Myriam Harry

Extraction and purification of microbial DNA from soil and sediment samples

Abstract Knowledge of the microbial diversity in natural ecosystems has long been limited because only a minority of naturally occurring microbes can be cultured using standard techniques. Several protocols for the extraction of nucleic acids directly from the environmental matrix have been recently developed to circumvent this problem and this review covers the major extraction procedures currently used to obtain microbial DNA from environmental samples. DNA extraction procedures can involve cell extraction or direct lysis, depending on whether or not the microbial cells are isolated from their matrix. An extraction protocol generally comprises three steps: cell lysis that can be chemical, mechanical and enzymatic, removal of cell fragments and nucleic acid precipitation and purification. Direct lysis methods are more often used than cell extraction ones because they are less time consuming and give a better recovery, resulting in an extracted DNA more representative of the whole microbial community present in the sample. However, with direct lysis, contaminants are also extracted which interfere with the DNA extract. As a consequence, a more extensive purification step is required. At least four types of purification are commonly used: cesium chloride density gradient ultracentrifugation, chromatography, electrophoresis and dialysis and filtration. To remove all contaminants, it could be recommended that several purification procedures be combined, depending on the environmental matrix. The efficiency of extraction/purification depends on the properties of the environmental sample, and each step of the extraction procedure must be adjusted for each sample. Moreover, each step of the procedure suffers from shortcomings, and each additional step inevitably induces a DNA loss. Thus, the choice of a protocol must be a compromise between the recovery of DNA that will be the most representative of the microbial community and the quality of the DNA obtained that is imposed by the objectives of the work, such as detection of specific organisms or assessment of the total microbial community structure. Nevertheless, molecular techniques, that could be used in combination with cultivation techniques, are powerful methods for surveying the microbial diversity in environmental samples, although investigators must be aware that such techniques are not exempt of methodological biases.

Biting by Anopheles funestus in broad daylight after use of long-lasting insecticidal nets: a new challenge to malaria elimination

BackgroundMalaria control is mainly based on indoor residual spraying and insecticide-treated bed nets. The efficacy of these tools depends on the behaviour of mosquitoes, which varies by species. With resistance to insecticides, mosquitoes adapt their behaviour to ensure their survival and reproduction. The aim of this study was to assess the biting behaviour of Anopheles funestus after the implementation of long-lasting insecticidal nets (LLINs).MethodsA study was conducted in Dielmo, a rural Senegalese village, after a second massive deployment of LLINs in July 2011. Adult mosquitoes were collected by human landing catch and by pyrethrum spray catch monthly between July 2011 and April 2013. Anophelines were identified by stereomicroscope and sub-species by PCR. The presence of circumsporozoite protein of Plasmodium falciparum and the blood meal origin were detected by ELISA.ResultsAnopheles funestus showed a behavioural change in biting activity after introduction of LLINs, remaining anthropophilic and endophilic, while adopting diurnal feeding, essentially on humans. Six times more An. funestus were captured in broad daylight than at night. Only one infected mosquito was found during day capture. The mean of day CSP rate was 1.28% while no positive An. funestus was found in night captures.ConclusionMosquito behaviour is an essential component for assessing vectorial capacity to transmit malaria. The emergence of new behavioural patterns of mosquitoes may significantly increase the risk for malaria transmission and represents a new challenge for malaria control. Additional vector control strategies are, therefore, necessary.

Candidate Chemosensory Genes in the Stemborer Sesamia nonagrioides

The stemborer Sesamia nonagrioides is an important pest of maize in the Mediterranean Basin. Like other moths, this noctuid uses its chemosensory system to efficiently interact with its environment. However, very little is known on the molecular mechanisms that underlie chemosensation in this species. Here, we used next-generation sequencing (454 and Illumina) on different tissues from adult and larvae, including chemosensory organs and female ovipositors, to describe the chemosensory transcriptome of S. nonagrioides and identify key molecular components of the pheromone production and detection systems. We identified a total of 68 candidate chemosensory genes in this species, including 31 candidate binding-proteins and 23 chemosensory receptors. In particular, we retrieved the three co-receptors Orco, IR25a and IR8a necessary for chemosensory receptor functioning. Focusing on the pheromonal communication system, we identified a new pheromone-binding protein in this species, four candidate pheromone receptors and 12 carboxylesterases as candidate acetate degrading enzymes. In addition, we identified enzymes putatively involved in S. nonagrioides pheromone biosynthesis, including a ∆11-desaturase and different acetyltransferases and reductases. RNAseq analyses and RT-PCR were combined to profile gene expression in different tissues. This study constitutes the first large scale description of chemosensory genes in S. nonagrioides.

