Patrick Ravanel

Long Lasting Persistence of Bacillus thuringiensis Subsp. israelensis ( Bti ) in Mosquito Natural Habitats

Background The detrimental effects of chemical insecticides on the environment and human health have lead to the call for biological alternatives. Today, one of the most promising solutions is the use of spray formulations based on Bacillus thuringiensis subsp. israelensis (Bti) in insect control programs. As a result, the amounts of Bti spread in the environment are expected to increase worldwide, whilst the common belief that commercial Bti is easily cleared from the ecosystem has not yet been clearly established. Methodology/Main Findings In this study, we aimed to determine the nature and origin of the high toxicity toward mosquito larvae found in decaying leaf litter collected in several natural mosquito breeding sites in the Rhône-Alpes region. From the toxic fraction of the leaf litter, we isolated B. cereus-like bacteria that were further characterized as B. thuringiensis subsp. israelensis using PCR amplification of specific toxin genes. Immunological analysis of these Bti strains showed that they belong to the H14 group. We finally used amplified length polymorphism (AFLP) markers to show that the strains isolated from the leaf litter were closely related to those present in the commercial insecticide used for field application, and differed from natural worldwide genotypes. Conclusions/Significance Our results raise the issue of the persistence, potential proliferation and environmental accumulation of human-spread Bti in natural mosquito habitats. Such Bti environmental persistence may lengthen the exposure time of insects to this bio-insecticide, thereby increasing the risk of resistance acquisition in target insects, and of a negative impact on non-target insects.

Interactions between immune and biotransformation systems in fish: A review

In animals biotransformation and immune system are not totally independent, there are numerous functional interrelationships between these two systems. They are both implicated in the capacity of organisms to resist to a wide variety of environmental components such as viruses, bacteria and xenobiotics. It is known for a long time that the immune system functions as a physiologic system and interacts with all the other components of the organism including nervous or endocrine ones. In the same manner, the biotransformation system (especially the cytochrome P450 monooxygenases) is involved in the regulation of numerous hormone productions. In this way, many studies in mammals have revealed the possible interaction between immune and biotransformation systems. Among these interactions, the capacity of the activation of host defense mechanisms to down-regulate microsomal cytochrome P450 and the role of biotransformation system in the xenobiotic-mediated immunotoxicity have been underlined. Advances in the basic knowledge of fish immune and biotransformation systems should lead to a better understanding of the possible interactions between both systems and should improve fish health monitoring which is a crucial ecotoxicological goal.

Response of Aedes aegypti (Diptera : Culicidae) larvae to three xenobiotic exposures: Larval tolerance and detoxifying enzyme activities

The ability of mosquito larvae to tolerate toxic compounds (temephos, Bacillus thuringiensis var. israelensis, toxic vegetable leaf litter) was examined on a laboratory larval strain of Aedes aegypti L. Bioassays and detoxifying enzyme activity measurements were performed to compare tolerance/resistance capacities. The possibility of a functional plasticity of detoxifying equipment was investigated through experimental determination of the inductive effect of each xenobiotic within a given generation. In the same way, the selective effect of a toxic leaf litter was also investigated along successive generations. Results revealed that differential cytochrome P450 monooxygenase, esterase, and glutathione S-transferase activity levels correlated with the bioassay results. Both induction and selection increased larval tolerance to the xenobiotics used and increased the levels of larval detoxifying enzyme activities.

Effect of chlorophenols on isolated plant mitochondria activities: A QSAR study

Twenty-three chlorinated monophenols were investigated for their uncoupling and inhibitory properties on plant mitochondria. All the studied compounds had uncoupling properties but their activity was submitted to quantitative changes from 1- to 400-fold, according to the substitution. They also had inhibitory properties on the electron transfer at a level located upstream to the quinone pool. Three inhibitory types could be recognized: (a) chlorophenols inhibiting similarly the oxidation of every type of substrate (NADH, succinate, alpha-ketoglutarate, at pH 7.2; malate, at pH 7.5), (b) chlorophenols inhibiting first complex I, and (c) chlorophenols inhibiting first complex II. In this last case, the presence of substituents at the 2 and 6 positions increases selectively the inhibition of succinate oxidation. A quantitative structure activity relationship (QSAR) study was undertaken and showed that a good correlation appeared between the steric parameter, 1 chi v, and the inhibitory properties with NADH as substrate. This result could be explained by the binding of the chlorinated phenols with NADH dehydrogenase whose position, on the external surface of the mitochondrial membrane, is favorable to good accessibility of the xenobiotic. Equations for inhibitory properties with other substrates were clearly different. The relation between the uncoupling activity and the studied parameters was more complex but a good correlation occurred with the steric parameter (sigma D, A) and the electronic parameter sigma. Neither log P nor sigma alone gave equations of good quality. These results suggest a competition between the chlorophenol mobility in the inner membrane, necessary for uncoupling, and binding with different proteins in the same membrane on which the inhibitory activity is dependent.

A comparative study on the uptake and translocation of organochlorines by Phragmites australis

Organochlorines (OCs) are persistent chemicals found in various environmental compartments. The differences in the uptake of (14)C-labeled 1,4-dichlorobenzene (DCB), 1,2,4-trichlorobenzene (TCB) and γ-hexachlorocyclohexane (γHCH) by Phragmites australis were investigated under hydroponic conditions. The first step in sorption appears to be correlated with the hydrophobic nature of the compounds, since log-linear correlations were obtained between root concentration factor and partition coefficient (LogK(ow)). After 7 days of exposure, plant uptake of DCB, TCB, γHCH was significant with bioconcentration factors reaching 14, 19 and 15, respectively. Afterwards, uptake and translocation were seen to be more complex, with a loss of the simple relationship between uptake and LogK(ow). Linear correlations between the bioconcentration/translocation factors and the physico-chemical properties of OCs were shown, demonstrating that translocation from roots to shoots increases with solubility and volatility of the OCs. This suggests that OC-translocation inside plants might result from the combination of two processes, xylem sap flow and vapor fluxes. (14)C-phytovolatilization was measured and was correlated with the volatility of the compounds; the more volatile OCs being most the likely to be phytovolatilized from foliar surfaces (p=0.0008). Thus, OC-uptake/translocation appears to proceed at a rate that depends mostly on the OCs hydrophobicity, solubility and volatility.

