Methods and compositions for controlling fungus-growing ants

Abstract

The invention relates to a method for fighting fungus-growing ants, and to a formicide composition capable of destroying specifically the colonies of fungus-growing ants. The invention also relates a kit for use with said method.

Claims

1. A method for controlling fungus-growing ants, the method comprising administering on or around a nest of fungus-growing ants, near or on the fungus-growing ants, and/or in an area to be protected from fungus-growing ants a composition comprising at least one laccase inhibitor selected from the group consisting of ascorbic acid, citric acid, coumaric acid, ferulic acid, gallic acid, 6-1-palmitylascorbic acid, syringic acid, sodium ascorbate, calcium ascorbate, potassium ascorbate, butylhydroxyanisole, butylhydroxytoluol, cysteine, octyl gallate, dodecyl gallate, glutathione, sodium lactate, potassium lactate, calcium lactate, lecithins, lipoate, carotenoids, carotene, sodium tartrate, potassium tartrate, sodium thiosulfate, vitamin E, synthetic -tocopherol, synthetic -tocopherol, synthetic -tocopherol, selenium, hydroxylammonium chloride, ethylenediaminetetraacetic acid, calcium chloride, rhodotorulic acid, enterobactin, diethyldithiocarbamic acid, sodium azide, cetyltrimethylammonium bromide, sodium lauryl sulphate, sodium cyanide, sodium thiosulphate, oxalic acid, thioglycolic acid, diethyldithiocarbamic acid, Fe.sup.2+, Cu.sup.2+, Ag.sup.+, Li.sup.+, Sn.sup.+, Hg.sup.+, Mn.sup.2+, Zn.sup.2+, Al.sup.3+, thymol, and eugenol; wherein the fungus-growing ants are killed and/or forced to abandon the nest after coming into contact with the at least one laccase inhibitor.

2. The method according to claim 1, wherein the composition further comprises at least one antibacterial compound selected from the group consisting of an antibiotic, carvacrol, cinnamaldehyde, -terpineol, and terpinen-4-ol.

3. The method according to claim 2, wherein the antibiotic is selected from the group consisting of -lactams, tetracyclines, quinolones, macrolides, glycopeptides, and aminoglycosides.

4. The method according to claim 1, wherein the at least one laccase inhibitor is selected from the group consisting of ascorbic acid, citric acid, coumaric acid, ferulic acid, syringic acid, gallic acid, cysteine, glutathione, lecithins, lipoate, the carotenoid lutein the carotenoid luteolin, lycopene, sodium thiosulphate, vitamin E, -tocopherol, -tocopherol, -tocopherol, selenium and hydroxylammonium chloride.

5. The method according to claim 2, wherein the antibacterial compound is selected from the group consisting of cinnamaldehyde, thymol, eugenol, and carvacrol.

6. The method according to claim 1, wherein the at least one laccase inhibitor further possesses bactericidal and/or bacteriostatic activity against actinomycetes present in the fungus garden and on the ants.

7. The method according to claim 2, wherein the composition comprises (i) the at least one laccase inhibitor selected from the group consisting of (a) ascorbic acid, (b) thymol, (c) eugenol, (d) sodium thiosulfate, (e) L-cysteine, and (f) glutathione; or (ii) at least one combination of compounds selected from the group consisting of (g) thymol and L-cysteine, (h) eugenol and L-cysteine, (i) cinnamaldehyde and L-cysteine, (j) thymol and sodium thiosulfate, (k) eugenol and sodium thiosulfate, (l) cinnamaldehyde and sodium thiosulfate, (m) thymol and glutathione, (n) eugenol and glutathione, and (o) cinnamaldehyde and glutathione.

8. The method according to claim 1, wherein the composition further comprises at least one oligosaccharide feeding stimulant or at least one polysaccharide feeding stimulant.

9. The method according to claim 8, wherein the at least one oligosaccharide feeding stimulant or at least one polysaccharide feeding stimulant is selected from the group consisting of starch, amylose, laminarin, maltodextrin, and glycogen.

