Female Aedes aegypti mosquitoes are vectors of yellow fever, dengue and chikungunya viruses. Pre-exposure of Ae. aegypti larvae to the herbicide atrazine significantly reduced their sensitivity to the organophosphate insecticide temephos. Mosquito larvae pre-exposed for 48 h to non-lethal concentrations of atrazine ranging from 1 to 10 mu g/l commonly encountered in the field, appeared slightly less sensitive to temephos than non-pre-exposed larvae. The effect of a pre-exposure to atrazine on larval tolerance to temephos did not seem to be related to an induction of detoxification processes by this herbicide. Noimportant increase in glutathione transferase, or alpha- and beta-esterase activities was observed in pre-exposed larvae, while P450 monooxygenase activities increased.

Until recently, the chemical control of phytophagous larvae of the genus Agriotes was exclusively achieved through soil treatment with insecticides. Under such conditions, the larvae receive the active ingredient (a.i.) by integumental penetration whilst moving through the subsurface soil. The selectivity of such treatments is only based on the biochemical selectivity of the a.i. which is generally very low across soil invertebrates. More recent a.i.s from new chemical families such as fiprols show not only a new biochemical mode of action but also a particular mode of penetration which is restricted to the digestive pathway. This allows a new type of selectivity, based on penetration after feeding, when the a.i. is associated with food. We demonstrate that Agriotes species, which are important pests for several crops, feed almost exclusively on the seeds of plant species. This dietary selectivity is based on appetency, rather than attractivity. The appetent molecules are mostly present in the seed’s acetonic powders but there is also an effect of the small lipophilic molecules extracted by the solvent. Taking into account the meal size and the feeding behaviour of the larvae, we show that when an insecticide such as fipronil is associated with food as a seed coating, much lower amounts of a.i. are required than those necessary in a full-soil treatment. The theoretical basis of this treatment, and limits to control Agriotes populations by such seed coatings are discussed.

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.

In terrestrial ecosystems, higher plants are organized in two quite different spatial parts: a) a photosynthetic compartment, developed in the atmosphere, under light, and b) a soil compartment, specialized in the absorption of water and mineral salts, in the dark. An intense flux of water, of mineral salts and of organic compounds passes from the soil to the leaves. Small non-charged organic molecules with a medium lipophilicity can move passively in the transpiration stream, from soil water to the transpirating leaves. The composition of the soil solution is regulated by adsorption on the clay-humus complex and by exchanges with the microbial biomass. In the course of evolution, co-occuring microbes and plants have both developed, or maintained antitoxic properties allowing them to transform a large range of molecules, which are not among the group of essential compounds needed for microbial nor plant life. These antitoxic biochemical capabilities are generally not induced by the presence of a toxic property, but often only by foreign, unknown molecules. The case of phytoalexins seems to be an exception to this rule. Organic molecules reaching the soil are first submitted to microbial transformation, and plants absorb a mixture of molecules, of limited log P, either unchanged or partly transformed by microbes. During plant necrosis, the molecules which had been previously absorbed, and which are partly transformed by plant enzymes, are released into the soil and new transformations occur at this level, finally giving a complete mineralization. These mechanisms are illustrated by the case of two pesticides, atrazine and

Fipronil is a recently discovered insecticide of the phenylpyrazole series. It has a highly selective biochemical mode of action, which has led to its use in a large number of important agronomical, household, and veterinary applications. Previous studies have shown that, during exposure to light, fipronil is converted into a desulfurated derivative (desulfinyl-fipronil), which has slightly reduced insecticidal activity. In this study, the photodegradation of fipronil was studied in solution at low light intensities (sunlight or UV lamp). In addition to desulfinyl-fipronil, a large number of minor photoproducts were observed, including diversely substituted phenylpyrazole derivatives and aniline derivatives that had lost the pyrazole ring. Desulfinyl-fipronil itself was shown to be relatively stable under both UV light and sunlight, with only limited changes occurring in the substitution of the aromatic ring. Since this compound accumulated to levels corresponding to only 30-55% of the amount of fipronil degraded, it was concluded that one or more alternative pathways of photodegradation must be operating. On the basis of the structurally identified photoproducts, it is proposed that fipronil photodegradation occurs via at least two distinct pathways, one of which involves desulfuration at the 4-position of the pyrazole ring giving the desulfinyl derivative and the other of which involves a different modification of the 4-substituent, leading to cleavage of the pyrazole ring and the formation of aniline derivatives. The latter compounds do not accumulate to high levels and may, therefore, be degraded further. The ecological significance of these results is discussed, particularly with regard to the insecticidal activity of the photoproducts.

