Vincent Veron

Fine‐scale genetic structure and gene dispersal inferences in 10 Neotropical tree species

The extent of gene dispersal is a fundamental factor of the population and evolutionary dynamics of tropical tree species, but directly monitoring seed and pollen movement is a difficult task. However, indirect estimates of historical gene dispersal can be obtained from the fine‐scale spatial genetic structure of populations at drift–dispersal equilibrium. Using an approach that is based on the slope of the regression of pairwise kinship coefficients on spatial distance and estimates of the effective population density, we compare indirect gene dispersal estimates of sympatric populations of 10 tropical tree species. We re‐analysed 26 data sets consisting of mapped allozyme, SSR (simple sequence repeat), RAPD (random amplified polymorphic DNA) or AFLP (amplified fragment length polymorphism) genotypes from two rainforest sites in French Guiana. Gene dispersal estimates were obtained for at least one marker in each species, although the estimation procedure failed under insufficient marker polymorphism, limited sample size, or inappropriate sampling area. Estimates generally suffered low precision and were affected by assumptions regarding the effective population density. Averaging estimates over data sets, the extent of gene dispersal ranged from 150 m to 1200 m according to species. Smaller gene dispersal estimates were obtained in species with heavy diaspores, which are presumably not well dispersed, and in populations with high local adult density. We suggest that limited seed dispersal could indirectly limit effective pollen dispersal by creating higher local tree densities, thereby increasing the positive correlation between pollen and seed dispersal distances. We discuss the potential and limitations of our indirect estimation procedure and suggest guidelines for future studies.

Multiple independent introductions of Plasmodium falciparum in South America

The origin of Plasmodium falciparum in South America is controversial. Some studies suggest a recent introduction during the European colonizations and the transatlantic slave trade. Other evidence—archeological and genetic—suggests a much older origin. We collected and analyzed P. falciparum isolates from different regions of the world, encompassing the distribution range of the parasite, including populations from sub-Saharan Africa, the Middle East, Southeast Asia, and South America. Analyses of microsatellite and SNP polymorphisms show that the populations of P. falciparum in South America are subdivided in two main genetic clusters (northern and southern). Phylogenetic analyses, as well as Approximate Bayesian Computation methods suggest independent introductions of the two clusters from African sources. Our estimates of divergence time between the South American populations and their likely sources favor a likely introduction from Africa during the transatlantic slave trade.

Multiplex real-time PCR detection of P. falciparum, P. vivax and P. malariae in human blood samples

Two duplex real-time PCR assays were developed to diagnose three human parasites: Plasmodium falciparum, Plasmodium vivax and Plasmodium malariae. TaqMan duplex real-time PCR was evaluated in 263 blood samples of suspected malaria patients by comparing results against those obtained with microscopy and nested PCR. Compared with nested PCR, duplex real-time PCR assays showed 100% sensitivity and specificity. Duplex real-time PCR detected all mixtures of P. falciparum and P. vivax DNA, except at threshold detection limits for both parasites in which P. vivax was not amplified. Threshold detection limits of real-time PCR were 3.1, 0.3 and 0.8 parasites per microlitre of blood for P. falciparum, P. vivax and P. malariae, respectively. Duplex real-time PCR allows the detection of malarial cases, including mixed species infection, it simplifies analysis and reduces cost. Thus, this protocol may prove invaluable for use in the diagnosis of human infection, trial treatments and epidemiologic studies in which high-throughput analyses are often required.

Sensitive and Specific Detection of Trypanosoma cruzi DNA in Clinical Specimens Using a Multi-Target Real-Time PCR Approach

Background The laboratory diagnosis of Chagas disease is challenging because the usefulness of different diagnostic tests will depend on the stage of the disease. Serology is the preferred method for patients in the chronic phase, whereas PCR can be successfully used to diagnose acute and congenital cases. Here we present data using a combination of three TaqMan PCR assays to detect T. cruzi DNA in clinical specimens. Methods/Principal Findings Included in the analysis were DNA extracted from 320 EDTA blood specimens, 18 heart tissue specimens, 6 umbilical cord blood specimens, 2 skin tissue specimens and 3 CSF specimens. For the blood specimens both whole blood and buffy coat fraction were analyzed. The specimens were from patients living in the USA, with suspected exposure to T. cruzi through organ transplantation, contact with triatomine bugs or laboratory accidents, and from immunosuppressed patients with suspected Chagas disease reactivation. Real-time PCR was successfully used to diagnose acute and Chagas disease reactivation in 20 patients, including one case of organ-transmitted infection and one congenital case. Analysis of buffy coat fractions of EDTA blood led to faster diagnosis in six of these patients compared to whole blood analysis. The three real-time PCR assays produced identical results for 94% of the specimens. The major reason for discrepant results was variable sensitivity among the assays, but two of the real-time PCR assays also produced four false positive results. Conclusions/Significance These data strongly indicate that at least two PCR assays with different performances should be combined to increase the accuracy. This evaluation also highlights the benefit of extracting DNA from the blood specimens buffy coat to increase the sensitivity of PCR analysis.

