MOLYBDENUM-BASED FEED SUPPLEMENT FOR BEES

Abstract

Food supplements for bees that include molybdenum complexes and a method of administering the food supplements to the bees for preventing the infestation of bees and their larvae by the Varroa destructor mite. Also, the use of the food supplements to increase the production of honey and lower the winter mortality of bees.

Claims

1-19. (canceled)

20. A molybdenum complex of the following formula I: ##STR00011## wherein: A1 is cation selected from alkali metal cations, alkaline earth metal cations, transition metal cations, or a PPh.sub.4.sup.+ organic cation, E is selected from O or S, L is a ligand selected from the amino acids such as glycine (gly.sup.-), cysteine (L-cys.sup.2-) or histidine (L-his.sup.-) or polycarboxylate ligands such as oxalate (Ox.sup.2-), citrate (Cit.sup.3-), ethylenediaminetetraacetate (EDTA.sup.4-), nitrilotriacetate (HNTA.sup.2-), and iminodiacetate (IDA.sup.2-), m is the number of cations and is selected from 0, 1, 2, 3 and 4, and n is the number of ligands and is selected from 1 and 2.

21. The formula I molybdenum complex according to claim 20, wherein said molybdenum complex is represented by formula I(i) Al.sub.m—[Mo.sub.2O.sub.4]—L.sub.n or by formula I(ii) Al.sub.m—[Mo.sub.2O.sub.2S.sub.2]—L.sub.n.

22. The formula I molybdenum complex according to claim 20, wherein said complex corresponds to the following formula II: ##STR00012## wherein: A2 is a cation selected from the alkali metal cations, alkaline earth metal cations, or transition metal cations, E is selected from O or S, L is a ligand selected from the amino acids such as glycine (gly.sup.-), cysteine (L-cys.sup.2-) or histidine (L-his.sup.-) or polycarboxylate ligands such as oxalate (Ox.sup.2-), citrate (Cit.sup.3-), ethylenediaminetetraacetate (EDTA.sup.4-), nitrilotriacetate (HNTA.sup.2-), and iminodiacetate (IDA.sup.2-), m is the number of cations and is selected from 0, 1, 2, 3 and 4; n is the number of ligands and is selected from 1 and 2.

23. The formula I molybdenum complex according to claim 20, wherein said complex is selected from the group consisting of Na.sub.2[Mo.sub.2O.sub.4(EDTA)].xH.sub.2O (x = 5-6), Li.sub.2[Mo.sub.2O.sub.4(EDTA)].xH.sub.2O (x = 3-6), and (PPh.sub.4).sub.2[Mo.sub.2O.sub.4(EDTA)].xH.sub.2O (x = 2-5).

24. A composition comprising at least one formula I molybdenum complex according to claim 20.

25. The composition according to claim 24, wherein said at least one molybdenum complex corresponds to the following formula II: ##STR00013## wherein: A2 is a cation selected from the alkali metal cations, alkaline earth metal cations, or transition metal cations, E is selected from O or S, L is a ligand selected from the amino acids such as glycine (gly.sup.-), cysteine (L-cys.sup.2-) or histidine (L-his.sup.-) or polycarboxylate ligands such as oxalate (Ox.sup.2-), citrate (Cit.sup.3-), ethylenediaminetetraacetate (EDTA.sup.4-), nitrilotriacetate (HNTA.sup.2-), and iminodiacetate (IDA.sup.2-), m is the number of cations and is selected from 0, 1, 2, 3 and 4; n is the number of ligands and is selected from 1 and 2.

26. The composition according to claim 24, wherein said composition further comprises one or more foods for bees selected from water, pollen, nectar, honey, sugars, vitamins, minerals, lipids, and proteins.

27. A method for preventing the infestation of bees and/or bee larvae by Varroa Destructor mites, comprises feeding to a bee colony an effective amount of at least one formula I molybdenum complex according to claim 20 or a composition comprising said at least one formula I molybdenum complex.

28. The method according to claim 27, wherein the bee colony is fed an overall and annual amount of said at least one formula I molybdenum complex from 1 to 60 mg/colony.

29. The method according to claim 27, wherein the bee colony is fed said at least one formula I molybdenum complex or said composition at a single annual dose of 1 to 60 mg/colony.

30. A food supplement for bees, comprising a molybdenum complex of the following formula II: ##STR00014## wherein: A2 is a cation selected from alkali metal cations, alkaline earth metal cations, or transition metal cations E is selected from O or S, L is a ligand selected from amino acids such as glycine (gly-), cysteine (L-cys.sup.2-) or histidine (L-his.sup.-) or polycarboxylate ligands such as oxalate (Ox.sup.2-), citrate (Cit.sup.3-), ethylenediaminetetraacetate (EDTA.sup.4-), nitrilotriacetate (HNTA.sup.2-), and iminodiacetate (IDA.sup.2-) m is the number of cations and is selected from 0, 1, 2, 3 and 4, and n is the number of ligands and is selected from 1 and 2.