The resistance mechanism of vetiver (Chrysopogon zizanioides) to atrazine was investigated to evaluate its potential for phytoremediation of environment contaminated with the herbicide. Plants known to metabolise atrazine rely on hydroxylation mediated by benzoxazinones, conjugation catalyzed by glutathione-S-transferases and dealkylation probably mediated by cytochromes P450. All three possibilities were explored in mature vetiver grown in hydroponics during this research project. Here we report on the chemical role of benzoxazinones in the transformation of atrazine.Fresh vetiver roots and leaves were cut to extract and study their content in benzoxazinones known to hydroxylate atrazine, such as 2,4-dihydroxy-2H-1,4-benzoxazin-3(4H)one (DIBOA), 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA) and their mono- and di-glucosylated forms. Identification of benzoxazinones was performed by thin layer chromatography (TLC) and comparison of retention factors (Rf) and UV spectra with standards: although some products exhibited the same Rf as standards, UV spectra were different. Furthermore, in vitro hydroxylation of atrazine could not be detected in the presence of vetiver extracts. Finally, vetiver organs exposed to [(14)C]-atrazine did not produce any significant amount of hydroxylated products, such as hydroxyatrazine (HATR), hydroxydeethylatrazine (HDEA), and hydroxy-deisopropylatrazine (HDIA). Altogether, these metabolic features suggest that hydroxylation was not a major metabolic pathway of atrazine in vetiver.

The resistance mechanism of vetiver (Chrysopogon zizanioides Nash) to atrazine was investigated to evaluate its potential for phytoremediation of environment contaminated with the herbicide. Plants known to metabolise atrazine rely on hydroxylation mediated by benzoxazinones, conjugation catalyzed by glutathione-S-transferases (GST) and dealkylation probably mediated by cytochromes P450. All three possibilities were explored in mature vetiver grown in hydroponics during this research project. Here we report on the role of glutathione-S-transferase in the detoxification of atrazine, as determined in vetiver leaf and root of 5-week- and 8-month-old plants grown in hydroponics.Fresh vetiver roots and leaves were cut to extract and study their GST activities toward 1-chloro-2,4-dinitrobenzene (CDNB) and atrazine, using HPLC to quantify the biosynthesis of atrazine conjugates. The global GST activity was three orders of magnitude higher than that of GST isoform able to conjugate atrazine. In vitro activities of conjugation of CDNB were similar in all root and leaf vetiver extracts, whereas activities on atrazine were only detected in leaf extracts.Entire vetiver plants exposed to C-14-atrazine were found to accumulate radioactivity at the tip of leaves under moderate transpiring conditions (75% humidity). Vetiver transformed atrazine mainly into polar compounds, identified as conjugates by TLC analyses. After 20 days of exposure, the proportion of atrazine and metabolites compared to the total penetrated radioactivity into plants was: 50% of conjugates, 28% of atrazine plus possible dealkylates and 22% of unidentified products. The maximum conjugates production was observed at the tip of leaves (29 nmol g(-1) fresh biomass), as compared to roots (6 nmol g(-1) fresh biomass). Altogether, these metabolic features indicate that conjugation to glutathione was a major metabolic pathway to detoxify atrazine in vetiver. (c) 2005 Elsevier B.V. All rights reserved.

Several phenylpyrazole derivatives are selective inhibitors of chloride channel activities in insects. In this chemical family, fipronil is a powerful insecticide now widely used for several purposes. The dissipation of this molecule in a simplified aquatic ecosystem has been studied for 3 months, using (14)C-labeled fipronil. The main features of the complex process leading to fipronil transformation in this system were the following. The fipronil aqueous solution was submitted to two chemical transformations: the photodependent desulfuration of the side chain bound to the 4-position of the heterocyclic ring and the chemical hydrolysis of the nitrile function bound to the 3-position. Fipronil, rapidly transferred from the water solution to the organic matter, was protected from the previously mentioned chemical transformations but evolved to give two main metabolites, which were either reduced or oxidized in the side chain on the 4-position. These derivatives were powerful insecticides as shown by LC(50) measurements on Aedes aegypti larvae (LC(50) for CF(3)-S-R and CF(3)-SO(2)-R = 8.8 nM). During the course of this experiment, nitrile hydrolysis took place slowly, originating either from the chemical hydrolysis in the aqueous solution or from enzymatic hydrolysis inside the microbial biomass. The fipronil-amide (3-NH(2)-CO-R') derivative, although much more polar than fipronil itself, was mostly bound to the organic matter. Other more polar derivatives were also detected but in very small amounts. No (14)CO emission was observed during the experiment.