10. A fungus-growing ant formicidal composition consisting of (i) at least one laccase inhibitor selected from the group consisting of coumaric acid, 6-1-palmitylascorbic acid, syringic acid, sodium ascorbate, calcium ascorbate, potassium ascorbate, butylhydroxyanisole, butylhydroxytoluol, octyl gallate, dodecyl gallate, glutathione, sodium lactate, potassium lactate, calcium lactate, lipoate, carotenoids, carotenes, sodium tartrate, potassium tartrate, selenium, calcium chloride, rhodotorulic acid, enterobactin, diethyldithiocarbamic acid, sodium azide, sodium lauryl sulphate, sodium cyanide, sodium thiosulphate, oxalic acid, thioglycolic acid, diethyldithiocarbamic acid, thymol, eugenol, glutathione, Fe.sup.2+, Cu.sup.2+, Ag.sup.+, Sn.sup.+, Hg.sup.+, Mn.sup.2+, Zn.sup.2+, and Al.sup.3+; (ii) an antibacterial compound selected from the group consisting of an antibiotic, carvacrol, cinnamaldehyde, -terpineol, and terpinen-4-ol; and (iii) an oligosaccharide feeding stimulant or a polysaccharide feeding stimulant; wherein the formicidal composition exhibits colony destroying activity against the fungus-growing ants and/or killing activity against both the fungus-growing ants and the fungus.

11. The fungus-growing ant formicidal composition of claim 10, wherein the composition comprises at least one combination of compounds selected from the group consisting of (i) thymol and sodium thiosulfate, (ii) eugenol and sodium thiosulfate, (iii) cinnamaldehyde and sodium thiosulfate, (iv) thymol and glutathione, (v) eugenol and glutathione, and (vi) cinnamaldehyde and glutathione.

12. The fungus-growing ant formicidal composition according to claim 10, wherein the at least one oligosaccharide feeding stimulant or the at least one polysaccharide feeding stimulant is selected from the group consisting of starch, amylose, laminarin, maltodextrin, and glycogen.

13. The fungus-growing ant formicidal composition according to claim 10, wherein the at least one laccase inhibitor exhibits both (i) laccase inhibitor activity against laccases present in the fungus garden and in the ants, and (ii) bactericidal and/or bacteriostatic activity against actinomycetes present in the fungus garden and on the ants.

14. The formicidal composition according to claim 10, wherein the antibacterial compound is present in a concentration from 1 g/kg to 200 g/kg of the formicidal composition, and the at least one laccase inhibitor is present in a concentration from 1 g/kg to 200 g/kg of the formicidal composition.

15. The fungus-growing ant formicidal composition according to claim 10, wherein the oligosaccharide feeding stimulant or the polysaccharide feeding stimulant is present in a concentration from 600 g/kg to 999 g/kg of the formicidal composition.

16. The fungus-growing ant formicidal composition according to claim 10, wherein the oligosaccharide feeding stimulant or the polysaccharide feeding stimulant is present in a concentration from 800 g/kg to 999 g/kg of the formicidal composition, and the at least one laccase inhibitor is present in a concentration from 1 g/kg to 200 g/kg of the formicidal composition.

17. The method according to claim 1, wherein the at least one laccase inhibitor is selected from the group consisting of ascorbic acid, citric acid, coumaric acid, ferulic acid, syringic acid, gallic acid, 6-1-palmitylascorbic acid, sodium ascorbate, calcium ascorbate, potassium ascorbate, butylhydroxyanisole, butylehydroxytoluol, cysteine, octyl gallate, dodecyl gallate, glutathione, sodium lactate, potassium lactate, calcium lactate, lecithins, lipoate, carotenoids, carotene, sodium tartrate, sodium thiosulphate, vitamin E, synthetic -tocopherol, synthetic -tocopherol, synthetic -tocopherol, selenium, and hydroxylammonium chloride.

18. The method according to claim 6, wherein the at least one laccase inhibitor is thymol or eugenol.

19. The method according to claim 1, wherein the fungus-growing ants comprise a colony of fungus-growing ants, and the colony is destroyed after the fungus-growing ants come into contact with the at least one laccase inhibitor.

Description

FIGURES

(1) FIG. 1 shows a measurement of the anti-laccase activity of L-cysteine on a fungus garden sample (A) and an ant sample (B).

EXAMPLES

(2) 1. Materials and Methods

(3) 1.1. Measurement of Enzyme Activity

(4) 1.1.1 Sampling and Storage

(5) From a total of 18 nests, samples of fungus gardens and of ants were collected and stored at 20 C.

