Production of the bioinsecticide Bacillus thuringiensis subsp. israelensis with deltamethrin increases toxicity towards mosquito larvae.

UNLABELLED: Bacillus thuringiensis subsp. israelensis is a bioinsecticide used for larval mosquito control and it represents a safe alternative to chemical insecticides. Despite its environmental safety, it is less efficient and persistent than chemical insecticides. To bypass these limitations, we propose to combine the advantages of chemical and biological insecticides by producing Bti in a medium supplemented with a chemical insecticide (DDT, deltamethrin, permethrin, propoxur or temephos). Among the investigated insecticides, the addition of deltamethrin in the medium induced a higher toxicity (over 6.72-fold) of the composite deltamethrin-Bti towards mosquito larvae as compared to Bti alone. This was mainly due to the insertion of deltamethrin into the membranes of Bti spores, as evidenced by a quantification of membrane-extracted deltamethrin by HPLC. This composite larvicide is a promising tool to decrease the quantity of chemicals dispersed in the environment, to increase the efficacy of Bti and to facilitate its widespread use as a transition between chemical and biological insecticides. Further experiments are required to characterize the mechanisms that underline the incorporation of deltamethrin into Bti to optimize the production and the toxicity of this composite larvicide. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first report of an increased efficacy of the mosquitocidal bioinsecticide Bacillus thuringiensis subsp. israelensis (Bti) when produced with a chemical insecticide. The results clearly demonstrate that deltamethrin is able to synergize the insecticidal activity of Bti through inclusion into spore membranes, reducing off-target and nonspecific toxicity occurring when the chemical is used alone as sprays. This new composite chemical-biological insecticide can become an invaluable tool as an intermediate between single chemical usage and the widespread use of Bti, notably in developing countries with limited financial resources for intensive mosquito control campaigns.

Stability of floral specialization in Trollius europaeus in contrasting ecological environments.

Specialization of some plants on seed-eating pollinators is intriguing, especially when co-pollinators exclusively feeding on nectar are also present. We examined the stability of the morphological specialization of Trollius europaeus (L.) globeflowers with respect to Chiastocheta (Pokorny) flies by artificially opening the flowers. In the montane and subalpine environments studied, other visitors contributed 2% and 28% of all the visits, respectively, and visited open flowers nearly eight times more often than closed flowers, but in both environments their contribution to pollination did not compensate for Chiastocheta aversion against open phenotypes. Net seed set (female success) was slightly higher (+4%) and pollen export (male success) was much higher (+85%) for closed than open flowers. Selection in favour of the closed phenotype was even more intense in patches where open phenotypes were most common, precluding the evolution of open flowers in the study populations.

A spatial analysis method (SAM) to detect candidate loci for selection: towards a landscape genomics approach to adaptation.

The detection of adaptive loci in the genome is essential as it gives the possibility of understanding what proportion of a genome or which genes are being shaped by natural selection. Several statistical methods have been developed which make use of molecular data to reveal genomic regions under selection. In this paper, we propose an approach to address this issue from the environmental angle, in order to complement results obtained by population genetics. We introduce a new method to detect signatures of natural selection based on the application of spatial analysis, with the contribution of geographical information systems (GIS), environmental variables and molecular data. Multiple univariate logistic regressions were carried out to test for association between allelic frequencies at marker loci and environmental variables. This spatial analysis method (SAM) is similar to current population genomics approaches since it is designed to scan hundreds of markers to assess a putative association with hundreds of environmental variables. Here, by application to studies of pine weevils and breeds of sheep we demonstrate a strong correspondence between SAM results and those obtained using population genetics approaches. Statistical signals were found that associate loci with environmental parameters, and these loci behave atypically in comparison with the theoretical distribution for neutral loci. The contribution of this new tool is not only to permit the identification of loci under selection but also to establish hypotheses about ecological factors that could exert the selection pressure responsible. In the future, such an approach may accelerate the process of hunting for functional genes at the population level.

Genetic structure of the forest pest Hylobius abietis on conifer plantations at different spatial scales in Europe.

The distribution of genetic variation within and among 20 European sites infested by the large pine weevil, Hylobius abietis, was analysed using dominant amplified fragment length polymorphism markers. Analysis of molecular variance was performed at the European, regional and local scales. Most of the genetic variability was found within rather than among populations and the global fixation index averaged over loci was low (0.07). We found no evidence of genetic drift, even in relatively isolated sites. This genetic pattern tends to confirm the high dispersal ability of the weevil and the influence of human-mediated expansion of its range through conifer plantations across Europe since the 19th century. Assignment tests demonstrated that the regional forest is a pertinent geographic scale for defining populations in the large pine weevil. Testing the potential influence of the larval host-plant identity (Scot Pine vs Norway Spruce) on the genetic structure revealed a weak but significant effect in two of the three regions tested (in Ardèche and in Limousin but not in Finland). One locus varied with host-plant use in the two French regions, indicating a potential role in host-plant adaptation. However, host-race formation is not observed in H. abietis; we discuss this result in the light of our current knowledge of this insect's biology. Altogether, this study shows that the use of different host plants for development does not constitute a strong barrier to gene flow for H. abietis and confirms the high dispersal ability of this forest pest.

Mapping of resistance to vegetable polyphenols among Aedes taxa (Diptera, Culicidae) on a molecular phylogeny.

