Insecticide composition including thiamethoxam and a synergistic agent

Abstract

Insecticide composition wherein the active ingredient comprises the combination of a neonicotinoid insecticide, thiamethoxam as synthetic insecticide molecule, and at least one synergistic agent, which is chosen among the insect repellent agents such as DEET and/or IR3535 and present at a molar ratio of said synergistic agent to thiamethoxam comprised between O.OOI and 0.2 in the composition. Synergistic effect is observed at low doses. Use of the insecticide composition wherein said composition is sprayed or deposited on, or impregnated to a support, such as net, fabrics, cloth or tent, in the fight against insects which are harmful to human, to animals and/or to crops, and in particular against pyrethroid, carbanate and/or organophosphate resistant mosquitoes.

Claims

1. Insecticide composition comprising: an active ingredient that includes the combination of thiamethoxam as synthetic insecticidal molecule, and at least one synergistic agent selected from the group consisting of N,N-diethyl-3-methylbenzamide (DEET) at a concentration comprised between 3.10.sup.8 M and 3.10.sup.7 M in the composition, 3-[N-Butyl-N-acetyl]-aminopropionic acid ethyl ester (IR3535) at a concentration comprised between 3.10.sup.9 M and 3.10.sup.8 M in the composition, and a mixture thereof and present in the composition at a ratio of 0.001 and 0.2 between a molar amount of said synergistic agent to a molar amount of thiamethoxam.

2. Insecticide composition according to claim 1, wherein, in the present composition, the ratio between the molar amount of said synergist agent to the molar amount of thiamethoxam in the composition is 0.005 and 0.1.

3. Insecticide composition according to claim 1, wherein thiamethoxam is present at a concentration comprised between 10.sup.7 M and 3.10.sup.6 M in the composition.

4. Insecticide composition according to claim 2, wherein the molar ratio of said synergist agent to thiamethoxam in the composition is comprised between 0.01 and 0.1.

5. Insecticide composition according to claim 2, wherein the molar ratio of said synergist agent to thiamethoxam in the composition is comprised between 0.01 and 0.05.

6. Insecticide composition according to claim 1, wherein said composition is in a liquid form, the active ingredient being solubilized in an organic phase and/or encapsulated in nano- or micro-capsules.

7. Insecticide composition according to claim 1, wherein said composition is configured to be sprayed or deposited on, or impregnate a support, such as net, fabrics, cloth or tent.

8. Insecticide composition according to claim 1, wherein said composition is configured to be applied against insects which are harmful to human, to animals and/or to crops, including insects chosen from the group consisting of: diptera, dictyoptera, lepidoptera, orthoptera and hemiptera.

9. The insecticide composition as claimed in claim 8 wherein said insecticide composition is configured to be applied against pyrethroid resistant mosquitoes.

10. The insecticide composition as claimed in claim 8 wherein said insecticide composition is configured to be applied against mosquitos which are resistant to carbamate and/or organophosphate insecticide.

11. The insecticide composition as claimed in claim 8 wherein said insecticide composition is configured to be applied against Aedes aegypti or Anopheles gambiae.

12. The insecticide composition as claimed in claim 8 wherein said insecticide composition is configured to be applied for controlling mosquito-borne diseases.

Description

FIGURES

(1) The invention will be further described in the below embodiments given with reference to the accompanying drawings, in which:

(2) FIG. 1 illustrates the cockroach Periplaneta americana central nervous system used in the experiments;

(3) FIG. 2 (A to D) are curves showing the complex dose-dependent effects of IR3535 on DUM neuron intracellular calcium concentration;

(4) FIG. 3A and FIG. 3B are comparative histograms illustrating the effects of IR3535 used at 10.sup.8 M (A) and 10.sup.5 M (B) on the intracellular calcium concentration in the presence of different pharmacological agents;

(5) FIG. 4A illustrates the effect of IR3535 used at 10.sup.8 M on the DUM neuron spontaneous electrical activity and FIG. 4B shows comparative histogram of the effects of IR3535 used at 10.sup.8 M on action potential discharge frequency;

(6) FIG. 5 illustrates the effect of thiamethoxam-induced inward currents (10.sup.6 M), recorded under voltage-clamp condition, before (A) and after pretreatment with IR3535 used at 10.sup.8 M (B), (C) illustrates comparative histogram of the effect of IR3535 (10.sup.8 M) on the thiamethoxam-induced inward current amplitude;

(7) FIG. 6 is a comparative histogram of the effect of thiamethoxam at different concentrations on insect neuron after pretreatment with acetylcholine 10.sup.3M;

(8) FIG. 7 is an histogram illustrating the effects of IR3535/thiamethoxam mixture on insect neuron after pretreatment with acetylcholine 10.sup.3M.

