WO2012038945A2 - Lutte contre les acariens - Google Patents

Lutte contre les acariens Download PDF

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Publication number
WO2012038945A2
WO2012038945A2 PCT/IE2011/000049 IE2011000049W WO2012038945A2 WO 2012038945 A2 WO2012038945 A2 WO 2012038945A2 IE 2011000049 W IE2011000049 W IE 2011000049W WO 2012038945 A2 WO2012038945 A2 WO 2012038945A2
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WO
WIPO (PCT)
Prior art keywords
bees
bpq
buparvaquone
mites
composition
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Application number
PCT/IE2011/000049
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English (en)
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WO2012038945A3 (fr
Inventor
Nicholas Mchardy
Ronan Smith
Paul Declan Brady
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Bimeda International Pharmaceutical Limited
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Priority to GB1302471.6A priority Critical patent/GB2495683A/en
Publication of WO2012038945A2 publication Critical patent/WO2012038945A2/fr
Publication of WO2012038945A3 publication Critical patent/WO2012038945A3/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N35/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having two bonds to hetero atoms with at the most one bond to halogen, e.g. aldehyde radical
    • A01N35/06—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having two bonds to hetero atoms with at the most one bond to halogen, e.g. aldehyde radical containing keto or thioketo groups as part of a ring, e.g. cyclohexanone, quinone; Derivatives thereof, e.g. ketals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/14—Ectoparasiticides, e.g. scabicides

Definitions

  • the invention relates to controlling Varroa mites that parasitise honey bees.
  • Varroa mites are a severe health hazard to bees, killing or debilitating them, frequently causing the demise of the whole colony. This has a serious impact on honey production and the pollinating capacity of bees.
  • Varroa mites also transmit various viral, bacterial and fungal infections to bees. Thus, control of the mites can produce indirect benefits to the health and productivity of the bees.
  • miticides are applied to bees within the hive in a number of different ways.
  • the methods include sprays, smokes, aerosols, dusts, vaporisers, brushes, etc to apply them to the bees and 'brood combs' and thus to the mites that parasitise them. They are applied routinely as preventive treatments, mostly in the spring and autumn when the bees are confined to the hives. This also reduces the risk of contamination of honey and beeswax.
  • the aim is to minimise the infestation of bee larvae, which are particularly susceptible to such parasites. Once attached, the mites commonly remain on the bees for life.
  • mites may attach to each bee so the weight of mites may exceed 10% of the weight of the uninfested bee.
  • miticides are often applied therapeutically, regardless of the time of year, with greatly increased risk of contamination of honey and wax.
  • a method for controlling mites which comprises administering to the honeybees a non-toxic miticidally effective amount of burparvaquone.
  • said administering comprises feeding buparvaquone to the bees.
  • Said administering may comprise feeding buparvaquone in a concentrated sugar solution to the bees.
  • the buparvaquone is in the form of extremely finely divided particles.
  • the invention provides a composition for controlling mites parasitising honeybees comprising a commonly used diet supplement for honeybees which contains a miticidal amount of buparvaquone that is entirely non-toxic for bees and bee larvae.
  • the bee diet may comprise an aqueous solution of a sugar, most commonly sucrose, at a high concentration, commonly around 66%.
  • the diet may also contain other nutrients and other supplements such as minerals, vitamins and pharmaceutical substances, for example bactericides and fungicides.
  • the buparvaquone may be administered in a concentration of from 0.00005 to 0.1 ⁇ g/mL, preferably from 0.0005 to 0.1 ⁇ g/mL, most preferably from about 0.0005 to about 0.05 ⁇ g/mL.
  • the composition may comprise a carrier for buparvaquone.
  • the carrier may comprise a solvent such as N-methyl-pyrollidone (NMP).
  • NMP N-methyl-pyrollidone
  • the mean particle size of a 0.5 mcg/mL dispersion of Buparvaquone in 66% w/v sucrose is approximately 6 micron, while all particles are below 20 micron.
  • Buparvaquone is pre-diluted in a suitable solvent such as NMP to obtain an homogenous solution that can easily be dispensed and dispersed into the slightly thick sucrose syrup. As soon as the Buparvaquone solution gets in contact with water, it forms a fine yellowish precipitate (practically invisible) that is easily dispersed in the sucrose solution. This dispersion has been found to have particles of Buparvaquone with mean size at 6 micron, and all particles below 20 micron.
  • N-methylpyrrolidone is a suitable solvent.
  • other organic solvents could potentially be used to provide a pre-concentrated solution of buparvaquone.