Heavy metal contamination due to traffic was studied in the water basin of the Aiguebelette lake (Savoie, France) in the alpine chain. It is surrounded by mountains and crossed by a highway on a 6-km-distance. Contamination of lichens, mosses, barks and dead leaves litters were submitted to a comparative study. The quantities of six metals (Pb, Al, Cd, Zn, Mn, Ni) were estimated in each of these materials. Except for Al which was highly concentrated in Xanthoria parietina and to a lesser extent in mosses, all the matrices accumulated the metals in a relatively similar way. The hyperaccumulation factor varied from 2 to 258, depending on the sampling point on the studied metal and on the matrix. Bark represented a long-term accumulator and contained more lead than the other matrices. In the studied water basin, a specific atmospheric movement allowed to distribute the contaminants far away from the highway, especially on the west slope of the highest mountain.

The fipronil control of wireworms field populations (larval Agriotes sp., Coleoptera: Elateridae) is difficult because of the low integumental penetration rates of this insecticide into the target larval body. The main way of insecticide absorption being associated with food, analysis of the larval feeding behaviour is of key importance for designing a strategy of dietary chemical control of those subterranean pests. In this purpose, a standard method for assessing the long-term survival of field specimens in the laboratory was developed together with experimental designs for investigating food searching and choosing by larvae. Larval biological performances under those laboratory conditions were checked monthly. Three criteria were taken into account for measures: the duration of individual survival, the gain in average body weight, and the moulting rhythm. Experimentation revealed that seed flour was highly desired by larvae and that larval feeding choice was affected over short distances only. This suggests the involvement of dietary preference rather than attractivity in the larval feeding behaviour. This implies that, at the operational step, insecticide had better be associated to food, either as a coating on the seeds, or inside preferred baits.

Previously we described the mosquito larvicidal properties of decomposed leaf-litter from deciduous trees, especially the alder Alnus glutinosa (L) Gaertn., due to toxic polyphenols and other secondary compounds. To further examine the biocontrol potential of toxic leaf-litter for mosquito control, feeding rates of third-instar mosquito larvae were assessed for examples of three genera: Anopheles stephensi Liston, Aedes aegypti (L) and Culex pipiens L. (Diptera: Culicidae). When immersed in a suspension of non-toxic leaf-litter particles (approximately 0.4 mm), pre-starved larvae of all three species ingested sufficient material in 30 min to fill the anterior gut lumen (thorax plus two to three abdominal segments). Gut filling peaked after 1-2 h ingestion time, filling the intestine up to six to seven abdominal segments for Ae. aegypti, but maxima of five abdominal segments for Cx. pipiens and An. stephensi. Using three methods to quantify consumption of three materials by third-instar larvae of Ae. aegypti, the average amount of leaf-litter (non-toxic 0.4 mm particles) ingested during 3 h was determined as approximately 20 microg/larva (by dry weight and by lignin spectrophotometric assay). Consumption of humine (approximately 100 microm particles extracted from leaf-litter) during 3 h was approximately 80 microg/larva for Ae. aegypti, but only approximately 30 microg/larva for Cx. pipiens and 15 microg/larva for An. stephensi, with good concordance of determinations by dry weight and by radiometric assay. Cellulose consumption by Ae. aegypti was intermediate: approximately 40 microg/larva determined by radiometric assay. Apparent differences between the amounts of these materials ingested by Ae. aegypti larvae (humine four-fold, cellulose two-fold more than leaf-litter) may be attributed to contrasts in palatability (perhaps related to particle size or form), rather than technical discrepancies, because there was good concordance between results of both methods used to determine the amounts of humine and leaf-litter ingested. Bioassays of toxic leaf-litter (decomposed 10 months) with 4-h exposure period (ingestion time) ranked the order of sensitivity: Ae. aegypti (LC50 < 0.03 g/L) > An. stephensi (LC50 = 0.35 g/L) > Cx. pipiens (LC20 > 0.4 g/L). When immersed in the high concentration of 0.5 g/L toxic leaf-litter (0.4 mm particles), as little as 15-30 min ingestion time (exposure period) was sufficient to kill the majority of larvae of all three species, as soon as the gut lumen was filled for only the first few abdominal segments. Possibilities for mosquito larval control with toxic leaf-litter products and the need for standardized ingestion bioassays of larvicidal particles are discussed.