High or low dietary carbohydrate:protein ratios during first-feeding affect glucose metabolism and intestinal microbiota in juvenile rainbow trout

Based on the concept of nutritional programming in mammals, we tested whether an acute hyperglucidic–hypoproteic stimulus during first feeding could induce long-term changes in nutrient metabolism in rainbow trout. Trout alevins received during the five first days of exogenous feeding either a hyperglucidic (40% gelatinized starch + 20% glucose) and hypoproteic (20%) diet (VLP diet) or a high-protein (60%) glucose-free diet (HP diet, control). Following a common 105-day period on a commercial diet, both groups were then challenged (65 days) with a carbohydrate-rich diet (28%). Short- and long-term effects of the early stimuli were evaluated in terms of metabolic marker gene expressions and intestinal microbiota as initial gut colonisation is essential for regulating the development of the digestive system. In whole alevins (short term), diet VLP relative to HP rapidly increased gene expressions of glycolytic enzymes, while those involved in gluconeogenesis and amino acid catabolism decreased. However, none of these genes showed persistent molecular adaptation in the liver of challenged juveniles (long term). By contrast, muscle of challenged juveniles subjected previously to the VLP stimulus displayed downregulated expression of markers of glycolysis and glucose transport (not seen in the short term). These fish also had higher plasma glucose (9 h postprandial), suggesting impaired glucose homeostasis induced by the early stimulus. The early stimulus did not modify the expression of the analysed metabolism-related microRNAs, but had short- and long-term effects on intestinal fungi (not bacteria) profiles. In summary, our data show that a short hyperglucidic–hypoproteic stimulus during early life may have a long-term influence on muscle glucose metabolism and intestinal microbiota in trout.

Detection of Histoplasma capsulatum DNA in human samples by real-time polymerase chain reaction

The main aim of our study was to determine the added value of real-time polymerase chain reaction (PCR) for the diagnosis of Histoplasma capsulatum in routine biologic practice. No amplification signal was observed with the 18 non-H. capsulatum strains used to test the specificity of the protocol. The sensitivity threshold of the real-time PCR assay was about 10 fg of H. capsulatum DNA per microliter, tested with a 10-fold serial dilution of the positive control. We analyzed 348 human samples submitted for the routine diagnosis of systemic mycosis. Real-time PCR using the TaqMan system was evaluated against direct microscopic examination and culture. Among the 341 samples without PCR inhibition (n = 7), 66 tested positive by culture, whereas 74 tested positive by real-time PCR. Sensitivity of the real-time PCR assay was estimated at 95.4% and specificity at 96.0% with respect to culture, widely considered to be the gold standard method; however, the molecular approach in fact produced better sensitivity and specificity results. Moreover, for the 38 samples that tested negative by direct examination but positive by culture, the culture method took a mean of 31 days longer than the PCR method to generate results. The protocol presented here may be very useful for improving routine histoplasmosis diagnosis.

Genetic diversity of msp3α and msp1_b5 markers of Plasmodium vivax in French Guiana

BackgroundReliable molecular typing tools are required for a better understanding of the molecular epidemiology of Plasmodium vivax. The genes msp3a and msp1 _block5 are highly polymorphic and have been used as markers in many P. vivax population studies. These markers were used to assess the genetic diversity of P. vivax strains from French Guiana (South America) and to develop a molecular typing protocol.MethodsA total of 120 blood samples from 109 patients (including 10 patients suffered from more than one malaria episode, samples were collected during each episode) with P. vivax infection were genotyped. All samples were analysed by msp3a PCR-RFLP and msp1 _b5 gene sequencing was performed on 57 samples. Genotyping protocol applied to distinguish between new infection or relapse from heterologus hypnozoites and treatment failure or relapse from homologus hypnozoites was based on analysing first msp3a by PCR-RFLP and secondly, only if the genotypes of the two samples are identical, on sequencing the msp1 _b5 gene.Resultsmsp3a alleles of three sizes were amplified by PCR: types A, B and C. Eleven different genotypes were identified among the 109 samples analysed by msp3a PCR-RFLP. In 13.8% of cases, a mixed genotype infection was observed. The sequence of msp1_ b5 gene revealed 22 unique genotypes and 12.3% of cases with mixed infection. In the 57 samples analysed by both methods, 45 genotypes were found and 21% were mixed. Among ten patients with two or three malaria episodes, the protocol allowed to identify five new infections or relapses from heterologous hypnozoites and six treatment failures of relapses from homologous hypnozoites.ConclusionThe study showed a high diversity of msp3a and msp1_ b5 genetic markers among P. vivax strains in French Guiana with a low polyclonal infection rate. These results indicated that the P. vivax genotyping protocol presented has a good discrimination power and can be used in clinical drug trials or epidemiological studies.