EXAMPLES

(9) Materials and Methods

(10) Insect Neuronal Model

(11) Experiments were carried out on cockroach Dorsal Unpaired Median (DUM) neurons. Cockroach neuronal preparations are commonly used as biomedical models for vertebrates and invertebrates and DUM neurons are, furthermore, electrophysiologically and pharmacologically well characterized since most of the biophysical and pharmacological properties of ionic currents and receptors underlying and modulating their spontaneous action potentials have been established by using the well-known patch-clamp technique.

(12) Adult male cockroaches, Periplaneta americana (see FIG. 1), are taken from our laboratory colonies, which are maintained under standard conditions (29 C., photoperiod of 12 h light/12 h dark). Animals are immobilized dorsal-side up on a dissection dish. The dorsal cuticle, gut and some dorso-longitudinal muscles are removed to allow access to the ventral nerve cord. The abdominal nerve cord and its terminal abdominal ganglion (TAG), carefully dissected under a binocular microscope, are placed in normal saline. Animal care and handling procedures are in accordance with French institutional and national health guidelines.

(13) The ventral nerve cord and its terminal abdominal ganglion (TAG) are carefully dissected under a binocular microscope and placed in normal cockroach saline containing (in mM) 200 NaCl, 3.1 KCl, 5 CaCl2, 4 MgCl2, 50 sucrose, and 10 N-2-hydroxymethylpiperazine-N9-2-ethanesulfonic acid (HEPES); pH was adjusted to 7.4 with NaOH. Isolation of adult DUM neuron cell bodies are performed under sterile conditions using enzymatic digestion and mechanical dissociation of the median parts of the TAG as previously described in Lapied et al. (Ionic species involved in the electrical activity of single adult aminergic neurones isolated from sixth abdominal ganglion of cockroach Periplaneta americana J Exp Biol 144:535-49, 1989). The isolated neuron cell bodies are used for recordings 24 h after dissociation.

(14) Calcium Imaging

(15) Falcon 1006 Petri dishes with glass coverslips are coated with poly-D-lysine hydrobromide (mol. wt. 70,000-150,000), and isolated DUM neuron cell bodies are plated. External recording solution contains (in mM): 200 NaCl; 3.1 KCl; 5 CaCl2; 4 MgCl2, and 10 HEPES buffer; pH is adjusted to 7.4 with NaOH. The cells are incubated in the dark with 10 M Fura-2 pentakis (acetoxy-methyl) ester for 60 min at 37 C. After loading, cells are washed three times in saline. The glass coverslips are then mounted in a recording chamber (Warner Instruments, Hamden, Conn.) connected to a gravity perfusion system allowing drug application. Imaging experiments are performed with an inverted microscope (Nikon) equipped with epifluorescence. Excitation light is provided by a 75-W integral xenon lamp. Excitation wavelengths (340 nm and 380 nm) are applied using a computer driven a monochromator (Sutter Instruments Company, Lambda DG4) with a digital charge-coupled device (CCD) camera (Hamamatsu Orca R.sup.2) and they are recorded in the computer with calcium imaging software (Imaging Workbench 6, Indec BioSystem). Exposure times at 340 nm and 380 nm are usually 150 ms, and images are collected at various frequencies. Data are expressed as the ratio of emitted fluorescence (340 nm/380 nm). Different concentrations of the insect repellent IR3535 ranging from 10.sup.9 M to 10.sup.5 M have been tested.

(16) Electrophysiology and Whole-Cell Patch-Clamp Recordings

(17) Electrical activity and neonicotinod-induced inward currents are recorded using the patch clamp technique in the whole-cell recording configuration under current-clamp and voltage-clamp mode, respectively. Patch-clamp electrodes are pulled from borosilicate glass capillary tubes (GC150T-10) using a P-97 model puller. Patch pipettes have resistances ranging from 1 to 1.2 M when filled with internal pipette solution. The liquid junction potential between extracellular and intracellular solutions is always corrected before the formation of a giga Ohm seal (>3G). Signals are recorded with an Axopatch 200A amplifier. Ionic currents induced by thiamethoxam are displayed on a computer with software control pClamp connected to a digidata acquisition system (digidata 1320A). Under voltage-clamp conditions, DUM neuron somata are voltage-clamped at a steady state holding potential of 50 mV to measure the effects of thiamethoxam applied alone and after pretreatment with IR3535 (10.sup.8 M). Experiments are carried out at room temperature.

