Interactions between oral commensal Candida and oral bacterial communities in immunocompromised and healthy children.

Candida species are usually found as commensal microorganisms in the oral cavity of healthy people. During chemotherapy, cytostatic drugs lead to depletion of the oral flora with the emergence of a dominant bacterial species. The transition from commensal to pathogenic state, further associated with yeast colonization and oral mucositis implies a replacement of the dominant microorganism by Candida albicans. This process goes plausibly through cooperation between C. albicans and bacteria. This study focused on the first step of cooperation between microorganisms isolated from the same oral flora either of leukemic or healthy children. C. albicans isolated from 8/20 children were cultured to display their noninvasive blastosporic yeast form and mixed with their dominant bacteria to study the capacity of planktonic aggregation and the early state of biofilm formation. None of the dominant bacteria opposed the presence of yeast, on the contrary, an interesting cooperation was observed. This behavior is apparently different from that observed when mixing the type strains. In fact, three mutated C. albicans strains display, by their spontaneous ability to form filament, enhanced risks of virulence for leukemic ill carriers. Despite such risks, neither oral nor systemic pathology were observed in ill patients probably because the study was conducted during the first course of chemotherapy and Candida colonization is related to the number of chemotherapeutic cycles. The presence of C. albicans during the initial cycle represents, by its ability to interact with oral bacteria, an actual threat for further cures.

Shell shape of the land snail Cornu aspersum in North Africa: unexpected evidence of a phylogeographical splitting.

Anatomical and molecular characters used to differentiate populations of the land snail Cornu aspersum (Helix aspersa) exhibit, in the western Mediterranean, definite and concordant patterns of correlation with geography. Scenarios involving Pliocene geological changes and postglacial expansion during the Pleistocene were proposed in previous studies to account for the establishment of this geographical structure. In the present work, we have performed a spatial analysis of variation in shell morphometrics, after the partitioning of the overall variation into size and shape components by means of a principal component-based approach (Cadima and Jolliffe, 1996). In order to know if the same historical events have also structured shell variation, the analysis includes all the populations from North Africa which were investigated for anatomical and molecular surveys. Contrary to shell size, which shows a significant spatial heterogeneity essentially related to environmental pressures, variation in shell shape components splits the populations according to a geographical pattern reflective of hypotheses suggested for molecular markers and genital anatomy. This implies that the selective forces often invoked to explain spatial changes in shell shape are not the deciding factors in the present case. Moreover, within each of the two geographical clusters defined, Mantel correlograms show that the similarity between populations declines according to an isolation by distance model. Because of the different allometric relationships between shell size and genitalia measurements in Western and Eastern entities of North Africa, mechanical constraints, possibly leading to a precopulatory isolation in the contact zone, are involved.

Population genetic structure in a human-disturbed environment: a case study in the land snail Helix aspersa (Gastropoda: Pulmonata).

Local patterns of genetic variation were analysed in the land snail Helix aspersa for 32 populations sampled within a patchy agricultural landscape: the polders of the Bay of Mont-Saint-Michel (France). This investigation examined the allele frequencies at four enzymatic markers and five microsatellite loci through the genotyping of 580 individuals. A strongly significant population genetic substructuring (mean F(ST)=0.088, P<0.001) was found at the scale of the whole polders area (3050 ha) and both categories of markers displayed a similar magnitude of spatial genetic differentiation. We did not find any obvious effects of habitat fragmentation on the distribution of genetic variability. Despite the reality of habitat patchiness and environmental instability (related to farming practices), an isolation by distance process was clearly depicted, although selective pressures cannot be ruled out for one enzymatic locus. Overall, genetic drift, along with occasional long-distance episodes of gene flow, was presumably the most likely evolutionary force that shaped the observed pattern of genetic variation.

Spatial structure of shell polychromatism in populations of Cepaea nemoralis: new techniques for an old debate.

A conspicuous shell polychromatism is observed in colonies of Cepaea nemoralis from western France (Brittany). The present study is intended to search for a spatial structure of shell features at this scale and to infer evolutionary processes from the observed patterns. We used a database of morph frequencies (six composite phenotypes were retained) measured on 213 samples regularly distributed on the whole studied area. Data analysis was based on two distinct multivariate methods leading to the following steps: (i) to search for a structure without reference to environmental conditions with a method (global principal components analysis: GPCA) which takes into account the spatial information by means of a neighbouring relationship between sampling points (Delaunay triangulation); (ii) to test the structuring power of environmental conditions by means of two explanatory factors (distance from the sea, altitude) involved in a redundancy analysis (RDA); (iii) to search for a spatial structure using residuals of the previous analysis, ie, after removing effects of environmental conditions. Global covariance accounted for 26.4% of the total variance, leading to a highly significant autocorrelation for each phenotype (step 1). Geographical mapping of factorial scores resulting from global analysis showed a well structured littoral zone and a strong southern-northern inland differentiation. Sixteen percent of the total variance was expressed in RDA but all morphs were not equally concerned. After removing environmental effects, a significant spatial structure still remains but was essentially caused by random processes. We argue for the importance of these last phenomena.

