Moderate genetic drift is driven by extreme recruitment events in the invasive mollusk Crepidula fornicata.

Effective population size (Ne) is a measure of genetic drift and is thus a central parameter in evolution, conservation genetics and invasion biology. Interestingly, in native marine species, Ne is typically several orders of magnitude lower than the census size. This pattern has often been explained by high fecundity, variation in reproductive success and pronounced early mortality, resulting in genetic drift across generations. Data documenting genetic drift and/or Ne in marine invasive species are, however, still scarce. We examined the importance of genetic drift in the invasive species Crepidula fornicata by genotyping 681 juveniles sampled during each annual recruitment peak over nine consecutive years in the Bay of Morlaix (Brittany, France). Observed variations in genetic diversity were partially explained by variation in recruitment intensity. In addition, substantial temporal genetic differentiation was documented (that is, genetic drift), and was attributed to nonrandom variance in the reproductive success of different breeding groups across years in the study species. Using a set of single-sample and temporal estimators for Ne, we estimated Ne to be three or four orders of magnitude smaller than the census size (Nc). On one hand, this reduction in Ne relative to Nc appeared congruent with, although slight higher than, values commonly observed in native marine species. Particular life-history traits of this invasive species may play an important role in buffering genetic drift. On the other hand, Ne still remained far below Nc, hence, possibly reducing the efficiency of selection effects.

Optimising the hygiene of a liquid feeding system to improve the quality of liquid feed for pigs.

Poor feeding system hygiene may contribute to uncontrolled spontaneous fermentation in liquid pig feed and its associated undesirable effects. This study aimed to determine the effects of an intensive sanitisation programme in a grow-finisher liquid feeding system by monitoring microbiological and physico-chemical parameters of liquid feed and microbial colonisation of the feeding system surfaces. The sanitisation programme involved a combination of physical and chemical cleaning between batches of grow-finisher pigs, combined with nightly rinsing of the system with an organic acid blend. Improved hygiene of the internal surfaces of the mixing tank and feed pipeline, particularly until week 5 post-cleaning, was evidenced by reduced counts of lactic acid bacteria, total aerobes, Enterobacteriaceae, yeasts and moulds and decreased adenosine triphosphate concentrations. Enterobacteriaceae and moulds remained undetectable on pipeline surfaces for 10 weeks. Scanning electron microscopy of the feed pipelines confirmed these findings. Conversely, the impact on liquid feed microbiology was minimal and short-lived. However, acetic acid, ethanol and biogenic amine concentrations decreased in the feed post-cleaning and no gross energy losses were observed. Therefore, by controlling surface microbial communities on liquid feeding systems via implementation of the sanitisation programme developed in the current study, on-farm liquid feed quality should be improved.

Fine- and regional-scale genetic structure of the exotic ascidian Styela clava (Tunicata) in southwest England, 50 years after its introduction.

Styela clava, an ascidian native to the northwest Pacific, was first recorded in the Atlantic at Plymouth, southwest England, in 1953. It now ranges in the northeast Atlantic from Portugal to northern Denmark, and has colonized the east coast of North America. Within the region of first introduction, we aimed to characterize current genetic diversity in the species, elucidate the respective roles of human-aided vs. natural dispersal, and assess the extent of larval dispersal by looking for genetic differentiation at very small scales. Eight sites, mostly marinas, were studied along c. 200 km of coast in southwest England encompassing Plymouth. Five microsatellite loci were genotyped in 303 individuals to analyse gene flow at regional (among sites) and fine (within sites) scales. F-statistics and assignment tests were used to investigate regional genetic structure. At the fine scale, deviation from mutation-drift equilibrium was tested, and isolation by distance and genetic clustering analyses were undertaken. Significant genetic differentiation existed between sites, unrelated to geographical separation; migration between geographically distant marinas was inferred, highlighting the likely importance of human-mediated dispersal in range expansion and occupancy by S. clava. Fine-scale population structure was present within at least four sites, which may be explained by the limited dispersal ability of this ascidian and recruitment from differentiated pools of larvae. Populations in enclosed marinas had higher self-recruitment rates than those in open sites. Some marinas might therefore function as reservoirs of propagules for subsequent spread, whereas others might be sinks for migrants.

Comparative phylogeography among hydrothermal vent species along the East Pacific Rise reveals vicariant processes and population expansion in the South.