31. The food supplement for bees according to claim 30, wherein said molybdenum complex is represented by formula II(i) A2.sub.m—[Mo.sub.2O.sub.4]—L.sub.n or by formula II(ii) A2.sub.m-[Mo.sub.2O.sub.2S.sub.2]—L.sub.n.

32. The food supplement for bees according to claim 30, wherein A2 is a cation selected from the group consisting of Li.sup.+, Na.sup.+, K.sup.+, Ca.sup.2+, Mg.sup.2+ and Zn.sup.2+.

33. The food supplement for bees according to claim 30, wherein said complex is selected from the group consisting of Na.sub.2[Mo.sub.2O.sub.4(EDTA)].xH.sub.2O (x = 5-6), Li.sub.2[Mo.sub.2O.sub.4(EDTA)].xH.sub.2O (x = 3-6), K.sub.2[Mo.sub.2O.sub.2S.sub.2(LCys).sub.2].xH.sub.2O, Na.sub.2[Mo.sub.2O.sub.4(LCys).sub.2].xH.sub.2O, K.sub.2[Mo.sub.2O.sub.2S.sub.2](HNTA).sub.2].xH.sub.2O, K.sub.2[Mo.sub.2O.sub.2S.sub.2(EDTA)].xH.sub.2O, K.sub.2[Mo.sub.2O.sub.4(HNTA).sub.2].xH.sub.2O, Mg[Mo.sub.2O.sub.4(EDTA)].xH.sub.2O, Ca[Mo.sub.2O.sub.4(EDTA)].xH.sub.2O, and Zn[Mo.sub.2O.sub.4(EDTA)].xH.sub.2O, with x being between 3 and 6.

34. The nutritional supplement for bees according to claim 30, wherein said food supplement further comprises one or several foods for bees selected from the group consisting of water, pollen, nectar, honey, sugars, vitamins, minerals, lipids, and proteins.

35. The food supplement for bees according to claim 30, wherein said food supplement is in the form of a powder, syrup, paste or an aqueous solution.

36. A method for reducing bee colony mortality, comprising feeding to a bee colony an effective amount the food supplement for bees according to claim 30.

37. The method according to claim 36, wherein the bee colony is fed the food supplement to reduce winter mortality of the bee colony.

38. A method for increasing honey production in bees, comprising feeding to a bee colony an effective amount the food supplement for bees according to claim 30.

39. The method according claim 38, wherein the bee colony is fed the food supplement to increase honey production during a first harvest.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0149] The present invention is illustrated in more detail in the figures and examples below.

[0150] [FIG. 1]: Chemical structures of the cluster [Mo.sub.2O.sub.2S.sub.2].sup.2+ or [Mo.sub.2O.sub.4].sup.2+ supplemented by EDTA.sup.4-, HNTA.sup.2-, NTA.sup.3-, or L-Cys.sup.2-. In FIG. 1: Mo (gray, cross-hatching), O (gray, vertical hatching), S (gray, horizontal hatching), N (gray, oblique hatching), C (black), H (white).

[0151] [FIG. 2A]: Survival curves for bees of a known age, in cages, fed with 20 mg/L of Na-Mo.sub.2O.sub.4-EDTA in a sugary food solution.

[0152] The line with the symbol ■ corresponds to the group fed with a solution containing Na-Mo.sub.2O.sub.4-EDTA.

[0153] The line with the symbol □ corresponds to the control group fed with a sucrose solution.

[0154] The dotted line indicates the start of feeding with the solution containing Na-Mo.sub.2O.sub.4-EDTA for the treated group. (20 mg/L: n= 50+/-3 for each group).

[0155] [FIG. 2B]: Survival curves for bees of a known age, in cages, fed with 2 mg/L of Na-Mo.sub.2O.sub.4-EDTA in a sugary food solution.

[0156] The line with the symbol ■ corresponds to the group fed with a solution containing Na-Mo.sub.2O.sub.4-EDTA.

[0157] The line with the symbol □ corresponds to the control group fed with a sucrose solution.

[0158] The dotted line indicates the start of feeding with the solution containing Na-Mo.sub.2O.sub.4-EDTA for the treated group.

[0159] The experiment was repeated on 3 groups of bees. Each group had 50 bees.

DETAILED DESCRIPTION

Examples

1. Synthesis of the Formula I Molybdenum Complexes

[0160] The methods for synthesizing two complexes of formula I, Na.sub.2[Mo.sub.2O.sub.4(EDTA)].5H.sub.2O and Li.sub.2[MB.sub.2O.sub.4EDTA].6H.sub.2O, are described below. The synthesis of the compound (PPh.sub.4).sub.2[Mo.sub.2O.sub.4(EDTA)].2.5H.sub.2O, denoted PPh.sub.4-Mo.sub.2O.sub.4-EDTA, has been disclosed in patent MD4438.