Lipid digestion, absorption and uptake in Solea senegalensis.

Dietary lipids are the major energy source for metabolic purposes in most fish species, and improve dietary protein utilization for growth. In a previous study we have reported a low tolerance of Senegalese sole juveniles to dietary lipid levels and suggested a maximal dietary inclusion level of 8% lipids for both optimal growth and nutrient utilization. The mechanisms behind this apparent poor utilization of the dietary lipids are still to be elucidated. The primary aim of the present study was to investigate the overall process of digestion and lipid absorption in relation to dietary lipid levels. Triplicate groups of twenty fish (mean initial mass 29g) were fed two isonitrogenous diets (54% of protein dry matter basis) with different lipid levels (L4 and L17, 4 and 17% lipids dry matter basis), for 88days. Protein and lipid apparent digestibility coefficients as well as lipase activity were similar in both groups suggesting that Solea senegalensis has the ability to digest equally well a low fat or a high fat diet. Plasma triglyceride concentrations were significantly higher 5 and 16h after feeding in fish fed the L17 compared to those fed L4, following dietary lipid supply, demonstrating effective lipid absorption. Expression of proteins related to lipid transport (microsomal triglyceride transfer protein), trafficking (Fatty acid binding protein 11) and fatty acid uptake (VLDL-r) was significantly higher in liver of fish fed the high fat diet 16h after the meal, but remained unchanged in muscle. In conclusion, it seems that high fat diets do not impair lipid digestion and absorption.

Comparison of Glucose and Lipid Metabolic Gene Expressions between Fat and Lean Lines of Rainbow Trout after a Glucose Load

Two experimental rainbow trout lines developed through divergent selection for low (Lean ‘L’ line) or high (Fat ‘F’ line) muscle fat content were used as models to study the genetic determinism of fat depots. Previous nutritional studies suggested that the F line had a better capability to use glucose than the L line during feeding trials. Based on that, we put forward the hypothesis that F line has a greater metabolic ability to clear a glucose load effectively, compared to L line. In order to test this hypothesis, 250 mg/kg glucose was intraperitoneally injected to the two rainbow trout lines fasted for 48 h. Hyperglycemia was observed after glucose treatment in both lines without affecting the phosphorylation of AMPK (cellular energy sensor) and Akt-TOR (insulin signaling) components. Liver glucokinase and glucose-6-phosphate dehydrogenase expression levels were increased by glucose, whereas mRNA levels of β-oxidation enzymes (CPT1a, CPT1b, HOAD and ACO) were down-regulated in the white skeletal muscle of both lines. Regarding the genotype effect, concordant with normoglycemia at 12 h after glucose treatment, higher muscle glycogen was found in F line compared to L line which exhibited hyperglycemia. Moreover, mRNA levels of hepatic glycolytic enzymes (GK, 6PFK and PK), gluconeogenic enzyme PEPCK and muscle fatty acid oxidation enzymes (CPT1a, CPT1b and HOAD) were concurrently higher in the F line. Overall, these findings suggest that F line may have a better ability to maintain glucose homeostasis than L line.

Leishmania spp. identification by polymerase chain reaction–restriction fragment length polymorphism analysis and its applications in French Guiana

Leishmania (Viannia) guyanensis was for many years the only species commonly identified in French Guiana, but precise species identifications were quite rare. We describe a new restriction fragment length polymorphism-polymerase chain reaction technique using a 615-bp fragment of the RNA polymerase II gene and 2 restriction enzymes, TspRI and HgaI. Seven reference strains (Leishmania (Leishmania) amazonensis, Leishmania (Viannia) lainsoni, Leishmania (Viannia) braziliensis, L. (V.) guyanensis, Leishmania (Viannia) naiffi, Leishmania (Leishmania) major, Leishmania (Leishmania) infantum) and 112 clinical samples from positive lesions were used for the development of the technique. The rates of positive species identification were 85.7% for punch skin biopsy specimens, 93.1% for positive Giemsa-stained smears, and 100% for positive culture supernatants. In the framework of cutaneous leishmaniasis species surveillance for the 2006 to 2008 period, parasite identification was carried out for 199 samples from different patients. The prevalence of the various Leishmania spp. was 84.4% for L. (V.) guyanensis, 8.0% for L. (V.) braziliensis, 5.0% for L. (L.) amazonensis, and 2.6% for L. (V.) lainsoni. L. (V.) braziliensis seems to be locally an emerging pathogen.