The use of sequence polymorphism from individual mitochondrial genes to infer past demography has recently proved controversial because of the recurrence of selective sweeps acting over genes and the need for unlinked multilocus data sets. However, comparative analyses using several species for one gene and/or multiple genes for one species can serve as a test for potential selective effects and clarify our understanding of historical demographic effects. This study compares nucleotide polymorphisms in mitochondrial cytochrome oxidase I across seven deep-sea hydrothermal vent species that live along the volcanically active East Pacific Rise. Approximate Bayesian Computation (ABC) method, developed to trace shared vicariant events across species pairs, indicates the occurrence of two across species divergence times, and suggests that the present geographical patterns of genetic differentiation may be explained by two periods of significant population isolation. The oldest period dates back 11.6 Ma and is associated with the vent limpet Lepetodrilus elevatus, while the most recent period of isolation is 1.3 Ma, which apparently affected all species examined and coincides with a transition zone across the equator. Moreover, significant negative Tajima's D and star-like networks were observed for all southern lineages, suggesting that these lineages experienced a concomitant demographic and geographical expansion about 100 000-300 000 generations ago. This expansion may have initiated from a wave of range expansions during the secondary colonization of new sites along the Southern East Pacific Rise (founder effects below the equator) or recurrent bottleneck events because of the increase of eruptive phases associated with the higher spreading rates of the ridge in this region.

Population dynamics inferred from temporal variation at microsatellite loci in the selfing snail Bulinus truncatus.

We analyzed short-term forces acting on the genetics of subdivided populations based on a temporal survey of the microsatellite variability in the hermaphrodite freshwater snail Bulinus truncatus. This species inhabits temporary habitats, has a short generation time and exhibits variable rates of selfing. We studied the variability over three sampling dates in 12 Sahelian populations (1161 individuals). Classical genetic parameters (estimators of Ho, He, f, selfing rate and Fst) showed limited change over time whereas important temporal changes of allelic frequencies were detected for 10 of the ponds studied. These variations are not easily explained by selection, sampling drift and genetic drift alone and may be due to periodic migration. Indeed the habitats occupied by the populations studied are subject to large temporal fluctuations owing to annual cycles of drought and flood. In such ponds our results support a demographic model of population expansions and contractions under which available habitats, after the rainy season, are colonized by individuals originating from a smaller number of refuges (areas that never dry out in the deepest parts of the ponds). In contrast, selfing appeared to be an important force affecting the genetic structure in permanent ponds.

The spatial structure of sexual and cytonuclear polymorphism in the gynodioecious Beta vulgaris ssp. maritima: I/ at a local scale.

We have analyzed the spatial distribution of the sex phenotypes and of mitochondrial, chloroplast, and nuclear markers within two gynodioecious populations of Beta vulgaris ssp. maritima. Within both populations, sexual phenotype variation is controlled mainly by the cytoplasmic genotype, although in one study population a joint polymorphism of cytonuclear factors is clearly involved. In spite of contrasts in the ecology (mainly due to different habitats), a clear common feature in both populations is the highly patchy distribution of cytoplasmic haplotypes, contrasting with the wide distribution of nuclear diversity. This high contrast between cytoplasmic vs. nuclear spatial structure may have important consequences for the maintenance of gynodioecy. It provides opportunities for differential selection since nuclear restorer alleles are expected to be selected for in the presence of their specific cytoplasmic male sterile (CMS) type, but to be neutral (or selected against if there is a cost of restoration) in the absence of their CMS type. Selective processes in such a cytonuclear landscape may explain the polymorphism we observed at restorer loci for two CMS types.

Microsatellites and the genetics of highly selfing populations in the freshwater snail Bulinus truncatus.

Hermaphrodite tropical freshwater snails provide a good opportunity to study the effects of mating system and genetic drift on population genetic structure because they are self-fertile and they occupy transient patchily distributed habitats (ponds). Up to now the lack of detectable allozyme polymorphism prevented any intrapopulation studies. In this paper, we examine the consequences of selfing and bottlenecks on genetic polymorphism using microsatellite markers in 14 natural populations (under a hierarchical sampling design) of the hermaphrodite freshwater snail Bulinus truncatus. These population genetics data allowed us to discuss the currently available mutation models for microsatellite sequences. Microsatellite markers revealed an unexpectedly high levels of genetic variation with < or = 41 alleles for one locus and gene diversity of 0.20-0.75 among populations. The values of any estimator of Fis indicate high selfing rates in all populations. Linkage disequilibria observed at all loci for some populations may also indicate high levels of inbreeding. The large extent of genetic differentiation measured by Fst, Rst or by a test for homogeneity between genic distributions is explained by both selfing and bottlenecks. Despite a limited gene flow, migration events could be detected when comparing different populations within ponds.