Synthesis of Na.SUB.2.[Mo.SUB.2.O.SUB.4.(EDTA)].5H.SUB.2.O, Denoted Na-Mo.SUB.2.O.SUB.4.-EDTA; Mm = 680.15 g/mol

[0161] Sodium molybdate dihydrate Na.sub.2MoO.sub.4*2H.sub.2O (2.92 g, 12.07 mmol) and Na.sub.2H.sub.2EDTA*2H.sub.2O (2.25 g, 6.04 mmol) is dissolved in 30 mL of distilled water. An aqueous hydrazinium sulfate solution N.sub.2H.sub.4*H.sub.2SO.sub.4 (785.96 mg, 6.04 mmol/10 mL of water) is added and a 1 M HCl solution is used to adjust the pH to 3 in order to allow the reduction of [Mo.sup.(VI)O.sub.4].sup.2- into [Mo.sup.(V).sub.2O.sub.4].sup.2+. The dark red solution obtained is heated to 80° C. for one and a half hours with continuous stirring. After cooling to ambient temperature, the solution is transferred to a crystallizer and left to evaporate. After a few days, red crystals of the pure product Na.sub.2[Mo.sub.2O.sub.4(EDTA)].5H.sub.2O are collected by filtration, washed quickly in cold water and left to air dry. The yield is thus 65%. The product is characterized by FT-IR, elemental analysis, TGA, ESI-MS, EDX and RMN. FT-IR (v, cm.sup.-1): 3518 (m), 3300 (m), 1645 (s), 1445 (w), 1384 (m), 1350 (s, sh.), 1243 (w), 972 (w), 944 (m), 911 (w), 863 (w), 761 (w); Elemental analysis for Na.sub.2[Mo.sub.2O.sub.4(C.sub.10H.sub.12N.sub.2O.sub.8)].5H.sub.2O Calculated (found): C 17.66 (17.42) H 3.26 (2.92) N 4.12 (4.18); EDX: expected atomic ratio (found) Mo/Na 1.00 (1.05); ESI-MS m/z 272.05 (m/z calculated for [Mo.sub.2O.sub.4(EDTA).sub.2].sup.2- 271.93); TGA analysis shows 13.3% weight loss in the temperature range of 25-200° C. corresponding to the hydration water (calculated 13.2%); .sup.1H-NMR (δ ppm, 400 MHz, D.sub.2O) 2.61 (m, wide, 4H), 3.40 (m, wide, 8H).

Synthesis of Li.SUB.2.[Mo.SUB.2.O.SUB.4.(EDTA)).6H.SUB.2.O, Denoted Li-Mo.SUB.2.O.SUB.4.-EDTA; Mm = 666.07 g/mol

[0162] The compound Li.sub.2[Mo.sub.2O.sub.4(EDTA)].6H.sub.2O is obtained by exchanging Na.sup.+ cations of the compound Na.sub.2[Mo.sub.2O.sub.4(EDTA)].5H.sub.2O with Li.sup.+ on a DOWEX cation exchange resin loaded with Li.sup.+ cations. To do this, 1 g of Na.sub.2[Mo.sub.2O.sub.4(EDTA)].5H.sub.2O is dissolved in a minimum volume of water (10 mL) and this concentrated solution is passed over a resin-loaded column. An aqueous solution of the lithium salt Li.sub.2[Mo.sub.2O.sub.4(EDTA)] is obtained. This latter is spray dried under reduced pressure in a rotary evaporator to obtain the desired solid product. 90% yield. The product is characterized by FT-IR, elemental analysis, TGA, ESI-MS, EDX and RMN. (η = 90%). FT-IR (v, cm.sup.-1): 3412 (s, br.), 2983 (w), 1651 (s), 1625 (s), 1446 (w), 1392 (m), 1357 (m), 1244 (w), 964 (m), 948 (w), 933 (w), 909 (w), 861 (w), 759 (w), 475 (w); Elemental analysis for Li.sub.2[Mo.sub.2O.sub.4(C.sub.10H.sub.12N.sub.2O.sub.8)].6H.sub.2O Calculated (found): C 18.03 (17.79) H 3.63 (3.38) N 4.21 (4.10); EDX: expected atomic ratio (found) Mo 100% (100%). No trace of Na proving that the cation exchange definitely occurred; ESI-MS m/z 272.05 [Mo.sub.2O.sub.4(EDTA).sub.2].sup.2- (271.92); The TGA shows 14.83% weight loss in the temperature range of 25-200° C. corresponding to the hydration water (calculated 13.9%); .sup.1H-NMR (δ ppm, 300 MHz, D.sub.2O) 2.54 (m, wide, 4H), 3.33 (m, wide, 8H).

2. Toxicity of Formula I Complexes

[0163] The toxicity of formula I complexes was assessed using 3 different methods: in vitro tests on cells, on protozoa and directly on bees.