In order to examine ecological impact of fipronil use for larval culicine control in natural hydrosystems, toxicity and bioaccumulation of this new insecticide were analyzed on aquatic species representative of the nontarget arthropodan fauna (nonculicine larval Diptera: Chaoboridae, Chironomidae; planktonic Crustacea: Cladocera, Copepoda, Ostracoda) associated with target larval mosquito populations in the subalpine breeding sites. Standard toxicological bioassays using fipronil aqueous solutions from 1 to 2000 nM indicated different sensitivity levels among species. Insecticide bioaccumulation analyses, using [(14)C]fipronil solutions in simplified laboratory ecosystem, also indicated large differences among species. These differences may come from biological parameters characteristic of each species. Taking into account these nontarget effects of fipronil, a possible strategy of use of this insecticide for integrated mosquito control management was proposed, which is based upon selective dietary absorption of the insecticide by larval Culicidae.

The toxicity and bioaccumulation kinetics of fipronil were analyzed on fourth instar larvae of an Aedes aegypti laboratory strain in order to investigate the possible use of this new phenylpyrazole insecticide in the aquatic medium. Standard bioassays indicate that larvae fed with maize flour on which the fipronil has accumulated are about three times more susceptible to fipronil than unfed larvae because fed larvae ingest considerably more fipronil than unfed larvae. Bioaccumulation kinetic analysis, comparatively performed in simplified experimental microcosms, using [14C]fipronil solutions against dead larvae and living unfed larvae treated or not by Triton X-100, suggests that the insecticide penetrates mainly through the integument but also through the gut. This digestive route of fipronil accumulation is enhanced by feeding, suggesting that ingestion be the main and most selective way of insecticide administration to mosquito larvae. These biological effects of fipronil may offer a possible strategy of use of this insecticide in larval mosquito control.

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 I 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. (C) 2001 Editions scientifiques et medicales Elsevier SAS.

Soil thin-layer chromatography with water or water-methanol as solvents allows observation and measurement of the mobility of labelled pesticides through soil microstructures. Eleven different sieved matrices were studied: pure humine, pure clays, schists and soils. Ionized compounds (paraquat, glyphosate) were tightly bound to these matrices. The other compounds, lipophilic and generally non-ionized ones, migrated in the same order on most of the studied matrices, either mineral or organic: R(F) atrazine=isoproturon>diuron=fipronil>phenmedipham. This order was roughly correlated to log P but much more complex correlations were suggested. The rate of water movement, VWR, widely changed from one matrix to another. Therefore, the pesticide movement, M, in soil microstructures under the action of rain may be described by the equation M = WR R(F).

Several different matrices (water, n-butanol, n-octanol, lecithin, waxes, and suber) were chosen to measure [14C]atrazine diffusion rate and evaluate the specific diffusion parameter of this molecule. A simple experimen tal device was conceived for this purpose and two methods of calculation, deduced from Fick's law, were established and compared. The same device was used for diffusion measurements inside corn seedling fragments, either dead or alive. In inert matrices, the highest diffusion parameter found for atrazine was obtained for water (2.6 ± 0.9) 10−10 m2 s−1. For more lipophilic matrices, the value of the specific parameter decreased markedly, reaching only (1.2 ± 1) 10−12 m2 s−1 for glycerides and (2.5 ± 2.4) 10−13 m2 s−1 for paraffin. For dead corn roots or coleoptile, the diffusion parameter was close to that in water: (3.7 ± 2.1) 10−10 m2 s−1 and (9.6 ± 4) 10−10 m2 s−1. In living material, the movement of 14C-labeled compounds was much lower: (7.4 ± 4.6) 10−11 m2 s−1 and (1.6 ± 1.5) 10−11 m2 s−1. This was explained by atrazine hydroxylation in the presence of benzoxazinones, leading to a derivative which was accumulated inside the vacuole.

