Mosquito larval consumption of toxic arborescent leaf-litter, and its biocontrol potential.

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.

Toxicity and bioaccumulation of fipronil in the nontarget arthropodan fauna associated with subalpine mosquito breeding sites.

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.

Triticonazole distribution in dressed corn caryopsis and seedlings.

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.

Soil thin-layer chromatography and pesticide mobility through soil microstructures. New technical approach.

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).

Photosynthesis inhibition by phenylureas: a QSAR approach.

A series of 21 N'-phenyl-N,N-disubstituted ureas was studied for its effects on thylakoidal electron transfer, photophosphorylation, and photosynthetic O2 evolution in class A chloroplasts and leaf fragments. The concentrations of test compounds that produced 50% inhibition of PS II electron transfer and of photosynthesis in class A chloroplasts were highly correlated. Urea derivatives with a N-lipophilic substituent (three or four carbon chains) were uncouplers (i.e., neburon uncoupled by 50% at 7 microM). The efficiency of inhibition of PS II electron transfer was increased by the presence of a 3,4-disubstitution of the phenyl ring. The presence of N-lipophilic substituents was necessary for there to be an inhibitory effect, but an increase in the length of the chain lowered the activity. The best QSAR equations that described the inhibitory activity of the whole series were obtained when using 1 chi v or MR (probably representing dispersion forces) associated with parameters that described the steric hindrance at the p position of the phenyl ring (Verloop's B2(4), B4(4)) or in its neighborhood (such as the angular parameter A4). The inhibitory efficiency of the phenylurea series on wheat and spinach pieces led to the conclusion that some extrachloroplastic factors seemed to limit the accessibility of the D1-protein target.

Uncoupling properties of a chlorophenol series on Acer cell suspensions: a QSAR study.

A series of 22 chlorinated phenols was investigated for their uncoupling effect on Acer cell suspensions. All the studied molecules were able to enter the cell and had instantaneous uncoupling properties: pentachlorophenol was the best uncoupler of the series (the concentration required to uncouple by 50% was 5 microM), being 200-fold more effective than 3-Cl or 2-Cl phenol, which were the molecules having the lowest activity. The QSAR (quantitative structure-activity relationship) study gave good equations for the uncoupling activity with the steric and electronic parameters taken together. The electronic parameter sigma was always present with a positive sign, whereas A, the angular parameter, was always negative. The general steric parameters (sigma D, 1 chi v, MR) represented the third component of the equations and each of them played practically an equivalent role. Equations of quality (r greater than 0.9), which were statistically significant, could be obtained after four compounds in F (2,5-diCl phenol; 2,4,5-triCl phenol; 2,4-diCl 6-NO2 phenol; and 2-Cl 4,6-diNO2 phenol) had been excluded and when quadratic terms were not present. The best equation (r = 0.940) was obtained with sigma D, A, and sigma 1. The comparison of the significant equations with those previously established with isolated mitochondria led us to suppose that there were no selective limiting steps during the transfer of the studied compounds from the reaction medium to mitochondria inside the cells. The major difference between the uncoupling results obtained with isolated mitochondria and with cells was that the concentrations needed to uncouple by 50% were always higher in cells.

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.

Phytotoxicity of orellanine, a mushroom toxin.

Orellanine, a toxic principle of Cortinarius orellanus Fr., efficiently inhibited the photosynthetic activity of duckweed, Lemna minor L., at a concentration of 0.4 mM. A lower concentration (0.06 mM) blocked the O2 production in isolated spinach class A chloroplasts. However, no effect was observed on thylakoids (class C chloroplasts), showing that orellanine does not interfere with the chloroplastic electron transfer chain and that orellanine does not act like methylviologen (paraquat) in green plants, as previously suggested. An electrochemical study of orellanine and related compounds showed that orellanine can neither be reduced by electrons derived from H2O nor from NADPH, as is methylviologen in plant and animal cells, respectively.

Platanetin: A Potent Natural Uncoupler and Inhibitor of the Exogenous NADH Dehydrogenase in Intact Plant Mitochondria.

Platanetin is a 3,5,7,8-tetrahydroxy, 6-isoprenyl flavone isolated from the bud scales of the plane tree (Platanus acerifolia Willd.). Its effects on the oxidative activities of isolated potato and mung bean mitochondria have been studied. The most noticeable effect is the selective inhibitory effect of this compound on the activity of the external NADH dehydrogenase of the inner membrane. A 50% inhibition of the NADH oxidation rate is obtained at a 2 micromolar concentration. This activity is probably due to the flavonoid structure and the high lipophilicity of platanetin associated with the presence of the isoprenyl chain. Another important effect of platanetin is its uncoupling activity on oxidative phosphorylation. The presence of easily dissociable hydroxyl groups and the high lipophilicity of platanetin allow a potent H(+) transfer through the mitochondrial inner membrane. This uncoupling activity is comparable to that of carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Platanetin is therefore the most active natural uncoupler known at the present time (full uncoupling at 2 micromolar with succinate as substrate). At higher concentrations (10 micromolar and more), platanetin can transfer electrons from the mitochondrial inner membrane to O(2); the branching point of this KCN-salicylhydroxamic acid insensitive platanetin dependent oxidative pathway is located at the level of flavoproteins, no transfer occurring when succinate is the substrate. The redox properties of platanetin are in accord with such an activity.

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.

Effects of rotenoids on isolated plant mitochondria.

The effects of several rotenoids have been studied on potato (Solanum tuberosum L.) tuber and etiolated mung bean (Phaseolus aureus Roxb.) hypocotyls mitochondria. The selective inhibition of mitochondrial complex I is characterized by several tests: (a) no effect can be observed on exogenous NADH or succinate oxidation; (b) malate oxidation is inhibited at pH 7.5; (c) one-third decrease of ADP/O ratio appears during malate oxidation at pH 6.5 or during alpha-ketoglutarate, citrate, or pyruvate oxidation at a pH about 7; (d) during malate oxidation at pH 6.5, a transient inhibition appears which can be maintained by addition of exogenous oxaloacetate; (e) in potato mitochondria, the inhibition of malate oxidation disappears at pH 6.5 when NAD(+) is added. Then, a one-third decrease of the ADP/O ratio can be measured.Such a selective inhibition of complex I is obtained with deguelin, tephrosin, elliptone, OH-12 rotenone, and almost all the rotenoids extracted from Derris roots. The presence of the rings A, B, C, D, E seems to be necessary for the selective inhibition. Opening of the E ring and hydroxylation of the 9 position (rot-2'-enoic acid) give a rotenoid derivative with multisite inhibitory activities on flavoproteins, which are quite comparable to those of common flavonoids such as kaempferol (Ravanel et al. 1982 Plant Physiol 69: 375-378).

Effects of kaempferol on the oxidative properties of intact plant mitochondria.

The effects of kaempferol on the oxidative and phosphorylative properties of plant mitochondria from potato tubers and etiolated mung bean (Phaseolus aureus Roxb.) hypocotyls were investigated. Kaempferol inhibited the state 3 oxidation rate of malate, NADH, and succinate, but was without effect on the ascorbate-tetramethyl p-phenylenediamine oxidation rate. The inhibition was almost the same whether the mitochondria were in state 3 or in an uncoupled state 3. When 180 micromolar kaempferol was added during state 4, the tight coupling of succinate or NADH oxidation was not released. The results obtained indicate that kaempferol inhibits the mitochondrial electron flow at, or just after, the flavoprotein site.