发出黄绿色荧光.这些结果表明LCMV和OVA T细胞表位已成功表达.重组菌X4550(pYAGFPL-O)的获得为研究沙门氏菌载体携带外源抗原的T细胞应答规律及调控机理打下了重要基础.     

Assessment of phylogenetic signal in the germination ability of Phelipanche ramosa on Brassicaceae hosts

Phelipanche ramosa is a generalist parasitic weed known to cause yield losses in various crops, especially in winter oilseed rape, its new preferred host in France. This parasitic plant is also able to complete its life cycle on many Brassicaceae weeds, which are thus important alternative hosts. We studied a set of 14 common Brassicaceae weeds and characterised their ability to induce the germination of three genetically distinct pathovars of P. ramosa, based on in vitro experiments. We then investigated whether phylogenetic relatedness among Brassicaceae weeds could inform on their ability to induce germination of P. ramosa by testing for a phylogenetic signal in the germination rate of the parasite. In the presence of some phylogenetic signal, phylogenetic distances among species might be used as a surrogate to predict the ability to induce germination of potential additional Brassicaceae hosts. The three pathovars studied showed different germination patterns. Moreover, we found substantial variation in the germination rate of P. ramosa among the different Brassicaceae species, with a significant effect of the clustering of species into two ancient phylogenetic lineages. However, no significant phylogenetic signal was detected overall, that is we could not exclude that germination rates were randomly distributed over the phylogeny. We suggest that further analyses should be conducted across wider sets of potential hosts to better characterise the existence of a phylogenetic signal of the ability of weeds to induce the germination of P. ramosa.     

Acrylamide in French fries: influence of free amino acids and sugars

The free amino acid profile and sugar (fructose, glucose, and sucrose) composition were determined in potato samples selected to give a large range of variation (a total of 66 samples). From these samples French fries were produced in a laboratory-scale simulation of an industrial process followed by a finish fry at 180 degrees C for 3.5 min using a restaurant fryer. The final product was blast frozen and analyzed for acrylamide. Acrylamide was detected in all samples, but its concentration varied significantly from 50 to 1800 ng/g. For isotope dilution (13C3) acrylamide analysis, samples were extracted with water, cleaned up on HLB Oasis polymeric and Accucat mixed mode anion and cation exchange SPE columns, and analyzed by LC-MS/MS. Statistical analysis of the data indicates that the effect of sugars and asparagine on the concentration of acrylamide in French fries is positive and significant (p < 0.001). It appears that one of the ways acrylamide formation in French fries can be effectively controlled is by the use of raw products with low sugar (and to a lesser degree, asparagine) content.     

How social organization shapes crop diversity: an ecological anthropology approach among Tharaka farmers of Mount Kenya

The conservation of in situ crop diversity is a key issue to ensure food security. Understanding the processes that shape it is crucial for efficiently managing such diversity. In most rural societies, crop diversity patterns are affected by farmers’ practices of seed exchange, transmission, and selection, but the role of social organization in shaping those practices has been overlooked. This study proposes an ecological anthropology approach to investigate the relation between crop diversity patterns and the social organization of Tharaka farmers in Kenya. The Tharaka are organized in neighborhood-groups, clans, and age-sets. We quantified the influence of these three major social institutions on crop diversity patterns, for both crop species and sorghum landraces. General linear models were used to test the relations between crop species richness and each social factor, while the crop species and sorghum landraces compositions of cropping systems were compared separately through a between-class correspondence analysis. Crop species and sorghum landraces are not randomly distributed among farms, and neighborhood-groups constitute a significant factor organizing crop diversity at both specific and infraspecific levels. Adjacent neighborhood-groups present significantly different crop richness and composition. The results for species were consistent with those obtained for sorghum landraces, confirming that crop diversity was socially structured. The influence of social organization on seed networks and selection processes is discussed.     

Biotinylated DNA probes for detecting virus Y and aucuba mosaic virus in leaves and dormant tubers of potato

Summary Detection of potato virus Y (PVY) in dormant potato tubers using the enzyme-linked immunosorbent assay (ELISA) has been reported not to be accurate and reliable and it requires breaking of the dormancy of tubers prior to testing. We describe a simple, practical and highly sensitive hybridization method based on the use of biotinylated DNA probes for detecting PVY and potato aucuba mosaic virus (PAMV) in dilutions made of crude extracts of infected potato leaves and dormant tubers. As little as 50 fg of RNA can be detected by this method, and the probes are highly specific for their targets, even in crude plant extracts. The presence of one virus did not interfere with the detection of the other.     

Seasonal differences in the field performance of micropropagated potato under a short growing season in Quebec

Field performance comparisons between ex vitro plantlet-derived (EVPD) plants and seed tuber-derived (STD) plants ofRusset Burbank were conducted for two consecutive years under a short growing season in Quebec. The EVPD plants produced more potential seed tubers than STD plants although total tuber biomass of EVPD plants was less than that of STD plants. The frequency and weight distributions of tubers formed by the two types of propagates were different. The EVPD plants produced significantly more small sized tubers and fewer large sized tubers than the STD plants. Average tuber weight was less for EVPD than STD plants for each of five size categories. No significant differences were found in the fresh and dry weight of plants, leaves and stems at harvest time. However, plant height and growth habit differed between the two types of propagates. The EVPD plants had a unique appearance. They produced a single stem with extensive axillary bud development. The EVPD plants were apparently more susceptible to early water stress in the field than STD plants, probably due to an earlier production of stolons. No differences were found between ex vitro plantlet- or microtuber-derived plants, in plant growth or yield characteristics.     

Life history traits and trade-offs between two species of the ascochyta blight disease complex of pea

Plant disease complexes are a playground to investigate coinfections in natural or cultivated systems. Pathogens of such complexes may affect each other through direct and/or indirect interactions and lead to changes in virulence or aggressiveness, offspring production, and transmission. As coinfections by sympatric host-pathogens can strongly influence pathogen dynamics and their evolutionary trajectories, new insights into the mechanisms of their coexistence is thus of critical importance. In order to characterize differences in ecological niches liable to explain species coexistence on the same host, the inter- and intraspecific diversity of the life history traits in natural collections of the two main pathogens (Peyronellaea (formerly Didymella) pinodes [Dp] and Phoma medicaginis var. pinodella [Pmp]) of the ascochyta blight disease complex of pea was evaluated under controlled conditions. Dp strains developed 1.3 times faster and produced longer, mainly bicellular spores and in lower amounts (3.7 times less) than Pmp strains. Pmp strains were separated into two groups, one producing more pycnidiospores, mainly bicellular, with less resources, and the other with mainly unicellular ones. These three groups can be interpreted as three distinct life history strategies: pioneer colonizer (Dp), scavenger (large-spored Pmp), and intermediate (small-spored Pmp), allowing differentiation in access to and use of resources. While this experimental work provides new insight into coexistence of two species of the ascochyta blight disease complex of pea, it also raises the question of the benefit of having two distinct life history strategies for one species (Pmp).