Dispersion capacity of Triatoma sherlocki, Triatoma juazeirensis and laboratory-bred hybrids

Flight dispersion is recognized as one of the most important mechanisms for triatomine house infestation. Triatoma sherlocki and T. juazeirensis are closely related species that occur within the same ecotope and their possible reproductive boundaries are unknown. T. sherlocki has shorter wings than T. juazeirensis; a characteristic that possibly implies in reduced flight dispersion, however, this species has been found to invade and colonize homes in Bahia, Brazil. Here, we tested the flight potential of T. sherlocki, compared to that of T. juazeirensis and laboratory-bred hybrids. Insects were kept in an apparatus designed to distinguish flyers from nonflyers. Fifty-one and 53% of T. juazeirensis and hybrids were flyers respectively, whereas no T. sherlocki were recorded to fly. Morphometric analysis of the main structures associated with the locomotor abilities showed that hybrids exhibited intermediate size for most of characters. The width of pronotum of both hybrids and T. juazeirensis was significantly larger than T. sherlocki. We suggested that lack of flight ability of T. sherlocki is possibly a result of reduced wing size and distinct shape, combined with undeveloped flight muscles in a shorter thoracic box. The mobility of T. sherlocki might be compensated by its significantly longer legs, and may possibly increase its ability to invade human dwellings by active dispersion. What is more, this study showed that hybrids between T. sherloki and T. juazeirensis have intermediate morphological characters that may give them higher fitness than their parents, and thus may advance the process of house infestation by either fight or walking in case of an eventual natural hybridization.

Isolation and characterization of microsatellite markers in the bloodsucking bug Rhodnius pallescens (Heteroptera, Reduviidae).

extracting DNA from human nucleated cells. Nucleic Acids Research, 16, 1215. Petren K, Grant BR, Grant PR (1999) Low extra-pair paternity in the Cactus Finch (Geospiza scandens). The Auk, 116, in press. Primmer CR, Moller AP, Ellegren H (1995) Resolving genetic relationships with microsatellite markers: a parentage testing system for the swallow Hirundo rustica. Molecular Ecology, 4, 493Ð498. Primmer CR, Moller AP, Ellegren H (1996) A wide-range survey of cross-species microsatellite amplification in birds. Molecular Ecology, 5, 365Ð378. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor Press, New York.

Use of RAPD markers for the study of microbial community similarity from termite mounds and tropical soils

Abstract In this study, we test the use of the RAPD (Random Amplified Polymorphic DNA) molecular markers as a way to estimate the similarity of the microbial communities in various termite mounds and soils. In tropical ecosystems, termite activities induce changes in the chemical and physical properties of soil. The question then arises as to whether or not termites affect the presence of natural microbial communities. Successful 16S rDNA amplifications provided evidence of the occurrence of bacterial DNA in termite constructions including both soil feeder and fungus grower materials. A phenetic dendrogram using the similarity distance calculated from pairwise data including 88 polymorphic RAPD markers was reconstructed and bootstrap scores mapped. The microbial communities of the mounds of the four soil-feeding termites were clustered in the same clade, while those of the mounds of the fungus-growing species were distinct like those of control soils. Microbial changes in nests result from termite building behavior, depending on whether they include feces in their constructions for soil-feeders or use saliva as particle cement for fungus-growers. It is argued that RAPDs are useful markers to detect differences in microbial community structure not only between termitaries and control soils but also between mounds of soil-feeders.