A corn Seed dressing with the fungicide triticonazole at 760 nmol/seed prevents head smut disease. In resting seeds, the dressing treatment was followed by the penetration of 19% of the product, 9% inside the tegument and 7% inside the pedicel. In growing seedlings, the inner content increased in the storage organs (endosperm + scutellum) as well as in the growing organs. The partition Lipophilic phase/water certainly explains the high apparent fungicide concentration progressively reached inside endosperm and scutellum. However, no important transfer of fungicide from these organs to the growing parts seems to occur. It appears therefore that the fungicide transfer from the coating to the roots mostly occurs through dissolution of the product in the surrounding soil water and through root absorption. The efficient fungicide concentration inside the meristem is likely to be obtained during the early stages of development.

Among numerous tobacco transgenic lines expressing an altered gene encoding glyphosate-resistant EPSP synthase, two were chosen for detailed physiological studies, as they were both highly resistant to glyphosate in the growth chamber (HR and WR lines) while one of them (WR) was susceptible to the herbicide in the field. The two transformed lines and the susceptible line (S) grew similarly. Foliar applied glyphosate penetrated into the plants to the same extent in the three lines and phloem transfer from the source leaves to the sinks was also similar. In the transgenic lines, EPSP synthase was overexpressed in all leaves, with an average overexpression factor reaching 3 to 15, with the highest value in HR. The averageI50for glyphosate of the leaf enzyme was approximatly 700-fold that from S for WR and 6 to 22 times lower for HR. The most physiologically significant difference between the two transgenic lines was that WR had a very low EPSP synthase content in its roots. Furthermore, the enzyme present in the roots was very sensitive to glyphosate. In contrast, the roots of the HR line contained a well-measurable pool of resistant enzyme. This difference probably explains why HR was resistant to glyphosate in the field, allowing a good rate of lignification to occur and ensuring high water movements inside the plant in order to compensate for the transpiration loss.

One of the possible detoxication pathways for atrazine in corn corresponds to a chemical hydroxylation process. The aim of this work was to describe the mechanism of this reaction. Under in vitro experimental conditions, a benzoxazinones mixture (10 mM) extracted from corn plantlets was able to transform 91% of atrazine (6 mu M) into 2-hydroxyatrazine during a 24-h period of incubation at 25 degrees C. This reaction was shown to be temperature-dependent; the half-life of atrazine was 67 h at 4 degrees C, 90 min at 25 degrees C, and only 30 min at 50 degrees C. However, at this temperature a rapid degradation of the active benzoxazinones occurred. The pH value of the incubation medium was shown to influence greatly the hydroxylation rate of atrazine (no hydroxylation process at pH 9, a relatively low rate at pH 7, and a maximum one at pH 5.5). The presence of an organic solvent (ethanol or acetone) in the reaction medium was responsible for a large decrease in hydroxylation activity. In a water medium, an optimal rate was obtained when the benzoxazinones concentration was close to 10 mM (average rate of hydroxylation: 2 to 3.10(-4) nmol h(-1) nmol(-1) benzoxazinones). For concentration values lower than 1 mM, the rates remained very low. Ln the presence of 10 mM benzoxazinones, the hydroxylation rate appeared not to be saturable for concentrations in atrazine varying between 6 and 100 mu M. The comparison of the hydroxylation rates obtained with purified benzoxazinones (DIMBOA, DIBOA, 2-monoglucosyl DIMBOA + 2-monoglucosyl DIBOA) suggested that the chemical reactivity of benzoxazinones toward atrazine involved the 4-N-OH of the heterocycle and not the 2-C-OH. This hypothesis was reinforced by the fact that the atrazine hydroxylating activity of DIMBOA or DIBOA remained unchanged even in the presence of AlCl3, (a chelator of the 2-OH group). The natural concentration of benzoxazinones in the vacuolar sap of corn seedlings (greater than or equal to 10 mM) and the pH of this solution (close to 5.5) contribute to explain the high rate of atrazine chemical hydroxylation in vivo. (C) 1997 Academic Press.