Complete cytogenetic conversion in chronic myelocytic leukemia patients undergoing interferon alpha therapy: follow-up with reverse polymerase chain reaction.

Fifteen chronic myelocytic leukemia patients in durable complete cytogenetic conversion (CCC) under interferon therapy, were monitored every three to six months by bone marrow (BM) karyotypes and/or reverse-transcription polymerase chain reaction (RT-PCR) on peripheral blood (PB) leukocytes (by a nested primer approach using two rounds of amplification, 30 cycles each). Special care was taken to minimize the risk of contamination. The median time of follow-up after first CCC was 12 months (range, 6-30). Thirty five BM karyotypes were performed. Only three patients demonstrated the transient reappearance of a few Philadelphia-positive metaphases, while other patients remained in CCC. Forty five PB samples were studied by RT-PCR. In two patients, no BCR/ABL transcript could be detected in three consecutive samples. In the 13 other cases, RT-PCR was intermittently negative, indicating a level of residual leukemic cells close to the threshold of sensitivity of the technique.

Can c-myc amplification reliably discriminate postradiation from primary angiosarcoma of the breast?

PURPOSE: Breast angiosarcomas are rare vascular malignancies that arise secondary to irradiation or de novo as primary tumours. The aim of this study is to know whether c-myc amplification can reliably discriminate these two entities. MATERIEL AND METHODS: Forty-seven patients treated for breast angiosarcomas were studied. Thirty-two patients were diagnosed with postradiation angiosarcomas after breast cancer treatment and 15 patients with primary angiosarcomas. Interphase fluorescence in situ hybridization (FISH) was performed by hybridization of probes covering C-MYC (chromosome 8q24.21) and CEP8 on tissue sections. RESULTS: Amplification (5- to 20-fold) of the c-myc oncogene was found in all breast radiation-induced angiosarcomas (32 tumours) but in none of the 15 primary angiosarcomas except one (7%). CONCLUSION: This study reinforces that there are true pathogenetic differences between the two types of breast angiosarcomas which are morphologically indistinguishable. These data point the pathways preferentially involved in the pathogenesis of post radiation angiosarcomas of the breast and may provide the basis for an additional targeted therapy.

Human adenovirus 2 temperature-sensitive mutant 112 contains three mutations in the protein IIIa gene.

The temperature-sensitive (ts) mutant 112 of human adenovirus 2 is defective in the late stage of virus maturation. The region of functional mutation has been localised by marker rescue. It was observed that the ts mutation can be rescued by the left-hand part of the wild-type gene (nucleotides 12,301-12,891). By nucleotide sequencing, two mutations, both C to T (at position 12,386 and 12,741), were found in this region. The first one, in the glycine 20 codon, is silent, whereas the second changes alanine 145 to valine. A third mutation, which changed C to A (nucleotide 13,613), was identified in the right-hand part of the gene, resulting in the replacement of alanine-436 by threonine.

Gene flow in the European corn borer Ostrinia nubilalis: implications for the sustainability of transgenic insecticidal maize.

Strategies proposed for delaying resistance to Bacillus thuringiensis toxins expressed by transgenic maize require intense gene flow between individuals that grew on transgenic and on normal (referred to as refuges) plants. To investigate gene flow in the European corn borer, Ostrinia nubilalis (Hübner), the genetic variability at 29 sampled sites from France was studied by comparing allozyme frequencies at six polymorphic loci. Almost no deviations from Hardy-Weinberg expectations occurred, and a high stability of allelic distribution was found among samples collected in the same site over two or three different generations, indicating a high stability of the genetic structure over time. The overall genetic differentiation was low at the region and whole country level, suggesting a high and homogeneous gene flow. These results are discussed in relation to the sustainability of transgenic insecticidal maize.

Host-plant diversity of the European corn borer Ostrinia nubilalis: what value for sustainable transgenic insecticidal Bt maize?