(6) 1.1.2. Obtaining Enzyme Extracts

(7) Crushing was performed in mortars placed on ice, using pestles. Ants (2 g) and fungus garden (2 g) were crushed in 10 mL of distilled water refrigerated at 4 C. After obtaining crushed matter of homogeneous texture, the whole was centrifuged. Centrifugation was performed at 4 C., 15,000 rpm for 20 min.

(8) For protein precipitation, the supernatant was 80% saturated with ammonium sulphate ((NH.sub.4).sub.2SO.sub.4). After total dissolution of the ammonium sulphate, the solution obtained is placed at 4 C. for 12 hours. The solution was then centrifuged at a temperature of 4 C., at 15,000 rpm, for 20 min. The pellet obtained is redissolved in a minimum of cold distilled water and then dialysed against 5 litres of distilled water at 4 C. for 12 hours. The extract thus obtained constitutes the crude enzyme extract. These extracts were aliquoted and then stored at 20 C.

(9) 1.1.3. Enzyme Substrates Analysed

(10) Enzyme activity was evaluated in relation to the degradation capacity of 16 substrates:

(11) five natural oligosaccharides (cellobiose, gentiobiose, lactose, maltose, sucrose); six synthetic heterosides (PNP-- and --glucopyranosides, PNP-- and --galactopyranoside, PNP--xyloside and PNP-N-acetyl-glucosamine); four polysaccharides (starch, carboxymethylcellulose (CMC), pullulan and xylan).

(12) 1.1.4. Enzyme Analyses

(13) Enzyme solutions were incubated with substrates at 37 C. for 30 min in Mcllvain buffer solution (pH 5.2). All enzyme and control assays were performed in 3 replicates to ensure repeatability of the results. To express the results, the average of the three repetitions was taken into account.

(14) Oligosaccharidase activity was determined by the glucose oxidase (GOD) assay method according to Werner et al. (Chem. (1970) 252, 224-228).

(15) Heterosidase activity was indirectly evaluated by measuring the amount of para-nitrophenol (PNP) released, according to the method described by Nunan et al. (Metabolising old soil carbon: Simply a matter of simple organic matter, Soil Biology & Biochemistry 88 (2015) 128-136).

(16) The reduced sugars produced by polysaccharide hydrolysis are assessed by the Somogyi-Nelson microassay method with the cupro-alkaline reagent (Williams et al., -Galactosidases II, III and IV from Seeds of Trifolium repens. (1978) Biochem. J. 175, 1069-1077).

(17) Laccase activity was evaluated using 2,2-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) as oxidation control substrate. Briefly, ABTS is diluted to 27.4 mg/L in Mcllvain buffer solution (pH 5.2). 150 L of ABTS solution is incubated with 100 L of test solution and then incubated for 30 min at 37 C. The measurement of OD at 405 nm is used to determine the amount of oxidised ABTS using Beer-Lambert's law (epsilon=36,000 mM).

(18) Proteins are assayed by a colorimetric assay for measuring total protein concentration Based on the color change of Coomassie brilliant blue G-250 dye in response to various concentrations of protein, sold under the trademark Bio-Rad reagent method. Bovine serum albumin was used to prepare the standards.

(19) 1.1.5. Laccase Inhibition

(20) The determination of laccase activity is based on a colorimetric method where the substrate ABTS (2,2-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) in reduced form (colourless) is oxidised (green colour) in the presence of the enzyme. The method was published by Bourbonnais et al. in 1990 (Oxidation of non-phenolic substrates. An expanded role for laccase in lignin biodegradation. FEBS, 1990, 267, 99-102). To test the inhibition of laccase activity, the molecule tested is added to the reaction mixture in amounts ranging from 0.1 mM to 100 mM. The inhibitors selected for analysis are compounds having antioxidant capacity.

(21) 1.2. Evaluation of Antibacterial Activity

(22) 1.2.1. Collection

(23) Bacterial strains adhering to the ant cuticle were collected using a dispersant solution corresponding to a 0.9% NaCl solution containing 10% polysorbate 80 or also called polyoxythylne (20) sorbitan monooleate sold under the trade mark Tween 80.

(24) 1.2.2. Isolation

(25) Actinomycete strains were isolated by successive plating on NA (composition: 2.5 g K.sub.2HPO.sub.4; 0.5 g sodium nitrate; 7.5 g agar; 2 g N-acetyl-glucosamine; qs 500 mL; pH 6.5) or PCA (composition: 5.0 g casein peptone; 2.5 g yeast extract; 1.0 g D(+)-glucose; 14.0 g agar; qs 1000 mL, pH 7) and morphology verification by optical microscopy.