Soil conservation for DNA preservation for bacterial molecular studies

The aim of this study was to select a method for preserving bacterial DNA in soil samples in cases where there are no possibilities of using freezing or cooling methods. To overcome this difficulty, we hypothesized that adding absolute ethanol to soil samples could be as successful to preserve bacteria as it is to preserve insect or tissue samples for molecular studies. In an attempt to test this assumption, we compared four conservation conditions. After inoculation of soil samples with Escherichia coli, they were either kept at 28 °C, stored in the cold (4 °C), dried at 60 °C, or treated with absolute ethanol. The relative effectiveness of the methods was evaluated by using both DNA recoveries and bacterial 16S rDNA amplification as criteria. Two kinds of soils, i.e. sandy clay and clayey soil, were used. Soil conservation was tested for seven time periods ranging from 2 d to 1 year after bacterial inoculation. Results showed that cold conservation or addition of absolute ethanol to the samples yielded similar DNA recoveries. For these treatments, successful amplifications are still obtained after one year of conservation. The ethanol conservation of soil samples appears to be the easiest method to preserve the bacterial DNA because of its reliability and field convenience.

SimAdapt: an individual-based genetic model for simulating landscape management impacts on populations

Summary 1. Simulation models are essential tools in landscape genetics to study how genetic processes are affected by landscape heterogeneity. However, there is still a need to develop different simulation approaches in landscape genetics, so that users may dispose of additional programs to explore further the impact of land-use and land-cover changes on population genetics. 2. We developed a spatially explicit, individual-based, forward-time, landscape-genetic simulation model combined with a landscape cellular automaton to represent evolutionary processes of adaptation and population dynamics in changing landscapes, using the NetLogo environment. 3. This simulation model represents a unique tool for scientists and scholars looking for a practical and pedagogical framework to explore both empirical and theoretical situations.

Tamandua tetradactyla Linnaeus, 1758 (Myrmecophagidae) and Rhodnius robustus Larrousse, 1927 (Triatominae) infection focus by Trypanosoma rangeli Tejera, 1920 (Trypanosomatidae) in Attalea phalerata Mart. ex Spreng (Arecaceae) palm tree in the Brazilian Amazon.

A sylvatic infection focus of Trypanosoma rangeli, whose cycle involves the anteater Tamandua tetradactyla and triatomine insect Rhodnius robustus was observed in a pasture-dominated landscape of the rural riparian community of São Tomé located along the Tapajós river in the municipal district of Aveiro (State of Pará, Brazil), the Brazilian Amazon region. During a field work campaign with the objective of Chagas disease diagnosis in the Tapajós region, an anteater and 31 triatomines were found inhabiting in the same Attalea phalerata palm tree crown. Collected triatomines were identified as R. robustus with morphological and molecular procedures. The analysis of infection by T. rangeli using the repetitive ARN nucleolar Cl1 (sno-RNA-Cl1) gene showed that 25 triatomines of all stages were infected by T. rangeli (total infection rate of 80.6%). Infection by Trypanosoma cruzi using mini-exon markers was not identified. Examination of the digestive content of the triatomines demonstrated that the only feeding source found was the anteater. These results demonstrate that T. tetradactyla can be an important reservoir for T. rangeli and a good vehicle of the parasite within the Brazilian Amazon region.

Influence of the palm tree species on the variability of Rhodnius nasutus Stål, 1859 (Hemiptera, Reduviidae, Triatominae)

This work evaluated the occurrence and genetic structure of Rhodnius nasutus sampled in two sites using morphometry and microsatellites. These sites, presented distinct abiotic features and palm trees: (i) nine Attalea speciosa palm trees, so called babaçu, were sampled from the Meruoca Mountain Ridge, a sloping region of reminiscent forest in the state of Ceará, Brazil, and (ii) 17 Copernicia prunifera palm trees, so called carnaúba, were sampled in the scrub savanna region (Sobral district) that surrounds the mountain ridge. Of the twenty-six palm trees dissected, 70.6% of carnauba and 88.9% of babaçu were infested by R. nasutus. The micro-climatic data where R. nasutus were sheltered demonstrated that the babaçu and carnaúba palm trees presented significant differences (p < 0.05) in relation to the external environment, except for temperature and relative humidity regulation, suggesting that the architecture of the babaçu crown keeps a more stable micro-environment. The morphometric studies of the F1 generation demonstrated that insects from the babaçu (A. speciosa) were significantly larger (p = 0.000) than those collected in carnaúba (C. prunifera) palm trees. Also, microsatellite analysis demonstrated a high genetic differentiation between the two groups of R. nasutus (R(st) = -0.77). Our results suggest that the difference in size between the populations is probably related to an incipient process of genetic drift in populations associated to each palm tree, probably also driven by the different climatic features observed in these micro-environments.