To recover some evolutionary aspects of the interaction between culicine larvae and dietary polyphenols of the vegetation surrounding mosquito breeding sites, we constructed a phylogeny of the most common French Aedes species, chosen as reference species. We also evaluated the differential resistance of these larval taxa to the polyphenols of leaf litter from the riparian vegetation used as a food source. Mitochondrial DNA sequence analysis was performed among 14 different taxa and ecotypes (Aedes aegypti, Ae. albopictus, Ae. cantans, Ae. caspius, Ae. cataphylla, Ae. cinereus, Ae. detritus, Ae. geniculatus, Ae. mariae, Ae. pullatus, Ae. punctor, Ae. rusticus, Ae. sticticus, and Ae. vexans) through direct sequencing of a 763-base segment of the cytochrome oxidase subunit I gene. Phylogenetic analysis, based on nucleotide and amino acid sequences, was conducted by means of parsimony and distance methods. The differential tolerance of larvae to vegetable leaf litter was comparatively tested by use of 10-month-old alder leaf litter as an experimental standard. The absence of correlation between resistance to polyphenols and molecular phylogeny suggests that larval adaptation to polyphenol-rich vegetable breeding sites is a labile character. The acquisition of such resistance appears not to be ancestrally inherited, but rather to be a dynamic adaptation to the environment. Molecular data also support the classical morphological classification within the Aedes genus.

Mitochondrial genes of Schistosoma mansoni.

Two clones, totalling 8068 bp and spanning over half of the coding region of the mitochondrial genome of Schistosoma mansoni, have been sequenced. Complete sequences are presented of the large and small ribosomal RNA subunits, CO2, ND3, ND4, ND6 and ATPase 6 genes. Incomplete sequences were found for the CO1, ND2 and CytB genes. At least 10 tRNAs were also detected and alternative structures for some of these discussed. The gene order of S. mansoni is unique and differs from that of Fasciola hepatica, the only other trematode for which any information is available.

Systematics of the genus Capra inferred from mitochondrial DNA sequence data.

Traditional classification in the genus Capra is based mainly on horn morphology. However, previous investigations based on allozyme data are not consistent with this classification. We thus reexamined the evolutionary history of the genus by analyzing mitochondrial DNA (mtDNA) sequence variation. We collected bone samples from museums or dead animals found in the field. Thirty-four individuals were successfully sequenced for a portion of the mtDNA cytochrome b gene and control region (500 bp in total). We obtained a star-like phylogeny supporting a rapid radiation of the genus. In accordance with traditional classification, mtDNA data support the presence of two clades in the Caucasus and the hypothesis of a domestication event in the Fertile Crescent. However, in conflict with morphology, we found that C. aegagrus and C. ibex are polyphyletic species, and we propose a new scenario for Capra immigration into Europe.

ITS2 ribosomal RNA indicates Schistosoma hippopotami is a distinct species.

The internal transcribed spacer region of the ribosomal RNA, ITS2, was sequenced from a single specimen of S. hippopotami collected from a pulmonary artery of the hippopotamus, Hippopotamus amphibius in South Africa. The nucleotide sequence was aligned with those of S. mansoni, S. rodhaini, S. haematobium, S. intercalatum, S. curassoni, S. bovis and S. japonicum. Both maximum parsimony and genetic distance analyses were performed on these data sets. Using S. japonicum as outgroup to the African schistosomes, a single most-parasmonious tree was obtained of length 64 steps with a consistency index of 1-S. hippopotami was the sister-group to the remaining African species. This species has lateral-spined eggs and its basal position in the tree suggests that this condition is primitive and that terminal-spined eggs developed secondarily. Molecular data clearly show that S. hippopotami cannot be considered synonymous with S. mansoni. Assuming the hippopotamus is the normal host of S. hippopotami, phylogenetic analysis is consistent with an ancient association between schistosomes and ungulates.

Molecular characterization of mitochondrial DNA provides evidence for the recent introduction of Schistosoma mansoni into America.

Mitochondrial DNA was analyzed from 6 strains of Schistosoma mansoni with 12 restriction enzymes, corresponding to 40 restriction sites or about 1.5% of the coding region. An extensive length polymorphism among strains of S. mansoni was found, with size ranging from 16,500 bp to 24,900 bp. Five restriction sites among 40 were polymorphic; phylogenetic analysis using parsimony criteria supports the idea that American strains were introduced very recently from Africa. Two large fragments (2800 and 5400 bp), representing half of the coding region were cloned. The location of some genes was determined using heterospecific multigenes probes and suggests that this platyhelminth genome differs from all other known mtDNA gene organization.

Conserved secondary structures in the ITS2 of trematode pre-rRNA.

Biological functions of transcribed spacer regions in eucaryotic pre-rRNAs remain elusive so far. Utilization of the comparative approach to analyse their secondary structure has been impeded by the extensive sequence divergence observed among most of the specimens available to date. However, we have taken advantage of the recent derivation of a set of largely similar sequences for different schistosome species to look for the presence of constrained secondary structures within the internally transcribed spacer 2 (ITS2). We show that several common features of secondary structure are shared by these species despite sequence variation, with some of them supported by compensatory changes, suggesting a significant role of ITS2 as an RNA domain during ribosome biogenesis.

Molecular evidence linking hominid evolution to recent radiation of schistosomes (Platyhelminthes: Trematoda).

Molecular phylogenies for seven species of schistosomes, including four species infecting man, were constructed from PCR-amplified sequences of two ribosomal genes: one nuclear (internal transcribed spacer 2 in the ribosomal multigenic family) and one mitochondrial (16S rDNA). The two phylogenies obtained are congruent, and the data suggest that the mitochondrial sequence evolves about three times faster than the nuclear sequence. We propose a calibration of the phylogenetic tree of schistosomes that dates "human capture" of these parasites from other animal hosts (rodents and ruminants) in Africa to 1-10 million years ago, when the first hominids invaded savanna areas, which are the favorable environment for parasite transmission.