2.1 In Vitro Cytotoxicity Study

[0164] For cell culture, human cervical epithelial cells of the HeLa line, human epithelial pancreatic adenocarcinoma cells of the BxPC-3 line and healthy Madin-Darby canine kidney epithelial cells of the MDCK line were used in this study. The Resazurin cell proliferation test was applied according to the literature (S. Anoopkumar-Dukie, J. B. Carey, T. Conere, E. O’Sullivan, F. N. van Pelt, A. Allshire, Br J Radiol 2005, 78, 945, DOI: 10.1259/bjr/54004230). As an indicator of the effectiveness of the experimental compounds on cell proliferation, the median inhibitory concentration (IC.sub.50) was used. The lower the IC.sub.50 value, the higher the activity (in this case, the cytotoxicity). For IC.sub.50 values equal to and greater than 100 .Math.M, it is considered that there is no activity.

[0165] Five formula I complexes were tested in this study in comparison to two reference anti-cancer compounds (“Cisplatin” and Doxorubicin complex).

[0166] The results of the tests are given in Table 1 below.

TABLE-US-00001 Compound IC.sub.50, .Math.M MDCK BxPc-3 HeLa PPh.sub.4-Mo.sub.2O.sub.4-EDTA 60.72 ± 12.1 25.09 ± 7.02 ≥100 Na-Mo.sub.2O.sub.4-EDTA Inactive Inactive Inactive Li-Mo.sub.2O.sub.4-EDTA Inactive Inactive Inactive PPh.sub.4-Mo.sub.2O.sub.2S.sub.2-EDTA ≥100 59.81 ± 26.45 ≥100 K- Mo.sub.2O.sub.2S.sub.2-Cys Inactive Inactive Inactive Cisplatin (ref. 1) 30.9 ± 1.1 11.2 ± 1.2 3.99 ± 0.33 Doxorubicin (ref. 2) 1.48 ± 3.1 3.99 ± 7.9 1.31 ± 4.9 Cytotoxicity of compounds on healthy cells (MDCK), pancreatic cancer (BxPc-3) and cervical cancer cells (HeLa).

[0167] Table 1 shows that the compounds studied are mostly non-cytotoxic. Compounds with the counter cations, PPh.sub.4.sup.+, show a low activity related to the organic cation itself. Nevertheless, the IC.sub.50 gives concentrations at least 100 times greater than those used in hives (of the order of 0.2-0.6 .Math.M). Na-Mo.sub.2O.sub.4-EDTA and Li-Mo.sub.2O.sub.4-EDTA salts have no cytotoxicity.

2.2 Toxicity Study on Protozoa.

[0168] This study was also conducted by researchers of the Department of Chemistry from Moldova State University. Compounds which showed no in vitro toxicity on cells were used in other tests on simple single-cell microorganisms, namely protozoa. The spectrophotometric method was used to evaluate the toxicity of biologically active substances relative to a paramecium culture using the NR dye, according to the protocol described in the literature (1. Zhang, S.Z., Lipsky, M.M., Trump, B.F., Hsu, I.C. Neutral red (NR) assay for cell viability and xenobiotic-induced cytotoxicity in primary cultures of human and rat hepatocytes. Cell Biol Toxicol. 1990;6(2):219-34; 2. Repetto, G., del Peso, A., Zurita, J.L. Neutral red uptake assay for the estimation of cell viability/cytotoxicity. Nat Protoc 3: 1125-1131.). At the end, the LC.sub.50 value (median lethal concentration) is determined. The results of the tests are given in Table 2.

TABLE-US-00002 Compound LC.sub.50, .Math.M 24 hours 48 hours PPh.sub.4-Mo.sub.2O.sub.4-EDTA ≥100 ≥100 Na-Mo.sub.2O.sub.4-EDTA ≥100 ≥100 Li-Mo.sub.2O.sub.4-EDTA ≥100 ≥100 PPh.sub.4-Mo.sub.2O.sub.2S.sub.2-EDTA 40.25 ± 5.5 ≥100 K- Mo.sub.2O.sub.2S.sub.2-Cys ≥100 ≥100 Toxicity of compounds on Paramecium protozoa microorganisms

[0169] An LC.sub.50 less than 100 .Math.M reflects high toxicity. On the other hand, a value ≥100 indicates that there is no toxic effect on the development of Paramecium microorganisms. Of the 5 compounds tested, only one has slight activity that quickly disappears. The 5 compounds tested are deemed to be non-toxic.

2.3 Toxicity Study on Bees.

[0170] Additional tests on the impact on the lifespan of bees were carried out.

[0171] Fifty newly hatched bees (age = 1 day) were placed in a special cage with a wax strip, water and food, and were kept in the laboratory oven at a constant temperature of 33° C. and 50% humidity. A control group of bees was fed with a solution containing only sucrose; one group of bees was fed with a solution containing sucrose and 2 mg/L of Na-Mo.sub.2O.sub.4-EDTA; another group of bees was fed with a solution containing sucrose and 20 mg/L of Na-Mo.sub.2O.sub.4-EDTA. The consumption of water and food was checked every day. Dead bees were counted and removed. For statistical reasons, the experiments were repeated up to 3 times if necessary.