Spatial analysis of allozyme and microsatellite DNA polymorphisms in the land snail Helix aspersa (Gastropoda: Helicidae).

The genetic structure of the land snail Helix aspersa was investigated for 21 populations collected along a road located in the polders of the Bay of Mont-Saint-Michel (Brittany, France), following a sampling scheme the area of which did not exceed 900 m in length. A total of 369 individuals were genotyped for five enzymatic markers and seven microsatellite loci. We used sequential hierarchical F-statistics at different spatial scales and spatial autocorrelation statistics to explore recent historical patterns involved in the observed genetic distribution. Whatever the statistics used, congruent levels of spatial genetic substructuring across loci were demonstrated, excepted for one allozyme locus. Overall spatial genetic arrangement matched in a substantial fashion theoretical predictions based on the limited dispersal power of land snails. Positive autocorrelation over short-distance classes may result from the development of genetically distinct patches of individuals organized in family-structured colonies. Therefore, spatial signatures of average I correlograms can be viewed as the expression of a stepping-stone model of population structure, sometimes involving external migrational events. Overall, the revealed pattern of population subdivision on a microgeographical scale was suggestive of a neighbourhood structure. Finally, microsatellite loci are especially suitable for the detection of small genetic clustering, and combining different classes of markers offers the potential to gain further insight into the description of spatial genetic variability over short temporal and geographical scales.

Evolutionary history of the land snail Helix aspersa in the Western Mediterranean: preliminary results inferred from mitochondrial DNA sequences.

Intraspecific phylogeographic methods provide a means of examining the history of genetic exchange among populations. As part of a study of the history of Helix aspersa in the Western Mediterranean, we performed a phylogenetic analysis based on partial sequences of the mitochondrial large ribosomal subunit (16S) gene. Our samples include 31 H. a. aspersa populations from North Africa previously investigated for anatomical and biochemical characters. To clarify subspecific relationships, three individuals of the subspecies H. a. maxima were also studied. The molecular phylogeny inferred agrees largely with previous results, in splitting H. a. aspersa haplotypes into an eastern and a western group. H. a. maxima haplotypes form a third lineage arising before the H. a. aspersa groups. Divergence times estimated between the lineages suggest that dispersal during Pleistocene glaciation and vicariance events due to Pliocene geological changes in the western Mediterranean may both have played a significant part in the establishment of the present range of H. aspersa.

Analysis of a contact zone in the Forficula auricularia L. (Dermaptera: Forficulidae) species complex in the Pyrenean Mountains.

The taxon Forficula auricularia L. (Dermaptera: Forficulidae) is a complex of two sibling species that differ in life history (number of clutches per year and imaginal diapause) and that have diverged at the molecular level. The study of a contact zone in the Pyrenean Mountains, using the PCR-RFLP method on two mitochondrial regions (the 16S rRNA and the Cytochrome Oxidase intergenic region), revealed the coexistence of the sibling species at intermediate altitude (1200 m) whereas at lower and higher altitudes only one species was found. An allozyme study, conducted simultaneously and based on four polymorphic loci (PGI1, AAT1, Est-P1 and Est-P2), showed no sign of nuclear introgression. The apparent lack of hybridization in the field is consistent with a postzygotic barrier observed in the laboratory (a nearly complete failure to produce F(1) hybrids). This contact zone is probably a sympatric zone between two genetically differentiated species.

Comparisons of mitochondrial DNA (mtDNA) sequences (16S rRNA gene) between oviparous and viviparous strains of Lacerta vivipara: a preliminary study.

The lizard Lacerta vivipara has allopatric oviparous and viviparous populations. The mitochondrial DNA (mtDNA) gene coding for the 16S rRNA was sequenced for several viviparous lizard populations from France, Switzerland, Bulgaria, Czech Republic, The Netherlands, Sweden, and for oviparous lizard populations from the Pyrenean and Cantabric Mountains. Seven distinct groups (three oviparous and four viviparous) were identified. The net nucleotide divergence between oviparous and viviparous haplotypes was 1.3% +/- 0.5 (mean +/- standard deviation). These results on mtDNA, together with other data obtained previously, led us to formulate a biogeographical scenario that could be tested by further research.