(26) 1.2.3. Verification of Strain Identity

(27) 1.2.3.1. DNA Extraction

(28) Strains isolated from the external surface of ant bodies and selected were reactivated in 9 mL of culture medium (5 g peptone; 2.5 g yeast extract and 1 g glucose).

(29) The culture was incubated with shaking at 27 C. overnight. After bacterial growth, 2 mL culture samples were taken from each sample and placed respectively in Eppendorf tubes and then centrifuged at 10,000 g for 10 min. Bacterial lysis was performed using high-speed, digitally controlled benchtop homogenizer sold under the trademark FastPrep-24 (MP Biomedicals) and the PowerBead tubes included in the kit. The conditions were as follows: frequency 4.0 ms.sup.1, time 45 s. The aqueous solution of guanidine thiocyanate and garnet particles sold under the trademark PowerSoil DNA Isolation Kit (Mobio Laboratories) was used to extract total DNA from the pellets obtained after centrifugation. The protocol used is that recommended by the supplier. The DNA was eluted in 70 L of Tris-HCl buffer provided with the kit. The concentration of total DNA extracted was determined by spectrophotometry using the microvolume UV-Vis Spectrophotometer, sold under the trademark NanoDrop, and stored at 20 C. before subsequent analyses.

(30) 1.2.3.2 Amplification of a 16S rRNA Gene Region Specific to Bacteria

(31) A region specific to the bacteria-specific 16S rRNA gene was amplified by PCR under the following conditions:

(32) The primers used were 27F and 1390R (Mao et al. 2012). The amplicons were verified by 1% agarose gel electrophoresis (weight/volume of 0.5TAE buffer) containing 0.5SYBR Safe. The presence of bands was visualised by exposing the gel to ultraviolet (UV) light.

(33) 1.2.3.3. Sequencing and Phylogenetic Affiliation of Strains

(34) Sanger sequencing of amplicons was performed by Beckman Coulter Genomics. BioEdit software was used to align the sequences obtained. Comparison between these sequences and those available in the databases was performed using BLAST software on NCBI.

(35) 1.2.4. Measurement of Antibacterial Activity Using Discs in Petri Dishes

(36) The general principle is to test in solid medium the culture of the strain in the presence of a paper disc impregnated with the molecule to be tested. For each molecule tested at least three dilutions were prepared. In the presence of a molecule, a halo, the diameter of which is related to the intensity of the inhibitory effect, appears around the paper disc. This approach provides a simple way to select antibacterial molecules and to determine the minimum inhibitory concentration (MIC) of bacterial growth.

(37) Antibacterial activity can be measured in liquid medium (in a liquid bacterial culture) or in solid medium (on an agar Petri dish) by adding a known concentration of antibiotic. In liquid medium, inhibition is measured by an optical density (OD); in solid medium, growth inhibition is estimated by the diameter of the halo corresponding to the zone where no growth is observed.

(38) From the isolates, a sample is taken. This is suspended in 1 mL of physiological saline solution without measuring OD.

(39) Inoculation is performed with 100 L of suspension using a spreader. For undiluted essential oil tests, a disc soaked in essential oil is placed in the centre of the dish. For dilutions (; ), both discs are placed in the same dish. Dilutions were prepared by diluting the essential oils in sunflower oil. A control dish was prepared and consists of an inoculate in the absence of essential oil. A second control, this time with a disc impregnated with sunflower oil, was also prepared to confirm the absence of an effect. All products were sterilised and the experiments were performed under a hood in sterile conditions.

(40) After 4 days of growth, the inhibition diameters were measured.

(41) Erythromycin was used for this study and the study concerning the use of molecules.

(42) Erythromycin is a macrolide antibiotic with a similar or slightly broader antimicrobial spectrum than penicillins. This molecule serves as a reference.

(43) 1.3. Evaluation of Efficacy Against Fungus-Growing Ants Over 14 Days.

(44) 1.3.1. Preparation of the Composition

(45) The pellets (or bait) were prepared by incorporating 10% sodium thiosulphate or ascorbic acid into a wheat flour paste. The paste thus obtained was transformed into a pellet in the shape of a grain of rice. These pellets were then dried in an oven at a maximum temperature of 28 C.