Example 1

(18) 1.1Dose-Dependent Effect of the Insect Repellent IR3535 on Insect Neurons

(19) Using calcium imaging, it has been possible to study the effect of the insect repellent IR3535, on the intracellular calcium concentration in DUM neurons. Bath application of IR3535 induces a complex multiphasic dose-dependent effect on the intracellular calcium concentration (see FIG. 2).

(20) FIG. 2 A shows the dose-response curve illustrating changes in intracellular calcium concentration depending on the different concentrations of IR3535 tested. Intracellular calcium concentration variations (presented as ratios 340/380) have been calculated from mean values obtained for each IR3535 concentration tested (n=5). FIG. 2B to D represent spectrum of the intracellular calcium concentration rises induced by IR3535 tested at 10.sup.8 M, 3.10.sup.6 M and 10.sup.5 M, respectively.

(21) In the zone I (FIGS. 2A and 2B), it is possible to observe a transient increase of intracellular calcium concentration between 3.10.sup.9 M and 3.10.sup.8 M, reaching a maximum for IR3535 used at 10.sup.8 M, i.e. at a very low concentration.

(22) Zone II corresponds to an additional elevation of intracellular calcium concentration obtained for IR3535 used in the concentration range from 10.sup.7 M to 3.10.sup.6 M (FIGS. 2A and 2C). Finally, zone III corresponds to the maximum effect produced by IR3535 used at very high concentration (3.10.sup.5 M) (FIGS. 2A and 2D). These results demonstrate that the insect repellent IR3535 exerts its effect through an elevation of intracellular calcium concentration in insect neurons.

(23) 1.2Origin of the Intracellular Calcium Rise

(24) To determine the origin of the intracellular calcium concentration rise (i.e., intracellular and/or extracellular origin), different specific blockers and/or antagonists of calcium channels and membrane receptors have been tested. Histograms of FIG. 3 show the comparative effects of IR3535 used at 10.sup.8 M (FIG. 3A) and 10.sup.5 M (FIG. 3B) on the intracellular calcium concentration in the presence of different pharmacological agents such as caffeine, omegaconotoxin (-ctx) and pirenzepine (PZP).

(25) From these results inventors have determined that the effect of IR3535 used at 10.sup.8 M results from extracellular calcium through plasma membrane voltage-dependent calcium channels via M1/M3 mAChR sub-type modulation. For higher concentrations of IR3535, both extracellular calcium and calcium released from internal stores are involved in the effects of the repellent in insect neurons.

(26) 1.3Effect of the Insect Repellent IR3535 on DUM Neuron Spontaneous Action Potentials

(27) From the data presented just above, it appears that 10.sup.8 M is the lower concentration of IR3535, which produces a significant elevation of intracellular calcium concentration in DUM neurons. Consequently, the following experiments have been performed using 10.sup.8 M IR3535. Using the patch clamp technique in the whole-cell recording configuration, it has been possible to show that IR3535 (10.sup.8 M) induces a significant membrane depolarization associated with an increase of the spontaneous action potential discharge frequency (FIG. 4A). The corresponding FIG. 4B illustrates comparative histogram of the effect of IR3535 (10.sup.8 M) on the spontaneous action potential discharge frequency.

(28) According to the results presented above, using different pharmacological agents, calcium imaging and electrophysiological technique, and based on previous results obtained on the same neuronal preparation, it is possible to summarize the effect of IR3535 used at very low concentration on the intracellular calcium concentration in DUM neurons. IR3535, by acting on M1/M3 mAChR sub-types, inhibits background calcium-activated potassium channels resulting in the small depolarization observed. This membrane depolarization is sufficient to stimulate N-type high-voltage activated calcium channels involved in the calcium influx through the membrane.

Example 2Dose-Dependent Effect of the Insect Repellent DEET on Insect Neurons

(29) Similar experiments as example 1 have been made with DEET at different concentrations.