  • organic solvents such as propylene glycol, glycerol formal and dimethylacetamide could be potential candidates. Indeed, most organic solvents dissolve buparvaquone to some extent. The simplest ones, such as methanol or ethanol, dissolve a little, dimethyl sulphoxide is better, but NMP and similar solvents, such as dimethyl formamide, are best. In general, solvents that dissolve BPQ are readily miscible with oils.
  • buparvaquone is highly effective in killing the mites while being entirely safe for bees, both larvae and adults.
  • Buparvaquone can be applied either as a spray or by inclusion in the concentrated sugar solution that is fed to bees throughout the active season of the bees. As the bees ingest the medicated feed, they take up the buparvaquone, which then crosses from the gut lumen into the haemolymph. The mites ingest the buparvaquone and are affected by it, as they suck the haemolymph. Because buparvaquone is not present on the surface of the bees, as it would be if it was applied as a topical treatment such as a dust, and it is not present in a form such as an aerosol that would pervade the whole hive, the chances of it contaminating honey and wax are minimised.
  • any buparvaquone that is not absorbed from the digestive tract of the bee and which is voided in the faeces will not contaminate honey or wax because bees do not defecate on either the honey-bearing combs or brood combs, but elsewhere within or outside the hive.
  • Affected mites following treatment with buparvaquone, quickly detach from the bees and larvae. They may then survive for a few hours but they appear to be paralysed and are unable to reattach. This is in marked contrast to known products. Though such known products may kill some mites, those that detach and survive may recover and re-attach. The effect of some products, apparently, is solely to cause detachment but not death of the mites. These mites are readily able to re-attach, particularly if detachment occurs within the hive, and most importantly, if it occurs on or around the brood combs.
  • buparvaquone is highly effective for the prevention and treatment of Varroa mite infesations of bees, whilst being entirely without adverse effect on the bees themselves. It can be administered safely and conveniently simply by incorporation in bee diet. Furthermore, the risk of contamination of honey and beeswax with product residues is minimal and, in any case, the buparvaquone is of extremely low toxicity to mammals. Thus, it presents no hazard to beekeepers or consumers of honey or beeswax. In addition, because buparvaquone has no insecticidal or other toxic properties, its environmental effect on insects and other animals that may come into contact with it is negligible.
  • Fig. 2 bee mortality per cage at each concentration of product over time
  • Fig. 3 bee mortality per cage at different concentrations of solvent over time
  • Fig. 4 mean bee mortality at different treatment concentrations over time;
  • Fig. 5 mean been mortality at different solvent concentrations over time;
  • Fig. 6 the mean volume of product and solvent treated food consumed by cage bees over the 16 day treatment period
  • Fig. 7 the percentage survival of caged bees sprayed with different concentrations of BPQ A and solvent over a 7 day period
  • Fig. 8 the mean number of bees surviving when sprayed with an aqueous solution of product or solvent over a 7 day period;
  • Fig. 9 mean survival rate of Varroa mites over time when treated with different concentrations of BPQ;
  • Fig. 10 the mean percentage efficacy of BPQ fed in a sucrose solution at different concentrations
  • Fig. 1 1 the percentage efficacy of BPQ fed in a sucrose solution at different concentrations in each of the replicates;
  • Fig. 12 the number of bees surviving on day 5 in each of the replicates at different concentrations;
  • Fig. 13 the mean percentage efficacy of BPQ sprayed as an aqueous solution
  • Fig. 14 the percentage efficacy of the BPQ against the Varroa mite when sprayed on adult bees as an aqueous solution
  • Fig. 15 is a chart of the mean volume of food consumed during a seven day trial period in each of 6 test groups.
  • Fig. 16 is a chart of the percentage survival of bees in cages at different test concentrations during a seven day trial period.
  • the active ingredient in the invention is buparvaquone (BPQ), a hydroxynaphthoquone compound that has been used for the treatment of certain tick-transmitted diseases of farm livestock.
  • BPQ may be supplied already formulated into sugar-based bee diet or as a solution in an organic solvent such as N-methyl-pyrrolidone (NMP) or dimethylsulphoxide (DMSO) for incorporation into bee diet by diet suppliers or bee owners.
  • NMP N-methyl-pyrrolidone
  • DMSO dimethylsulphoxide
  • Bee diet is typically a 66% aqueous solution of sucrose.
  • Commercially-supplied bee diets may contain a number of other additives, including minerals, vitamins, other medicaments etc.