(46) 1.3.2. Tests Against Fungus-Growing Ants

(47) 1.3.2.1. Anti-Laccase

(48) Initial tests were performed to acquire initial data concerning the use of pellets impregnated with various laccase inhibitors.

(49) The nests are visited for the first time 2 days after the pellets are deposited, and then at 4, 6, 8, 9, 10, 12 and 14 days. Observation is made of the presence or absence of pellets (10 g) which, placed in a small plastic bag, were deposited near a dome. At each visit the bag is replaced whether or not the pellets were harvested by the ants. Pellets impregnated with ascorbic acid or sodium thiosulfate are alternated during each visit.

(50) A colony activity test is performed using a wooden rod. The latter is inserted into the ground at various points around the cone; a lack of resistance means that the rod is inserted into the cavity containing the fungus garden. Within a few seconds several tens of ants disturbed by the insertion of the rod into the fungus garden cavity climb up the rod, thus attesting to the vitality of the colony.

(51) 1.3.2.2. Anti-Laccase+Antibacterial

(52) The pellets may also contain a mixture of anti-laccase and antibacterial molecules.

(53) 1.4. Evaluation of Efficacy Against Fungus-Growing Ants Over Several Months.

(54) 1.4.1. Experimental Sites.

(55) An in situ experiment was conducted in the French West Indies, on 44 nests.

(56) 1.4.2. Compositions Tested.

(57) Several compositions of the invention were tested: thymol and L-cysteine, eugenol and L-cysteine, cinnamaldehyde and L-cysteine, thymol and sodium thiosulfate, eugenol and sodium thiosulfate, cinnamaldehyde and sodium thiosulfate, thymol and glutathione, eugenol and glutathione, cinnamaldehyde and glutathione, thymol, eugenol, cinnamaldehyde, sodium thiosulfate, L-cysteine, and glutathione.

(58) The compositions were prepared as mentioned in section 1.3.1. Each active compound represented 10% of the composition (by weight of the composition), the remainder being wheat flour paste.

(59) Each of the above-mentioned compositions was tested on two or three nests. At each nest, 5 bags each containing 25 g of one of the above-mentioned compositions were deposited at the same time.

(60) 2. Results

(61) 2.1. Measurement of Enzyme Activity

(62) Results of enzyme activity measurements are presented in Table 1.

(63) TABLE-US-00001 TABLE 1 Enzyme activities measured in the fungus-growing ant Acromyrmex octospinosus and the fungus Leucophorus gongylophorus Ant Fungus Oligosaccharidases (g glucose min.sup.1 mg.sup.1 protein) Cellobiase 41.3 4.5 Gentiobiase 62.4 8.8 Lactase 12.0 5.0 Maltase 988.7 5.2 Sucrase 790.7 0.9 Heterosidases (g phenol min.sup.1 mg.sup.1 protein) -Galactosidase 48.5 14.2 -Galactosidase 12.0 10.3 -Glucosidase 2706.5 9.3 -Glucosidase 46.9 12.9 -Xylosidase 22.1 14.0 N-Acetyl-glucosaminidase 23.5 3.2 Polysaccharidase (g glucose equivalent min.sup.1 mg.sup.1 protein) Amylase 385.6 26.5 Cellulase 75.9 1.7 Pullulanase 14.1 0.8 Xylanase 96.6 13.1 Phenol oxidase (g fluorescein min.sup.1 mg.sup.1 protein) Laccase 38830.5 8538.1

(64) The major result of this enzyme screening is the demonstration of very high laccase activity both in the fungus garden and in the ants. Indeed, this enzyme represents more than 90% of all measured activities. Laccases or polyphenol oxidases catalyse the oxidation of phenolic substances (Thurston, The structure and function of fungal laccases, Microbiology (1994) 140, 19-26). In fungi, laccases, in addition to their oxidative action on polyphenolic compounds (involvement in the degradation of lignin), also play a detoxifying role with respect to plant defence substances.

(65) The results of the glycosidase spectrum study show that the ants' spectrum is characterised by amylolytic activity. Thus, the ants' maltase (starch dimer) activity is not only very much higher than that detected in the fungus garden but it is also one of the majority enzymes. The same applies to the activities measured on -glucoside. These high starch degradation activities are confirmed by high amylase activities. Starch therefore appears as an important carbon source for Acromyrmex octospinosus, contrary to what can be observed in the fungus garden.