The strategies proposed for delaying the development of resistance to the Bacillus thuringiensis toxins produced by transgenic maize require high levels of gene flow between individuals feeding on transgenic and refuge plants. The European corn borer Ostrinia nubilalis (Hübner) may be found on several host plants, which may act as natural refuges. The genetic variability of samples collected on sagebrush (Artemisia sp.), hop (Humulus lupulus L.) and maize (Zea mays L.) was studied by comparing the allozyme frequencies for six polymorphic loci. We found a high level of gene flow within and between samples collected on the same host plant. The level of gene flow between the sagebrush and hop insect samples appeared to be sufficiently high for these populations to be considered a single genetic panmictic unit. Conversely, the samples collected on maize were genetically different from those collected on sagebrush and hop. Three of the six loci considered displayed greater between-host-plant than within-host-plant differentiation in comparisons of the group of samples collected on sagebrush or hop with the group of samples collected on maize. This indicates that either there is genetic isolation of the insects feeding on maize or that there is host-plant divergent selection at these three loci or at linked loci. These results have important implications for the potential sustainability of transgenic insecticidal maize.

Evidence for gene flow via seed dispersal from crop to wild relatives in Beta vulgaris (Chenopodiaceae): consequences for the release of genetically modified crop species with weedy lineages.

Gene flow and introgression from cultivated to wild plant populations have important evolutionary and ecological consequences and require detailed investigations for risk assessments of transgene escape into natural ecosystems. Sugar beets (Beta vulgaris ssp. vulgaris) are of particular concern because: (i) they are cross-compatible with their wild relatives (the sea beet, B. vulgaris ssp. maritima); (ii) crop-to-wild gene flow is likely to occur via weedy lineages resulting from hybridization events and locally infesting fields. Using a chloroplastic marker and a set of nuclear microsatellite loci, the occurrence of crop-to-wild gene flow was investigated in the French sugar beet production area within a 'contact-zone' in between coastal wild populations and sugar beet fields. The results did not reveal large pollen dispersal from weed to wild beets. However, several pieces of evidence clearly show an escape of weedy lineages from fields via seed flow. Since most studies involving the assessment of transgene escape from crops to wild outcrossing relatives generally focused only on pollen dispersal, this last result was unexpected: it points out the key role of a long-lived seed bank and highlights support for transgene escape via man-mediated long-distance dispersal events.

Serial cytogenetic studies in allografted patients with chronic myeloid leukemia.

Serial marrow karyotyping was performed in 31 chronic myeloid leukemia (CML) patients treated with allogeneic bone marrow transplantation (BMT). Of 11 hematological relapses, seven were heralded for up to 20 months by a cytogenetic relapse (characterized by increasing percentages of Philadelphia (Ph)-positive metaphases, seen on serial karyotypes). Chromosomal abnormalities additional to the Ph, seen before BMT, were not found again at relapse. Relapses were characterized by clonal evolutions of the Ph-positive cells, likely corresponding to cytogenetic patterns of treatment-induced leukemia [del(5q), del(7q), complex karyotypes] and were different from those generally found in CML evolution. Involvement of chromosome 1 was also frequent. Sporadic Ph-positive metaphases (not seen in repeated karyotypes) were seen only during the first 8 months after BMT.

Mixed chimerism after sex-mismatched allogeneic BMT: evaluation of two molecular techniques.

Two different molecular techniques were used to monitor chimerism following 17 non-T cell-depleted BMTs from female donors to male recipients: pHY10, a Y chromosome-specific probe (Southern or slot blots), and a set of primers for Y chromosome sequence-specific amplification by the polymerase chain reaction (PCR). On Southern blots, male DNA was detectable at a level less than 1% of 10 micrograms DNA while cross-reactivity with autosomal sequences was avoided. On slot blots, male DNA was reliably detectable at levels less than 0.5%, even in small sample (0.5 microgram DNA). With the PCR technique, male DNA was detectable at levels of 1:10(6) to 1:10(7) of 0.5 microgram DNA. Slot blot and PCR results were concordant in 19 of 23 samples. Both techniques demonstrated a constant small mixed chimerism during the first year after BMT and in four of nine patients, this chimerism persisted even longer (up to 29 months after BMT).

Crop-weed interactions in the Beta vulgaris complex at a local scale: allelic diversity and gene flow within sugar beet fields.

Crop-wild hybrids and weed beets are the main source of agronomic concern for sugar beet production all over Europe. In order to understand the dynamics of crop-wild interactions and the evolution of weediness in Beta vulgaris, we investigated genetic features of bolting individuals occurring at a local scale, i.e. within two sugar beet fields of the French northern area of sugar beet production. By analysing ploidy level, mitochondrial DNA and microsatellite polymorphism, the genetic diversity and the genetic relationships among three different classes of individuals (variety, in-row and out-row weed-beets) from a given field were examined. Such genetic analyses provide a unique opportunity to obtain evidence for the weeds origin and the evolutionary hypotheses previously stated. All the individuals shared in common the Svulg mitochondrial haplotype, and thus a common maternal origin. Conversely, the large genetic diversity at microsatellite loci highlighted the large diversity of the pollinator plants (cultivated and wild plants) during the-seed production process, as well as during the further evolution of weed beets in the sugar production area.

