Maintenance of permanent sexual activity throughout the year in seasonal bucks using short photoperiodic cycles in open barns.

Seasonality of reproductive activity in rams and bucks is the major constraint in temperate and subtropical zones. Rapid alternation between 1 month of short days and 1 month of long days (LD) over three years in lightproof buildings eliminates this seasonality. We examined if this would also work in open barns, using only supplementary light. Over two years, one group of bucks (n = 7) was subjected to alternate 1 month of LD and 1 month of permanent light (LD-LL) and another group (n = 7) to alternate 1 month of LD and 1 month of natural light (LD-NL). A simultaneous control group, used for both experiments (CG1, n = 6; CG2, n = 6), remained under natural photoperiod. BW, testis weight (TW), plasma testosterone (T) and cortisol (C) were evaluated in all bucks. CG1 and CG2 bucks showed identical dramatic seasonal variations in BW (stable or decreasing in summer), TW (from 85 ± 12 g in February to 127 ± 7 g in July) and T (from 2.7 ± 1.2 ng/mL in January-April to 24.3 ± 3.2 ng/mL in June-October). By contrast, BW of LD-LL and LD-NL bucks increased regularly during the experiment. From 5 and 9 months after the experiment onset, LD-LL and LD-NL bucks, respectively, maintained constant TW of 115 ± 5 g until the experiment end. After the first 3 months <5 ng/mL, T of LD-LL bucks remained constant (5-10 ng/mL) until the experiment end. By contrast, T of LD-NL bucks showed four periods of low (<5 ng/mL) and two periods of high concentrations (18.1 ± 2.6 and 11.9 ± 3.4 ng/mL). Plasma C remained low (5-8 ng/mL) and did not change with group or light treatment. These results show for the first time in any seasonal photoperiodic species that it is possible to maintain the sexual activity of males all year round in open buildings using alternating periods of LD and LL. By contrast, return to NL instead of LL every other month does not prevent seasonality in T concentration. These results raise interesting questions about the photoperiodic control of neuroendocrine regulation of seasonal sexual activity and suggest that these treatments can be used to manage males in open barns in farms and in artificial insemination centres. (Spanish and French versions of the full text are available as Supplementary Materials S1 and S2).

Genome-wide association study for age at puberty and resumption of cyclicity in a crossbred dairy cattle population.

Fertility is of primary economic importance in dairy cattle and the most common reason for involuntary culling. However, standard fertility traits have very low heritability that renders genetic selection slow and difficult. In this study, we explored fertility from an endocrine standpoint. A total of 1,163 crossbred Holstein-Normande females in a 3-generation familial design were studied for progesterone level measured every 10 d to determine age at puberty (PUB) and commencement of postpartum luteal activity (CPLA). Genetic parameters were estimated using REML with WOMBAT software. The heritability estimates were 0.38 ± 0.10 and 0.16 ± 0.07 for PUB and CPLA, respectively. Moreover, the 2 traits were genetically correlated (0.45 ± 0.23), suggesting a partially common determinism. Because of the family structure, a linkage disequilibrium and linkage analysis approach was preferred over standard genome-wide association study to map genomic regions associated with these traits. Ten quantitative trait loci (QTL) were detected for PUB on chromosomes 1, 3, 11, 13, 14, 21, and 29, whereas 3 QTL were associated with CPLA on chromosomes 21 and 26. Only the QTL on chromosome 21 was common to both traits. Four functional candidate genes (NCOA2, GAS2, OVOL1, and FOSL1) were identified in the detected regions. These findings will contribute to a clearer understanding of fertility determinism and enhance the value of introducing endocrinological data in fertility studies.

A bovine-specific FSH enzyme immunoassay and its application to study the role of FSH in ovarian follicle development during the postnatal period.