(30) Application of the insect repellent DEET onto insect neurons produces a biphasic effect on the intracellular calcium concentration changes. In the low concentration range (from 10.sup.9 M to 10.sup.7 M), DEET induces an elevation of the intracellular calcium concentration reaching a maximum at 10.sup.7 M DEET (ratio of emitted fluorescence (340 nm/380 nm) ranging from 0.4 to 0.6 with a maximum value of 0.7).

(31) For higher concentrations than 10.sup.7 M, DEET produces an opposite effect (i.e., an important decrease of the intracellular calcium concentration).

(32) Therefore, preferred concentrations of DEET as synergist agent should be chosen between 3. 10-8 M and 3. 10-7 M where the ratio of emitted fluorescence (340 nm/380 nm) is above 0.6.

Example 3Synergistic Effect Occurring Between IR3535/Thiamethoxam

(33) The neonicotinod insecticide thiamethoxam has been tested alone and after pretreatment with IR3535 (10.sup.8 M) on DUM neurons using the patch-clamp technique, under voltage clamp condition (FIG. 5).

(34) Application of thiamethoxam (10.sup.6 M) alone induces an inward current with small amplitudes (see FIGS. 5A and 5C: recorded under voltage-clamp condition, at a holding potential of 50 mV, means+S.E.M., **, p<0.01; n=3-4).

(35) By contrast, after pretreatment of DUM neuron with the repellent IR3535 used at 10.sup.8 M, an important increase of the thiamethoxam-induced inward current amplitude is observed. In this case, the mean current amplitude is about 6-fold more important than the current amplitude obtained with thiamethoxam applied alone (FIGS. 5B and 5C).

(36) In conclusion, the complementary approaches such as calcium imaging and electrophysiology reveal that the neonicotinod insecticide thiamethoxam acts like an agonist able to induce an inward current with a small amplitude. When DUM neurons are pretreated with low concentration of IR3535, the inward current amplitude produces by thiamethoxam is more important. This confirms the role of IR3535 as synergistic agent, which can increase the effect of thiamethoxam via an increase of intracellular calcium concentration. These results confirm that combining the repellent IR3535 with the molecule thiamethoxam could be an interesting alternative to 1) circumvent resistance mechanisms developed by mosquitoes-borne diseases and 2) increase insecticide efficacy while reducing doses.

Example 4Pretreatment with Acetylcholine

(37) Acetylcholine at a concentration of 10.sup.3M has been applied to DUM neurons during one second, three minutes before testing thiamethoxam at 10.sup.7M, 10.sup.6M and 10.sup.5M on DUM neurons under the same protocol as example 3.

(38) Although no current has been recorded with thiamethoxam alone, thiamethoxam after pretreatment with acetylcholine induces a current the amplitude of which amplitude is reported in histogram of FIG. 6. This suggests that acetylcholine, acting on the cholinenergic receptors of the neurons, leads to an increase of the intercellular calcium that induces the effect of thiamethoxam shown by the observed current.

(39) FIG. 6 shows that the current amplitude is at the highest value for thiamethoxam 10.sup.7M and the lowest value for thiamethoxam 10.sup.6M.

(40) However, at this concentration of thiamethoxam 10.sup.6M, when combined with IR3535 at 10.sup.8M, (after a pretreatment with acetylcholine 10.sup.3M), a synergist effect is observed (see histogram of FIG. 7).

(41) From the above results, it can be concluded that the well know insect repellent IR3535 is able to potentiate the neurotoxic effect of the neonicotinoid thiamethoxam. Example 4 clearly indicates that the potentiation is more effective when the neurons are pretreated with the endogenous neurotransmitter acetylcholine. In fact, acetylcholine is known to increase the intracellular calcium concentration. Pre-treatments with both acetylcholine and IR3535 reinforce the synergistic effect, via the intracellular calcium rise. These results are very interesting since the endogenous acetylcholine level and/or the density of targets affected by thiamethoxam seem to be different between the wild population of mosquito A. gambiae (named Kis) and mosquitoes resistant to insecticides such as organophosphates and/or carbamates (named AcerKis). In this case, application of such IR3535/thiamethoxam mixture on resistant mosquitoes AcerKis will be very efficient to increase the mortality rate of these mosquitoes and to overcome the resistance mechanism.

(42) In other words, the above results may indicate that the repellent/insecticide composition of the present invention would be more efficient against resistant mosquitoes than wild mosquitoes.