[0172] There is no significant difference between the control group and the treated group fed respectively with a solution containing 2 mg/L of Na-Mo.sub.2O.sub.4-EDTA (p>0.05, X.sup.2= 3.36, ddl=1) (FIG. 2B) or a solution containing 20 mg/L of Na-Mo.sub.2O.sub.4-EDTA (p>0.05, X.sup.2= 0.04, ddl=1) (FIG. 2A).

[0173] This study confirms that Na-Mo.sub.2O.sub.4-EDTA does not induce bee mortality at 2 mg/L nor at 20 mg/L.

[0174] This test clearly shows the non-toxicity of the complex [Mo.sub.2O.sub.4(EDTA)].sup.2- on bees.

3. Chemical Stability of Formula I Complexes

[0175] In solid form, the compounds obtained are perfectly stable in air and can be kept without any special conditions for years without any alteration.

[0176] The stabilities of the complexes in solution are studied by different techniques (.sup.1H-NMR or UV-vis spectroscopy), the objective being to study their behavior in different media in terms of stability.

[0177] The stability experiments in water in the 5×10.sup.-3 - 2×10.sup.-7 M concentration range reveal the fact that EDTA complexes ([Mo.sub.2O.sub.2S.sub.2-EDTA].sup.2- and [Mo.sub.2O.sub.4-EDTA].sup.2-) show no sign of decomposition in water, which suggests very high chemical stability. In the case of K-Mo.sub.2O.sub.2S.sub.2-LCys, it is also possible to reduce down to micromolar concentrations without any breakdown, which also suggests good chemical stability. This means that these compounds do not undergo structural modifications under high dilution conditions. The aqueous solutions of these complexes prove to be particularly stable over time.

[0178] In a physiological medium or in a sugar syrup with 50% by weight, the complexes [Mo.sub.2O.sub.2S.sub.2-EDTA].sup.2- and [Mo.sub.2O.sub.4-EDTA].sup.2- (Na and Li salts) and K-Mo.sub.2O.sub.2S.sub.2-L-Cys also show good stability at very low concentrations.

[0179] In conclusion, the formula I complexes prove to be particularly stable in different media and at high dilution.

4. Tests on Formula I Complexes in Hives

4.1. 1.SUP.st Test in Hives

Protocols Used, Parameters Monitored, and Methods Used

[0180] Biological field tests were performed on families of European domestic bees of the species Apis Mellifera Carpatica in an experimental apiary located in the Ghidighici forestry district (Republic of Moldova). Feeding with the complex of the invention took place in spring. The biological tests were monitored up to the month of September.

[0181] To administer the compounds to the bees, a very small amount of bioactive substance was added to two types of sugary feeding products for bees: candy in the form of a paste (70% sucrose, 30% honey) or syrup (50% sucrose, 50% water). The bees were fed by adding the candy/syrup to the feeder located in the inner upper part of the hive. The volume of syrup was adjusted based on the number of bee frames at the initial time, more precisely 200 g of candy per frame and 100 mL of syrup per frame, that is 2 kg of candy and 1 L of syrup per hive of 10 frames.

[0182] For the first feeding step, candy in the form of a paste was only given once. Next, for the main feed, determined volumes of syrup were used several times to feed the bees every two days for a period of two weeks, which corresponds to an annual feed quantity of approximately 2 mg to 6 mg of each compound for each hive. From the start of the test to the end (after the second harvest), all the bees were monitored for several key morpho-producing parameters that reflect the development level of the colony and the vital aspects of the bees. The parameters studied are: the prolificity of the queen (fertility), the power of the colonies, the production of honey and the production of wax. These parameters were monitored in line with the zootechnical standards concerning the assessment of bee families, breeding and certification of beekeeping parental material approved by government decision No. 306 dated Apr. 28, 2011 of the Republic of Moldova.

Assessment of Honey Production:

[0183] The production of honey (in kg) was determined for each family of bees by adding the amount of honey produced (honey from supers), extracted during the harvest season, to the amount of honey built up in the honeycomb (brood chamber) and left as food for the bees for the winter period (brood honey). The amount of honey produced will be determined for each family, for each extraction, by weighing the frames/honeycomb before and after extraction (with an accuracy of 0.1 kg), the weight difference being the quantity of honey produced that was extracted. The amount of honey left in the honeycomb to feed the bees was determined at the autumn visit (September), by weighing the honey frames and deducting (from their total weight) the total weight of the standard honeycomb frames; for Dadant type frames (435×300 mm) - 0.6 kg, for Langstroth frames (435×230 mm) - 0.5 kg.

Assessment of the Power of the Colony:

[0184] The power of the bee colony represents the number of bees present in the honeycomb at the time of the assessment. The assessment is performed three times per year: at the spring visit (March-April), at the end of spring (20-31 May) and at the visit in autumn (September). After these three assessments, the average power of the bee family is determined. The amount of bees (kg) is determined by multiplying the number of intervals between the frames, uniformly occupied by the bees, by the coefficient 0.25 for the standard Dadant frame (435×300 mm) and by 0.2 for the standard Langstroth frame (435×230 mm).