The primary aim of this study was to develop a FSH enzyme immunoassay (EIA) for the bovine species. The newly developed EIA was validated for FSH determination in bovine plasma by comparison with an existing bovine FSH radioimmunoassay. The EIA detected bovine FSH with a high sensitivity (0.1 ng/ml). Cross-reactivity of the EIA was 0.01% with bovine LH, 51% with ovine FSH, <0.1% with porcine FSH and <0.01% with equine FSH. Using this EIA on different time series of plasma in cows, we have confirmed the presence of a FSH pre-ovulatory peak at estrus, of periodic FSH fluctuations accompanying the waves of terminal follicular development, and of FSH pulses, mainly asynchronous with LH ones, in the peri-ovulatory phase of the cycle. In a second objective, the EIA was used to assess the role of FSH in regulating the development of ovarian follicles up to the small antral stage in young calves. To answer this question, six calves were submitted to weekly blood sampling during their first 3 months of life, and FSH changes were studied concomitantly to those of anti-Müllerian hormone (AMH), a well-established endocrine marker of the ovarian population of small antral follicles in cows. In the ovaries of 3-month calves, the population of 3 to 5 mm follicles contained the highest intra-follicular AMH amounts, and the number of 3 to 5 mm follicles on ovaries was closely correlated with AMH concentrations in the plasma of calves at this age (rs = 0.94). Before 3 months of age, only two out of six calves showed a clear postnatal FSH peak in plasma, and no correlation was found between plasma FSH and AMH concentrations. These results indicate that female calves undergo different patterns of FSH secretion and that postnatal activation of follicular growth up to the small antral stage appears independent and not directly related to circulating FSH levels.

Enhanced oral delivery of celecoxib via the development of a supersaturable amorphous formulation utilising mesoporous silica and co-loaded HPMCAS.

Stabilization of amorphous formulations via mesoporous silica has gained considerable attention for oral delivery of poorly soluble drugs. The release of the drug from the silica is expected to generate supersaturation which is often associated with subsequent precipitation. The aim of the study was hence to develop a novel supersaturable amorphous formulation through the co-loading of a BCS class II drug Celecoxib (CXB) with a precipitation inhibitor hydroxypropyl methylcellulose acetate succinate (HPMCAS) onto the silica. The addition of HPMCAS did not hamper the adsorption but on the contrary promoted the complete solid state conversion of the drug as proved by DSC analysis. In an in vitro pH shift assay, the CXB-HPMCAS co-loaded silica achieved a 5-fold solubility increase over the crystalline CXB and over the CXB-loaded silica blended with HPMCAS which did not show any enhancement. The drug co-loaded silica was then suspended in an aqueous vehicle facilitating the dosing to animals. The CXB-HPMCAS co-loaded silica suspension achieved 15-fold solubility increase in vitro over the crystalline counterpart which translated in 1.35-fold Cmax increase in vivo after oral dosing in rats. This approach represents a novel formulation strategy to maximize in vivo exposure of poorly soluble drugs critical for discovery studies.

A hyperparasite affects the population dynamics of a wild plant pathogen.

Assessing the impact of natural enemies of plant and animal pathogens on their host's population dynamics is needed to determine the role of hyperparasites in affecting disease dynamics, and their potential for use in efficient control strategies of pathogens. Here, we focus on the long-term study describing metapopulation dynamics of an obligate pathogen, the powdery mildew (Podosphaera plantaginis) naturally infecting its wild host plant (Plantago lanceolata) in the fragmented landscape of the Åland archipelago (southwest Finland). Regionally, the pathogen persists through a balance of extinctions and colonizations, yet factors affecting extinction rates remain poorly understood. Mycoparasites of the genus Ampelomyces appear as good candidates for testing the role of a hyperparasite, i.e. a parasite of other parasites, in the regulation of their fungal hosts' population dynamics. For this purpose, we first designed a quantitative PCR assay for detection of Ampelomyces spp. in field-collected samples. This newly developed molecular test was then applied to a large-scale sampling within the Åland archipelago, revealing that Ampelomyces is a widespread hyperparasite in this system, with high variability in prevalence among populations. We found that the hyperparasite was more common on leaves where multiple powdery mildew strains coexist, a pattern that may be attributed to differential exposure. Moreover, the prevalence of Ampelomyces at the plant level negatively affected the overwinter survival of its fungal host. We conclude that this hyperparasite may likely impact on its host population dynamics and argue for increased focus on the role of hyperparasites in disease dynamics.

Conventional versus stealth lipid nanoparticles: formulation and in vivo fate prediction through FRET monitoring.