Vaccination against Eimeria magna coccidiosis using spray dispersion of precocious line oocysts in the nest box.

Coccidiosis mostly affects young rabbits just after weaning (5- to 6-week-old animals). Prevention of this disease must therefore be initiated before weaning. 'Precocious lines', derived from field species display good immunogenicity, though not pathogenic when administered at the right dose. In the present work, we tested the vaccination method of the whole litter at 25 days of age by spray dispersion of oocysts of a precocious line of Eimeria magna in the nest box. Three doses were tested, d1 = 4 x 10(2) oocysts, d2 = 4 x 10(3) oocysts, d3 = 4 x 10(4) oocysts. The animals were challenged individually with 10(4) oocysts of a wild strain of E. magna at 35 days of age, 5 days after weaning. The oocyst output and weight gain were recorded after vaccination and after challenge. The vaccinated animals did not display any vaccine reaction. The dose d3 was totally effective on oocyst output and on weight gain under our experimental conditions. As our challenge conditions were severe, we think that a lower vaccine dose could be used. This method proved to be efficient, and quick and easy to use because it did not require animal manipulation.

Acquired protection of the rabbit (Oryctolagus cuniculus) against coccidiosis using a precocious line of Eimeria magna: effect of vaccine dose and age at vaccination.

Sucklings were vaccinated orally once at 25, 27 or 29 days of age with a precocious line of Eimeria magna. Each group received two doses varying from 3.5 x 10(2) to 3.5 x 10(4) oocysts. At 36 days of age, animals received a challenge inoculation with 10(4) oocysts of the wild strain of E.magna. Vaccination reduced oocyst output 10 to 1000 times after the challenge inoculation and prevented the decrease in the weight gain observed in non vaccinated challenged animals. When the vaccination was performed more than 9 days before challenge, full protection was obtained. An individual oral vaccination performed with 3500 oocysts gave total protection whatever the age at vaccination between 25 and 29 days of age.

Eimeria magna: pathogenicity, immunogenicity and selection of a precocious line.

A precocious line (PrEmag) of Eimeria magna in rabbits was obtained by selecting for early development of oocysts. The prepatent period was shortened by 46 h. The pathogenicity of PrEmag was substantially reduced and its reproductive potential was much lower (500 times) than that of the parent strain. Rabbits given 2500 oocysts of PrEmag were almost totally protected against a challenge with the parent strain. As in other precocious lines of coccidia from the rabbit, PrEmag showed morphological anomalies of the sporulated oocysts. Each sporocyst harboured one large refractile body instead of the two smaller ones in the parent strain.

Dynamics and responsiveness of T-lymphocytes in secondary lymphoid organs of rabbits developing immunity to Eimeria intestinalis.

Primary infection with Eimeria intestinalis confers very effective immunity against further infections in rabbits. This study was designed to determine the onset of the immune response in primary-infected rabbits and to characterise the immune status of protected rabbits. Variations in kinetics of CD4+ and CD8+ T-cell subpopulations were followed after primary infection at the intestinal sites of penetration (duodenum) and development (ileum), in mesenteric lymph nodes (MLN) and in the spleen. The response against the parasite was measured by specific lymphocyte proliferation in the spleen and MLN and by determining specific IgG titres in serum. The mucosal immune response was strong after primary infection and was characterised by (i) transient increase in the percentages of intestinal CD4+ lymphocytes and MLN CD8+ lymphocytes 14 days PI and (ii) strong increase in the percentages of intestinal CD8+ lymphocytes from 14 days PI persisting throughout further infections. Extensive infiltration of the lamina propria with CD8+ lymphocytes was observed 14 days PI. The specific proliferative response started between 7 and 14 days PI in MLN but remained undetectable in spleens for up to 21 days, in contrast to "immunised" rabbits. The fact that systemic immune responses were low after primary infection, in contrast to indicators of mucosal immune responsiveness, suggests that protection of rabbits against E. intestinalis infection is due to an effective mucosal immune response, and that systemic responses that increase after successive infections are only reflections of repeated encounters with parasite antigens.