Assessment of the Prolificity of the Queen:

[0185] The prolificity of the queen (eggs/24 hours) is determined during the visit at the end of the spring (20-31 May) by dividing the number of cells with the brood by 12 (duration of development of the brood, days), the number of eggs laid in 24 hours. The number of cells in the honeycomb is determined using the Netz frame by measuring the number of squares (5×5 cm) occupied by the brood, which is multiplied by 100, to give the total number of cells with the brood covered.

Assessment of the Degree of Infestation With Varroa

[0186] The assessment consists in counting the number of Varroa on a sample of adult bees. The value from this count is used as an index for monitoring the level of parasitism in the colony.

[0187] The method uses icing sugar and a “shaker” jar made of transparent glass with a capacity of 1 kg. The cover is a mesh made of galvanized steel of the type placed at the bottom of the hive, with a mesh size of 3 mm. The mesh allows Varroa mites to pass through but retains the honey bees.

[0188] The method is implemented according to the following steps: 40 to 50 g of honey bees are collected in the jar (approximately 400 bees), 100 g of icing sugar is added and the shaker jar is rolled for 1 minute so as to cover every bee in icing sugar. The icing sugar then makes it possible to separate the Varroa mites from the body of the host bee so that they drop off. Leave to rest for 1 minute: bees have a delousing behavior, which helps the Varroa mites to drop off. The icing sugar is sieved over a sieve which makes it possible to retain the Varroa mites and then count them. The result is given as the number of mites per 10 g of bees (approximately 100 bees) and can be expressed as a %. This rate is indicative of the Varroa burden in the colony. A rate between 1 and 2 indicates low Varroa burden. A moderate rate is between 2 and 5 and, in this case, treatment of the colonies must be scheduled. Finally, a rate greater than 5, requires emergency treatment of the colony. Our tests give a result of around 2% for worker bees.

Results

[0189] The molecules tested, their concentration in solution and their annual overall doses are listed below: [0190] 1. (PPh.sub.4).sub.2[Mo.sub.2O.sub.4(EDTA)].2,5H.sub.2O, abbreviated as PPh.sub.4-Mo.sub.2O.sub.4-EDTA. The feeding is carried out with 2000 g of candy containing 0.8 mg of complex then 6 to 7 L of syrup containing 0.2 mg/L of complex. The concentration of the complex in the syrup: 1.5×10.sup.-7 mol/L. Overall and annual product dose: 2 mg of complex per colony [0191] 2. Na.sub.2[Mo.sub.2O.sub.4(EDTA)].5H.sub.2O, abbreviated as Na-Mo.sub.2O.sub.4-EDTA. The feeding is carried out with 2000 g of candy containing 0.8 mg of complex then 6 to 7 L of syrup containing 0.2 mg/L of complex. The concentration of the complex in syrup: 3.0×10.sup.-7 mol/L. Overall and annual product dose: 2 mg of complex per colony [0192] 3. Li.sub.2[Mo.sub.2O.sub.4(EDTA)].6H.sub.2O, abbreviated as Li-Mo.sub.2O.sub.4-EDTA. The feeding is carried out with 2000 g of candy containing 0.8 mg of complex then 6 to 7 L of syrup containing 0.2 mg/L of complex. The concentration of the complex in syrup: 3.2×10.sup.-7 mol/L; the Lithium concentration C(Li) = 6.4×10.sup.-7 mol/L that is 4.5×10.sup.-6 g/L. Overall and annual product dose: 2 mg of complex per colony [0193] 4. LiCH.sub.3COO.H.sub.2 O as a positive control, abbreviated as Li-Ac The feeding is carried out with 2000 g of candy containing 0.8 mg of positive control product, then 6 to 7 L of syrup containing 0.2 mg/L of positive control product. The concentration of the product in syrup: 2.36×10.sup.-6 mol/L that is 1.67×10.sup.-5 g/L. Overall and annual product dose: 2 mg of product per colony

[0194] The results are illustrated in Table 3 below.

TABLE-US-00003 Morpho-productive characters Batch I, control (N* = 10) Batch II, PPh.sub.4-Mo.sub.2O.sub.4-EDTA Overall dose 2 mg (N* = 10) Batch III, Na-Mo.sub.2O.sub.4-EDTA Overall dose 2 mg (N* = 10) Batch IV, Li-Mo.sub.2O.sub.4-EDTA Overall dose 2 mg (N* = 10) Batch V, Li-Ac Overall dose 2 mg (N* = 10) % % relative to batch I % relative to batch I % relative to batch I % relative to batch I Prolificity of the queen 100 107.1 113.3 111.7 108.8 Power of the bee family 100 102.1 101.2 103.3 98.3 Quantity of honey at the first harvest 100 113.3 (+13.3%) 149.9 (+49.9%) 143.1 (+43.1%) 122.9 (+22.9%) Degree of infestation with Varroa 100.sup.a 78.7 (-21.3%) 86.5 (-13.5%) 57.3 (-42.7%) 58.4 (-41.6%) N* - number of hives. a) mean initial infestation rate of 1.78 mite/10 g of bees (approximately 100 bees) that is 1.78%. This value is set at 100 as a reference for our tests.