(66) 2.2. Measurement of Anti-Laccase Activity

(67) Results of anti-laccase activity measurements on ant or fungus garden samples are presented in Table 2.

(68) TABLE-US-00002 TABLE 2 IC.sub.50 Acromyrmex IC.sub.50 Leucophorus octospinosus gongylophorus L-Cysteine <0.25 mM <0.25 mM Coumaric acid <0.25 mM <0.25 mM Sodium thiosulfate <0.25 mM <0.25 mM Ferulic acid <0.1 mM <0.05 mM Syringic acid <0.25 mM <0.1 mM Ascorbic acid <0.25 mM <0.25 mM Gallic acid <0.05 mM <0.05 mM

(69) The results of inhibition of the laccase activity present in the ants show that the listed compounds inhibit at least 50% of the laccase activity of Acromyrmex octospinosus and of Leucophorus gongylophorus at concentrations below 0.25 mM.

(70) Moreover, L-cysteine, at concentrations above 0.5 mM, inhibits more than 80% of the activity (FIG. 1). Ascorbic acid and gallic acid, in turn, inhibit more than 90% of the activity at concentrations of 0.25 mM. With ferulic acid and syringic acid, this inhibition percentage is obtained with a concentration of 0.5 mM (results not shown).

(71) The inhibition results obtained are very homogeneous for fungus garden and ant samples. All of the molecules used to test inhibitory capacity are known for their ability to react as antioxidants. These compounds have a very high inhibitory capacity.

(72) 2.3. Isolation of Actinomycete Strains

(73) This step made it possible to isolate, from ants belonging to the species Acromyrmex octospinosus, 15 actinomycete strains including 12 belonging to the genus Tsukumurella, two to the genus Streptomyces and one to the genus Gordonia. In total, 5 strains belonging to the genera Streptomyces, Tsukumurella and Gordonia were selected for the analysis of antibacterial substances.

(74) 2.4. Measurement of Antibacterial Activity

(75) The results obtained on strain Streptomyces NA2B3 (Table 3) show that essential oils of thyme, cinnamon, oregano and clove have an inhibitory effect on growth. Considering that inhibition resulting in a diameter of 1.5 cm or greater is a selection criteria, the inventors identified as preferred solutions essential oils of oregano and clove causing inhibition with dilutions up to . Thyme and cinnamon are effective up to a dilution of .

(76) On average, on strains isolated from fungus-growing ants (Table 4), essential oils of thyme, oregano, cinnamon and clove inhibit growth. With the exception of thyme essential oil, these oils have a deleterious action for dilutions.

(77) It is interesting to note that erythromycin gives similar results with regard to these two strains, namely that this antibiotic gives overall results comparable to those obtained with essential oils of thyme, cinnamon, oregano and clove.

(78) TABLE-US-00003 TABLE 3 Streptomyces (NA 2B3): inhibition diameter in cm Thyme 4.5 0.6 Erythromycin 20% 3.6 0.4 Oregano 3.2 0.4 Cinnamon 2.9 0.6 Clove 2.4 0.3 Erythromycin 10% 2.2 0.3

(79) TABLE-US-00004 TABLE 4 Average inhibition diameters (cm) observed on the 5 actinomycete strains isolated from fungus-growing ants Pure thyme 6.1 1.2 Pure oregano 5.8 1.1 Pure cinnamon 5.4 0.6 Cinnamon 4.9 0.4 Oregano 4.0 0.3 Erythromycin 20% 3.6 0.4 Thyme 3.6 0.3 Erythromycin 10% 3.2 0.4 Clove 2.9 0.2

(80) The results obtained from the essential oils tested show that 3 of them (thyme, oregano and cinnamon), when used undiluted, have inhibition diameters between 5.4 and 6.1 cm. Diluted to , the cinnamon and oregano essential oils result in growth inhibition superior to that obtained with 20% erythromycin, thereby attesting to the efficacy of these oils.