The determination of the nanocarrier fate in preclinical models is required before any translation from laboratory to clinical trials. Modern fluorescent imaging techniques have gained considerable advances becoming a powerful technology for non-invasive visualization in living subjects. Among them, Forster (fluorescence) resonance energy transfer (FRET) is a particular fluorescence imaging which involves energy transfer between 2 fluorophores in a distance-dependent manner. Considering this feature, the encapsulation of an acceptor/donor pair in lipid nanoparticles (LNEs: lipid nanoemulsions, LNCs: lipid nanocapsules) allowed the carrier integrity to be tracked. Accordingly, we used this FRET technique to evaluate the behavior of LNEs, conventional LNCs and newly designed stealth LNCs. After the development through a one-step (OS) PEGylation process of these stealth LNCs (OS LNCs), in vitro guest exchange dynamics and release kinetics were evaluated for both LNC formulations. We thereafter assessed in vivo biodistribution of all types of lipid nanoparticles. Results showed enhanced stability of encapsulation in OS LNCs in comparison to conventional LNCs. Additionally, the presence of the long PEG chains on the lipid nanoparticle surface altered the biodistribution pattern. Despite different release kinetic profiles, OS LNCs and LNEs showed extended blood circulation time associated with a good structure stability over several hours after intravenous injection.

Investigating the production of sexual resting structures in a plant pathogen reveals unexpected self-fertility and genotype-by-environment effects.

The sexual stage of pathogens governs recombination patterns and often also provides means of surviving the off-season. Despite its importance for evolutionary potential and between-season epidemiology, sexual systems have not been carefully investigated for many important pathogens, and what generates variation in successful sexual reproduction of pathogens remains unexplored. We surveyed the sexually produced resting structures (chasmothecia) across 86 natural populations of fungal pathogen Podosphaera plantaginis (Ascomycota) naturally infecting Plantago lanceolata in the Åland archipelago, southwestern Finland. For this pathosystem, these resting structures are a key life-history stage, as more than half of the local pathogen populations go extinct every winter. We uncovered substantial variation in the level of chasmothecia produced among populations, ranging from complete absence to presence on all infected leaves. We found that chasmothecia developed within clonal isolates (single-strain cultures). Additionally, these clonal isolates all contained both MAT1-1-1 and MAT1-2-1 genes that characterize mating types in Ascomycetes. Hence, contrary to expectations, we conclude that this species is capable of haploid selfing. In controlled inoculations, we discovered that pathogen genotypes varied in their tendency to produce chasmothecia. Production of chasmothecia was also affected by ambient temperature (E) and by the interaction between temperature and pathogen genotype (G × E). These G, E and G × E effects found both at a European scale and within the Åland archipelago may partly explain the high variability observed among populations in chasmothecia levels. Consequently, they may be key drivers of the evolutionary potential and epidemiology of this highly dynamic pathosystem.

Variation in infectivity and aggressiveness in space and time in wild host-pathogen systems: causes and consequences.

Variation in host resistance and in the ability of pathogens to infect and grow (i.e. pathogenicity) is important as it provides the raw material for antagonistic (co)evolution and therefore underlies risks of disease spread, disease evolution and host shifts. Moreover, the distribution of this variation in space and time may inform us about the mode of coevolutionary selection (arms race vs. fluctuating selection dynamics) and the relative roles of G × G interactions, gene flow, selection and genetic drift in shaping coevolutionary processes. Although variation in host resistance has recently been reviewed, little is known about overall patterns in the frequency and scale of variation in pathogenicity, particularly in natural systems. Using 48 studies from 30 distinct host-pathogen systems, this review demonstrates that variation in pathogenicity is ubiquitous across multiple spatial and temporal scales. Quantitative analysis of a subset of extensively studied plant-pathogen systems shows that the magnitude of within-population variation in pathogenicity is large relative to among-population variation and that the distribution of pathogenicity partly mirrors the distribution of host resistance. At least part of the variation in pathogenicity found at a given spatial scale is adaptive, as evidenced by studies that have examined local adaptation at scales ranging from single hosts through metapopulations to entire continents and - to a lesser extent - by comparisons of pathogenicity with neutral genetic variation. Together, these results support coevolutionary selection through fluctuating selection dynamics. We end by outlining several promising directions for future research.

Spatiotemporal structure of host-pathogen interactions in a metapopulation.