[0195] We can observe that the three formula I molybdenum complexes show a significant protective effect against the infestation of bees by Varroa destructor relative to the untreated batch I.

[0196] At a lithium concentration almost 4 times lower, the Li-Mo.sub.2O.sub.4-EDTA complex shows a protective capacity comparable to that of the lithium acetate used as a positive control. This amount of lithium is much lower than the minimum effective amount of 2 mM used in the publication by B. Ziegelmann et al. (Scientific Reports 2018, 8, DOI: https://doi.org/10.1038/s41598-017-19137-5).

[0197] Moreover, two molecules of formula II tested (Na-Mo.sub.2O.sub.4-EDTA and Li-Mo.sub.2O.sub.4-EDTA) show higher effects compared to PPh.sub.4-MB.sub.2O.sub.4-EDTA, both for the prolificity of the queen and the amount of honey collected.

[0198] The honeys produced in the spring and summer for batches 1 to 4 were analyzed in a COFRAC-accredited laboratory. The results of these analyses show that the honey composition is not altered by the treatment with the complexes of the invention. The honey produced comply with European standards and no trace of Mo was found in the honey, and therefore a total absence of the complexes in the honey produced.

4.2. 2.SUP.nd test in the hives

[0199] The 2.sup.nd test was performed in the same apiary the following year. Feeding with the complex of the invention took place in spring. The biological tests were monitored up to the month of September.

[0200] The molecules tested, their concentration in solution and their annual overall doses are listed below: [0201] 1. (PPh.sub.4).sub.2[Mo.sub.2O.sub.4(EDTA)].2,5H.sub.2O, abbreviated as PPh.sub.4-Mo.sub.2O.sub.4-EDTA, The feeding is carried out with 2000 g of candy containing 0.8 mg of complex then 6 to 7 L of syrup containing 0.2 mg/L of complex. The concentration of the complex in syrup: 1.5×10.sup.-7 mol/L. Overall product dose: 2 mg/colony [0202] 2. Li.sub.2[Mo.sub.2O.sub.4(EDTA)].6H.sub.2O, abbreviated as Li-Mo.sub.2O.sub.4-EDTA. The feeding is carried out with 2000 g of candy containing 0.8 mg of complex then 6 to 7 L of syrup containing 0.2 mg/L of complex. The concentration of complex in the syrup: 3.2×10.sup.-7 mol/L; the Lithium concentration C(Li) = 6.4×10.sup.- .sup.7 mol/L that is 4.5×10.sup.-6 g/L; O□erall complex dose: 2 mg/colony [0203] 3. Li.sub.2[Mo.sub.2O.sub.4EDTA)].6H.sub.2O, abbreviated as Li-MO.sub.2O.sub.4-EDTA. The feeding is carried out with 2000 g of candy containing 2.4 mg of complex then 6 L of syrup containing 0.6 mg/L of complex. The concentration of complex in the syrup: 9.6×10.sup.-7 mol/L; the Lithium concentration C(Li) = 1.92×10.sup.-6 mol/L that is 1.32×10.sup.-5 g/L. Overall complex dose: 6 mg/colony [0204] 4. LiCH.sub.3COO.H.sub.2O for comparison, abbreviated as Li—Ac

[0205] The feeding is carried out with 2000 g of candy containing 0.8 mg of product then 6 to 7 L of syrup containing 0.2 mg/L of product. The concentration of product in the syrup: 2.36×10.sup.-6 mol/L that is 1.67×10.sup.-5 g/L. Overall product dose: 2 mg/colony

[0206] The results are illustrated in Table 4 below.

TABLE-US-00004 Morpho-productive characters Batch I, control (N* = 10) Batch II, PPh.sub.4-Mo.sub.2O.sub.4-EDTA Overall dose 2 mg/hive (N* = 10) Batch III, Li-Mo.sub.2O.sub.4-EDTA Overall dose 2 mg/hive (N* = 10) Batch IV, Li-Mo.sub.2O.sub.4-EDTA Overall dose 6 mg/hive (N* = 10) Batch V, Li-Ac Overall dose 2 mg/hive (N* = 10) % % relative to batch I % relative to batch I % relative to batch I % relative to batch I Power of the bee family 100 120 120 Not measured 97.5 Degree of infestation with Varroa 100.sup.a 60.8 (-39.2%) 52.6 (-47.4%) 38.4 (-62.6%) 69.0 (-31%) Degree of larvae infestation with Varroa 100.sup.b 56.0 (-44.0%) 42.6 (-57.4%) 18.4 (-81.6%) 64.9 (-35.1%) N* - number of hives. a) mean initial infestation rate of 2.32 mite/10 g of bees (approximately 100 bees), that is 2.32%; b) this reference □alue of 100 corresponds to a mean initial larvae infestation rate of 28.2% measured by directly counting the infected larvae after opening one hundred cells per hive.