(81) TABLE-US-00005 TABLE 5 Streptomyces (NA 2B3): inhibition diameter in cm Erythromycin 20% 4.3 0.3 Erythromycin 10% 3.5 0.5 Cinnamaldehyde 20% 3.0 1.5 Carvacrol 20% 2.2 0.3 Thymol 20% 1.7 0.8

(82) TABLE-US-00006 TABLE 6 Average inhibition diameters observed on 5 actinomycete strains isolated from fungus-growing ants Cinnamaldehyde 20% 4.5 0.6 Erythromycin 20% 3.6 0.4 Erythromycin 10% 3.2 0.4 Cinnamaldehyde 10% 2.9 0.6 Eugenol 20% 2.9 0.7 Carvacrol 20% 2.4 0.3 Thymol 20% 2.2 0.3

(83) The tests performed with the active molecules of the essential oils clearly show that cinnamaldehyde distinctly differs from the other molecules tested. Moreover, the results obtained with this molecule are comparable to those obtained with the reference antibiotic (erythromycin). However, the other molecules tested, namely eugenol, carvacrol and thymol, also have high levels of inhibition.

(84) Quite surprisingly, anti-laccase activity was observed for eugenol and thymol, these compounds consequently being of particular interest for the compositions of the invention because they act on both targets identified by the inventors for controlling fungus-growing ants (data not shown).

(85) 2.5. In Situ Tests on Fungus-Growing Ants:

(86) 2.5.1. Tests of the Compositions of the Invention Over 14 Days:

(87) 2.5.1.1. Composition Comprising an Anti-Laccase Molecule:

(88) This experiment showed that the formicidal compositions (in the form of pellets formed with starch), regardless of the type of inhibitor, are taken by the ants and that they can induce an effect of wilting of the fungus garden (whitish, yellowish or pinkish zones).

(89) These experiments also made it possible to observe nest abandonment behaviour by the colony which could be accompanied by relocation of the fungus garden. This is confirmed by the observation of an absence of fungus garden or of fungus garden residues in the underground cavity of some nests.

(90) In these cases, the fungus garden can be reconstructed in a second nest at a distance from the first nest.

(91) 2.5.1.2. Composition Comprising an Anti-Laccase Molecule and an Antibacterial Molecule:

(92) Treating nests with compositions containing ascorbic acid (100 g/kg) and chloramphenicol (10 g/kg) causes all activity to disappear. Moreover, disappearance of the fungus garden and reappearance of the colony was not observed with this combination.

(93) 2.5.1.3. Effect of Carbohydrate Feeding Stimulant:

(94) Tests with the ants showed that the flour pellets were collected quickly by the ants and confirm that the flour (consisting mainly of starch) has an attractive nature and that the compositions do not have a repellent effect. Because the ants collect the bait and import it into the nests, there is no need to apply the compositions to the colony.

(95) 2.5.1.4. Tests of the Compositions of the Invention (See Section 1.4.2.) Over Several Months:

(96) Nests were monitored during the first two weeks (Table 7) after deposition of the formicidal combinations, then examined at 41 days (Table 8) and 4 months (Table 9) after deposition of the compositions.

(97) TABLE-US-00007 TABLE 7 Observation of nests in the 2 weeks after treatment Results at 2 weeks Nest observation period (number 10-14 of days after deposition) Number of nests observed 44 Number of nests with relocation 29 (66%) of the fungus garden (%)

(98) Normally, a salt and pepper colour on the top of the fungus garden, white in the middle part and brown in the lower part, attest to a good vitality of the fungus garden. At the end of a 14-day period, moving (relocation) of the fungus garden is observed in 66% of the nests on all the experimental sites. Moreover, a change in the colour of the relocated fungus garden was observed: the appearance of yellowish and pinkish zones, or a completely white fungus garden with no colour gradient, was observed.

(99) TABLE-US-00008 TABLE 8 Nest observation 41 days after treatment Results at 41 days Number of nests observed 38 Number of dead nests 10 (26%)

(100) Forty-one days after deposition of the compositions, dead nests were counted on the experimental sites (Table 8). Ten dead nests were observed on the experimental sites (26%).

(101) TABLE-US-00009 TABLE 9 Nests observation 4 months after treatment Results at 4 months Number of dead Number of live Number of nests that nests nests could not be observed 34 7 3

(102) Four months after treatment, 77% dead nests were observed on the experimental sites. On all the sites, it thus appears that 77% of the nests were destroyed by the treatment with the compositions of the invention.

(103) Therefore, the formicidal compositions comprising a laccase inhibitor, an antibacterial or bacteriostatic agent against commensal actinomycetes of fungus-growing ants or a combination thereof are effective in controlling fungus-growing ants by causing nest abandonment and/or destruction.