The ecological and evolutionary dynamics of species are influenced by spatiotemporal variation in population size. Unfortunately, we are usually limited in our ability to investigate the numerical dynamics of natural populations across large spatial scales and over long periods of time. Here we combine mechanistic and statistical approaches to reconstruct continuous-time infection dynamics of an obligate fungal pathogen on the basis of discrete-time occurrence data. The pathogen, Podosphaera plantaginis, infects its host plant, Plantago lanceolata, in a metapopulation setting where the presence of the pathogen has been recorded annually for 6 years in approximately 4,000 host populations across an area of 50 km x 70 km in Finland. The dynamics are driven by strong seasonality, with a high extinction rate during winter and epidemic expansion in summer for local pathogen populations. We are able to identify with our model the regions in the study area where overwintering has been most successful. These overwintering sites represent foci that initiate local epidemics during the growing season. There is striking heterogeneity at the regional scale in both the overwintering success of the pathogen and the encounter intensity between the host and the pathogen. Such heterogeneity has profound implications for the coevolutionary dynamics of the interaction.

The flowering locus Hr colocalizes with a major QTL affecting winter frost tolerance in Pisum sativum L.

An understanding of the genetic determinism of frost tolerance is a prerequisite for the development of frost tolerant cultivars for cold northern areas. In legumes, it is not known to which extent vernalization requirement or photoperiod responsiveness are necessary for the development of frost tolerance. In pea (Pisum sativum L.) however, the flowering locus Hr is suspected to influence winter frost tolerance by delaying floral initiation until after the main winter freezing periods have passed. The objective of this study was to dissect the genetic determinism of frost tolerance in pea by QTL analysis and to assess the genetic linkage between winter frost tolerance and the Hr locus. A population of 164 recombinant inbred lines (RILs), derived from the cross Champagne x Terese was evaluated both in the greenhouse and in field conditions to characterize the photoperiod response from which the allele at the Hr locus was inferred. In addition, the population was also assessed for winter frost tolerance in 11 field conditions. Six QTL were detected, among which three were consistent among the different experimental conditions, confirming an oligogenic determinism of frost tolerance in pea. The Hr locus was found to be the peak marker for the highest explanatory QTL of this study. This result supports the hypothesis of the prominent part played by the photoperiod responsiveness in the determinism of frost tolerance for this species. The consistency of three QTL makes these positions interesting targets for marker-assisted selection.

Pathogen fitness components and genotypes differ in their sensitivity to nutrient and temperature variation in a wild plant-pathogen association.

Understanding processes maintaining variation in pathogen life-history stages affecting infectivity and reproduction is a key challenge in evolutionary ecology. Models of host-parasite coevolution are based on the assumption that genetic variation for host-parasite interactions is a significant cause of variation in infection, and that variation in environmental conditions does not overwhelm the genetic basis. However, surprisingly little is known about the stability of genotype-genotype interactions under variable environmental conditions. Here, using a naturally occurring plant-pathogen interaction, I tested whether the two distinct aspects of the infection process - infectivity and transmission potential - vary over realistic nutrient and temperature gradients. I show that the initial pathogen infectivity and host resistance responses are robust over the environmental gradients. However, for compatible responses there were striking differences in how different pathogen life-history stages and host and pathogen genotypes responded to environmental variation. For some pathogen genotypes even slight changes in temperature arrested spore production, rendering the developing infection ineffectual. The response of pathogen genotypes to environmental gradients varied in magnitude and even direction, so that their rankings changed across the abiotic gradients. Hence, the variable environment of spatially structured host-parasite interactions may strongly influence the maintenance of polymorphism in pathogen life-history stages governing transmission, whereas evolutionary trajectories of infectivity may be unaffected by the surrounding environment.

Most significant genome regions involved in the control of earliness traits in bread wheat, as revealed by QTL meta-analysis.

Earliness is one of the most important adaptation traits in plant breeding. Our purpose was to identify the genome regions of bread wheat involved in the control of earliness and its three components: photoperiod sensitivity (PS), vernalization requirement (VR) and intrinsic earliness (IE). A QTL meta-analysis was carried out to examine the replicability of QTL across 13 independent studies and to propose meta-QTL (MQTL). Initial QTL were projected on a recent consensus map (2004). Quality criteria were proposed to assess the reliability of this projection. These criteria were based on the distances between markers in the QTL regions. Chromosomes of groups 2 and 5 had a greater incidence on earliness control as they carry the known, major genes Ppd and Vrn. Other chromosome regions played an intermediate role in earliness control: 4A [heading date (HD) Meta-QTL], 4B (HD MQTL), 2B (VR MQTL) and 5B (IE MQTL). Markers at this four MQTL should prove helpful in marker-assisted selection, to better control earliness.

Spatial scale of local adaptation in a plant-pathogen metapopulation.