[0207] The results of the 2.sup.nd test in the hives confirm the effects of the formula I complexes of the invention in protecting bees against Varroa infestation. Furthermore, according to the results illustrated in Table 4, this protective effect is increased when the dose is increased.

[0208] The results of the 2.sup.nd test particularly show that the formula I complex of the invention also protects the larvae from Varroa. To date, there is no known treatment to protect the larvae from Varroa.

4.3.3.SUP.rd Test in the Hives

[0209] A treatment campaign was carried out in the autumn-winter in apiaries located in California, near San Francisco. This region is particularly vulnerable to winter mortality of bee colonies which can reach as high as 80% in some years.

[0210] 151 hives, distributed over 6 different apiaries in California to the south of San Francisco were selected. The bees were of different types: Wildflower (VSH-Italian), Beeweaver, Carniolan Californian Bee (Pope Canyon), Californian bee Tom and Italian Californian bee (Sam).

[0211] The hives were Langstroth 2 body type hives (15 frames compared to 10 for Dadant hives in Europe). Each apiary was randomly divided into two populations which served as controls and those which received the product Li-Mo.sub.2O.sub.4-EDTA (76 controls, 75 test hives).

[0212] The product Li-Mo.sub.2O.sub.4-EDTA was introduced between 25 and 28 Oct. 2019 by syringe in the form of a concentrated aqueous solution (8 g/L) in a sugar syrup into the feeding frame. 0.5 mL of this solution was introduced and dispersed in 3.5 liters of sugar syrup at 65% sugar and introduced into the hive. Each hive thus received a single overall dose of 4 mg of Li-Mo.sub.2O.sub.4-EDTA. The control hives only received sugar syrup.

[0213] The surviving colonies were counted on Jan. 7, 2020. A total of 73 colonies out of the initial 151 died, that is an overall mortality of 48.3%.

[0214] The mortality of control hives amounted to 47 colonies lost out of 76, that is 61.8% winter mortality, which is a standard result in this region.

[0215] The mortality of the test hives treated with 4 mg of Li-Mo.sub.2O.sub.4-EDTA was 26 colonies lost out of the initial 75, that is 34.7% winter mortality, which is a very significant drop in comparison to the control hives.

[0216] In conclusion, the molybdenum complex of the present invention makes it possible to significantly lower the winter mortality of bee colonies.

4.4. 4.SUP.th Test in the Hives

[0217] A second treatment campaign was carried out in the autumn-winter 2020 in apiaries located in California, near San Francisco. This region is particularly vulnerable to winter mortality of bee colonies which can reach as high as 80% in some years.

[0218] 220 hives distributed over 13 different apiaries in California to the south of San Francisco were selected. Each apiary comprised between 8 and 24 hives. The bees were of different types: 165 hives of Wildflower bees (VSH-Italian) and 55 hives of Beeweaver bees (VSH-Carniolan).

[0219] The hives were Langstroth 2 body type hives (15 frames compared to 10 for Dadant hives in Europe). Each apiary was randomly divided into 4 batches of hives having a total of: [0220] Batch: 55 test hives [0221] Batch 2: 55 hives that received Li-Mo.sub.2O.sub.4-EDTA on Sep. 20, 2020 [0222] Batch 3: 55 hives that received Li-Mo.sub.2O.sub.4-EDTA on Oct. 12, 2020 [0223] Batch 4: 55 hives that received Li-Mo.sub.2O.sub.4-EDTA on Sep. 20, 2020 and Oct. 12, 2020.

[0224] 1 apiary only contained Wildflower type bees. 2 apiaries only contained Beeweaver type bees. The other 10 apiaries comprised Wildflower and Beeweaver bees that were evenly distributed between the 4 batches.

[0225] The product Li-Mo.sub.2O.sub.4-EDTA was introduced on Sep. 20, 2020, and/or on Oct. 12, 2020 by syringe in the form of a concentrated aqueous solution (0.8 g/L) in a sugar syrup into the frame feeder. 10 mL of this solution were introduced and dispersed in 3.5 liters of sugar syrup at 65% sugar and introduced into the hive. Each hive thus received a single overall dose of 8 mg of Li-Mo.sub.2O.sub.4-EDTA by feeding. The control hives only received sugar syrup.

[0226] The dead colonies were counted on Dec. 31, 2020. A total of 28 colonies were dead on 31/12/2020, that is 12.73% of the total. The mortality rate is identical for Wildflower bees (21/165) and Beeweaver bees (7/55). The mortality per batch is distributed as follows: [0227] Batch1: 15 hives/55 hives, that is 27.3% losses [0228] Batch 2: 0 hive/55 hives, that is 0% losses [0229] Batch 3: 10 hives/55 hives, that is 18.2% losses [0230] Batch 4: 3 hives/55 hives, that is 5.5% losses.

[0231] In conclusion, the molybdenum complex of the present invention makes it possible to significantly lower the winter mortality of the bee colonies in California at an overall dose of 8 mg per colony. Starting feeding early enough in September makes it possible to obtain significantly higher effects to feeding later.