Microspatial genetic structure in the land snail helix aspersa (Gastropoda: helicidae)

The microspatial genetic structure of allele frequencies at seven isozyme loci was examined for 15 populations of the land snail Helix aspersa sampled in a village from Brittany (north-western France). Spatial heterogeneity of allele frequencies was highly significant (P < 0.001). Fixation indices reflected nonrandom mating within neighbourhoods and a slight but consistent differentiation between colonies (FST=0.044; P < 0.01). Analyses of gene flow or genetic distances failed to reveal a significant relationship with geographical distance, probably because of the complexity of environmental heterogeneity. However, matrix comparisons between genetic distances and connectivity networks among adjacent colonies (Gabriel-connected graph) yielded a significant correlation in every case, indicating a 'step-by-step' relationship between neighbouring localities. Moreover, most of the allozymes were spatially structured and showed (i) a gradual isolation of colonies with increasing geographical distances, and (ii), for some correlogram profiles, a circular gradient illustrating a multidirectional colonization of the village. The probable existence of disperser individuals allowed us to suggest a metapopulation model which would explain the maintenance of such animals in fragmented habitats where anthropogenic disturbances and extinction/recolonization events are commonly observed.

Isotope dilution high-performance liquid chromatography-electrospray tandem mass spectrometry assay for the measurement of 8-oxo-7,8-dihydro-2'-deoxyguanosine in biological samples.

A sensitive and specific assay aimed at measuring 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) has been developed by associating a reversed-phase liquid chromatographic separation with an electrospray tandem mass spectrometric detection. The HPLC-MS approach in the single ion monitoring (SIM) mode and the HPLC-MS/MS assay in the multiple reaction monitoring (MRM) mode have been compared, using isotopically labeled [M+4] 8-oxodGuo as the internal standard. The limit of detection of 8-oxodGuo was found to be around 5 pmol and 20 fmol for the HPLC-MS and HPLC-MS/MS methods, respectively. The HPLC-MS/MS assay is sensitive enough to allow the determination of the level of 8-oxodGuo in cellular liver DNA and in urine samples.

Genetic distances and ordination: the land snail Helix aspersa in north Africa as a test case.

We examined the efficiencies of ordination methods in the treatment of gene frequency data at intraspecific level, using metric and nonmetric distance measures (Nei's and Rogers' genetic distances, chi 2 distance). We assessed initial processes responsible for the geographical distribution of the Mediterranean land snail Helix aspersa. Seventeen enzyme loci from 30 North African snail populations were considered in the present analysis. Five combinations of distance/multivariate analysis were compared: correspondence analysis (CA), nonmetric multidimensional scaling (NMDS) on Nei's, Rogers', and chi 2 distances, and principal coordinates analysis on Rogers' distances. Configuration of the objects resulting from ordination was projected onto three-dimensional graphics with the minimum spanning tree or the relative neighborhood graph superimposed. Pre- and postordination or clustering distance matrices were compared by means of correlation methods. As expected, all combinations led to a clear west versus east pattern of variation. However, the intraregional relationships and degree of connectivity between pairs of operational taxonomic units were not necessarily constant from one method to another. Ordination methods when applied with Nei's and Rogers' distances provided the best fit, with original distances (r = 0.98) compared with UPGMA clustering (r approximately 0.75). The Nei/NMDS combination seems to be a good compromise (distortion index dt = 10%) between Rogers/NMDS, which produces a more confusing pattern of differentiation (dt = 24%), and chi 2/CA, which tends to distort large distances (dt = 31%). NMDS obviously provides a powerful method to summarize relationships between populations, when neither hierarchical structure nor phylogenetic inference are required. These findings led the discussion on the good performance of NMDS, the appropriate distances to be used, and the potential application of this method to other types of allelic data (such as microsatellite loci) or data on nucleotide sequences of genes.

Extreme divergence of mitochondrial DNA within species of pulmonate land snails.

Mitochondrial DNA, inherited predominantly through the female line, has been exceptionally useful for reconstructing phylogenies (Avise, in Molecular markers, natural history and evolution. New York: Chapman and Hall (1994)). However, at the lowest taxonomic level, if there are polymorphisms within species the lineages of mitochondria need not correspond to the lineages of the species (Avise, in Phil. Trans. R. Soc. Lond. B 312, 325-342 (1986)). We find that a classic organism in ecological genetics, Cepaea nemoralis, has the most extreme intraspecific variation and polymorphism so far recorded, and that at least one other pulmonate land mollusc also has very high levels of mitochondrial diversity. Making the simplest assumptions, the data suggest times of divergence as long ago as 20 million years between haplotypes now coexisting within a single population. There are four overlapping explanations of the diversity: (i) that mitochondrial evolution in pulmonates is exceptionally fast; (ii) that the morphs have differentiated in isolated 'refuges' and then come together; (iii) that natural selection has acted to preserve the variation; and (iv) that the population structure of pulmonates favours the persistence of ancient haplotypes. We argue for the importance of the last explanation.