The rate and scale of gene flow can strongly affect patterns of local adaptation in host-parasite interactions. I used data on regional pathogen occurrence to infer the scale of pathogen dispersal and to identify pathogen metapopulations in the interaction between Plantago lanceolata and its specialist phytopathogen, Podosphaera plantaginis. Frequent extinctions and colonizations were recorded in the metapopulations, suggesting substantial gene flow at this spatial scale. The level of pathogen local adaptation was assessed in a laboratory inoculation experiment at three different scales: in sympatric host populations, in sympatric host metapopulations and in allopatric host metapopulations. I found evidence for adaptation to sympatric host populations, as well as evidence indicating that local adaptation may extend to the scale of the sympatric host metapopulation. There was also variation among the metapopulations in the degree of pathogen local adaptation. This may be explained by regional differences in the rate of migration.

Partial sequences of nitrogen metabolism genes in hexaploid wheat.

Our objective was to partially sequence genes controlling nitrogen metabolism in wheat species in order to find sequence polymorphism that would enable their mapping. Primers were designed for nitrate reductase, nitrite reductase, glutamate dehydrogenase and glutamate synthase (GOGAT), and gene fragments were amplified on Triticum aestivum, T. durum, T. monococcum, T. speltoides and T. tauschii. We obtained more than 8 kb of gene sequences, mainly as coding regions (60%). Polymorphism was quantified by comparing two-by-two the three genomes of the hexaploid cultivar Arche and genomes of diploid wheat species. On average, the polymorphism rate was higher for non-coding regions, where it ranged from 1/60 to 1/23, than for coding regions (range: 1/110-1/40) except when the hexaploid D genome was compared to that of T. tauschii (1/800 and 1/816, respectively). Genome-specific primers were devised for the ferredoxin-dependent (Fd)-GOGAT gene, and they enabled the mapping of this gene on homoeologous chromosomes of group 2 using Chinese Spring deletion lines. A single nucleotide polymorphism (SNP) detected between the two hexaploid wheat cultivars Arche and Recital was used to genetically map Fd-GOGAT on chromosome 2D using a population of dihaploid lines. Fd-GOGAT-specific primers were used to estimate the SNP rate on a set of 11 hexaploid and nine Durum wheat genotypes leading to the estimate of 1 SNP/515 bp. We demonstrate that polymorphism detection enables heterologous, homeologous and even paralogous copies to be assigned, even if the elaboration of specific primer pairs is time-consuming and expensive because of the sequencing.

Genetic diversity within Pisum sativum using protein- and PCR-based markers.

A collection of 148 Pisum accessions, mostly from Western Europe, and including both primitive germplasm and cultivated types, was structured using 121 protein- and PCR-based markers. This molecular marker-based classification allowed us to trace back major lineages of pea breeding in Western Europe over the last decades, and to follow the main breeding objectives: increase of seed weight, introduction of the afila foliage type and white flowers, and improvement of frost tolerance for winter-sown peas. The classification was largely consistent with the available pedigree data, and clearly resolved the different main varietal types according to their end-uses (fodder, food and feed peas) from exotic types and wild forms. Fodder types were further separated into two sub-groups. Feed peas, corresponding to either spring-sown or winter-sown types, were also separated, with two apparently different gene pools for winter-sown peas. The garden pea group was the most difficult to structure, probably due to a continuum in breeding of feed peas from garden types. The classification also stressed the paradox between the narrowness of the genetic basis of recent cultivars and the very large diversity available within P. sativum. A sub-collection of 43 accessions representing 96% of the whole allelic variability is proposed as a starting point for the construction of a core collection.

Advanced biological unit processes for domestic water recycling.

The potential of advanced biological unit operations for the recycling of grey and black waters has been evaluated. The membrane bioreactor (MBR) demonstrated the greatest efficacy towards water recycling in terms of all the quality determinants. Both the biologically aerated filter (BAF) and the MBR were able to effectively treat the organic and physical pollutants in all the types of wastewater tested. The main difference was observed in terms of the microbiological quality, measured as total coliforms. The open bed structure of the BAF enabled passage of coliforms whereas the complete barrier of the MBR produced a non detectable level in the effluent. The MBR process complied with commonly adopted water recycling quality standards for the all determinants during the grey water trials and failed only in terms of total coliform counts once black water had been introduced into the feed. The MBR was seen as a particularly suitable advanced biological process as it was very effective at stabilising out the considerable load variations encountered during the trial.