Effects of flavone on the oxidative properties of intact plant mitochondria

Abstract The effects of flavone on the oxidative and phosphorylative properties of plant mitochondria from potato tubers and etiolated mung bean hypocotyls were investigated. Flavone inhibited the state 3 oxidation rates of malate, NADH and, to a lesser extent, succinate but was without effect on the ascorbate-TMPD oxidation rate. The inhibition was the same whether the mitochondria were in state 3 or in an uncoupled state 3. When 100 μM flavone was added during the state 4, the tight coupling of succinate or NADH oxidation was not released. In the electron transfer chain, flavone inhibition appeared to be located in the flavoprotein region. All forms of NADH dehydrogenases seemed to be affected but the greatest inhibition appeared when exogenous NADH was used.

Differential sensitivity to Bacillus thuringiensis var. israelensis and temephos in field mosquito populations of Ochlerotatus cataphylla (Diptera: Culicidae): toward resistance?

In the present study, four populations of the same field mosquito species, Ochlerotatus cataphylla, were sampled over the Rh6ne-Alpes region (France), and their respective sensitivity to the organophosphate temephos and the bacterio-insecticide Bacillus thuringiensis var. israelensis (Bti) was measured. The results obtained in toxicological tests showed significant differences in the larval sensitivities of the four populations for both insecticides. These differences appeared to be related to the activity of the three main families of detoxifying enzymes: Cytochrome P450 monooxygenases, glutathione-S-transferases (GSTs), and esterases. All three enzyme families were significantly overexpressed in the less susceptible larval population, and after multiple regressions, GSTs and esterases came out as the most explicative variables of the larval sensitivity. Considering these results and the chemical history of the sites in terms of insecticide treatments, the hypothesis of cross-effects of insecticides leading to resistance acquisition to Bti in field organisms emerges.

Atrazine metabolism in corn seedlings

Atrazine is a herbicide widely used in corn culture. Corn seedlings metabolize this active ingredient readily in three ways. Two of them are catalysed by enzymes and another is purely chemical. In seedlings, the precise role played by each of these was still not clearly understood. Our work demonstrates that the chemical pathway leading to the formation of the inactive OH-2 atrazine is the pre-eminent form of metabolization inside the roots and, during the first week, inside the leaves. As shown by the kinetics of accumulation of the benzoxazinones responsible for this metabolization, a high potential of atrazine hydroxylation remains effective inside the leaves for at least 1 month. The OH-2 atrazine, which seems to accumulate inside the cell vacuoles, cannot move freely inside the plant and therefore cannot be abundantly transported from roots to leaves. The formation of a glutathione-atrazine conjugate, due to the activity of a glutathione-S-transferase (GST) isoenzyme, represents only a very small part of the total GST in corn. It is mostly present in the aerial parts and is highly effective only after a 1-week culture. When analysing the mixture of the metabolites formed in isolated corn leaves, which is very complex (nine products, among which six were major products), it seems that the metabolization pattern of corn leaves is the combined result of the three pathways and of the transformation of the conjugate into more simple derivatives.

Effects of chlorophenols on isolated class A chloroplasts and thylakoids: a QSAR study.

A series of 22 chlorinated phenols was studied for their effects on photosynthesis of isolated chloroplasts. Each chlorophenol was an uncoupler, but the uncoupling activity depended upon substitution. The di-, tri-, or pentachloro substitution greatly enhanced the uncoupling activity. However, 2,6 substitution was not favorable to the uncoupling activity. Fifty percent uncoupling of photophosphorylations was obtained for concentrations between 4 mM for phenol itself, and 20 microM for pentachlorophenol. The quantitative structure-activity relationship (QSAR) study indicated a good relationship between, on the one hand, steric and electronic parameters, and, on the other hand, the uncoupling activity. In such equations, the A parameter describing ortho-substitution, always presented a negative sign. No good equation could be obtained with only log P and sigma. At concentrations which generally induced uncoupling, the chlorinated phenols inhibited the electron transfer in thylakoids. Di, tri, or penta substitution by chlorine enhanced the inhibition. A good relation appeared between this effect and the steric parameters MR and A. The study of isolated class A chloroplasts demonstrated that the uncoupling and the inhibition of the electron transfer in thylakoids could explain the effect of the chlorophenols on the whole photosynthetic mechanism. The effects of phenols on isolated chloroplasts were compared to those obtained with the same series on mitochondria. To explain the differences between QSAR equations, on the one hand for chloroplasts, and, on the other hand, for mitochondria, we suggested a selective binding of chlorinated phenols to proteins of the biological membranes.

Repartition des flavonols dans l'epaisseur des feuilles de quelques vegetaux vasculaires

Abstract The tissue localisation of flavonoids has been studied in leaves of Betula , Corylus , Fagus , Fraxinus , Pisum , Platanus , Quercus , Spinacia and Tilia and scales of onion bulbs. All these species contain flavonols which are, for the most part, located in the upper epidermis of the leaves. In the onion bulb, flavonols are exclusively in the epidermis. The flavonols are glycosylated and dissolved in the vacuoles. The leaves were fractionated by an original technique of abrasion of the frozen material. The physiological significance of such a distribution of flavonoids in the adult leaves or scales is discussed.