  • Buparvaquone is extremely stable and non-reactive, and is therefore unlikely to react in any way with any other additives in the diet.
  • BPQ is completely insoluble in water and it immediately falls out of solution in organic solvents on contact with water. If applied to Varroa mites in its solid form it has no miticidal effect because the particles are too large to pass across the cuticle of the mites. Similarly, if fed to bees in its solid form, it is not absorbed into the haemolymph in sufficient amounts to kill mites that ingest the bee's haemolymph.
  • the buparvaquone is presented as a solution in, for example, NMP because in this form, when used as a spray, it readily crosses the mite's cuticle or if ingested by the bee it would enter the haemolymph readily.
  • the BPQ composition of the invention is preferably supplied as an aqueous suspension of extremely finely-divided particles of BPQ, or a nano-suspension of BPQ which behaves almost as a solution.
  • Particles of sufficient fineness can be prepared in several ways, commonly used in pharmaceutical formulations, such as micronisation. However, the nature of BPQ lends itself to a simpler and less expensive process, which is preferred.
  • the BPQ is first dissolved in a solvent such as NMP or DMSO and this is sprayed into water. On contact with the water, the BPQ immediately precipitates as fine particles.
  • the fineness of the particles can be varied in at least three ways: concentration of BPQ in the solvent; rate of addition of solution of BPQ to the water; and/or aggitation of the water by e.g. mechanical or ultrasonic means.
  • the finest particles can be generally produced by spraying a dilute solution of BPQ into rapidly agitated water. Coalescence of these ultra-fine particles into larger particles can be prevented by known methods.
  • Some of the efficacy and safety studies conducted to date have used aqueous suspensions of BPQ produced crudely in this way. Refinement and standardisation of the production method may significantly improve product performance by minimising particle size, thereby increasing its concentration in haemolymph when fed to bees, or by maximising the amount of BPQ absorbed from the bee's intestine into the haemolymph thereby minimising the amount of BPQ that must be ingested by the bees, or by increasing penetration of mite cuticle or entry into respiratory spiracles when applied as a spray.
  • BPQ miticidal effect
  • BPQ When used as a treatment for tick-transmitted protozoal diseses of livestock, its action is as an electron transport inhibitor but the electron transport systems of protozoa and mites are very dissimilar. Mites that detach from bees following treatment with BPQ become paralysed before death. This might suggest a neurological effect (in common with that of other varroacides such as pyrethroids and organophosphates).
  • BPQ has no insecticidal effect.
  • the miticidal action of BPQ therefore, may be unique, but its nature remains unknown.
  • the toxicology of BPQ to mammals has revealed no neurotoxic effect whatsoever, even at dosages many thousands of times higher than those in the present invention.
  • the active ingredient is buparvaquone, (BPQ), supplied as a formulated product (BPQ A) and as a powder (BPQ B).
  • BPQ buparvaquone
  • BPQ was presented in two forms: (a) a liquid at a concentration of 50mg/mL, (BPQ A), and
  • Example 1 Toxicity of BPQ A to honeybees when fed in a sugar syrup solution
  • Hoarding cages consisted of plastic tumblers (600mL) with an opening diameter of 84mm. The tumblers were paired with standard plastic petri-dishes and glass scintillation vials. To allow sufficient ventilation, 33 holes, approximately 3mm in diameter were made in the sides of each tumbler, in addition to two 30mm x 30mm holes fitted with nylon mesh. A circular hole was cut out of the bottom of the cup, which produced an opening large enough to allow a lidded scintillation vial to pass through the hole. Two holes were drilled into the lids of the scintillation vials with a 1mm bit and these vials were filled a 1 : 1 sugar solution, together with different concentrations of the solvent and product. A piece of disposable incontinence underpad was cut to be slightly larger than the petri-dish so that drips from the scintillation vial would be absorbed.
  • Solvent in suganwater feed (equivalent solvent concentration to that in BPQ A-sugar/water solutions)
  • BPQ A treated groups peaks in bee mortality were recorded on Day 8 and Day 14 (Fig. 2, 4), which was the day after administering the product. This was not as apparent in the solvent treated group, but mortality did increase over time reaching a peak on Day 1 1 - 14 (Fig. 3, 5). Mortality also increased in the untreated control group at the end of the treatment period. Caged bees consumed the solvent and syrup (control diet) at a slightly faster rate than the caged bees fed BPQ A, consequently required feeding on Day 5, Day 12 and Day 16. BPQ A treated bees were fed on Days 7, 13 and 16. The total consumption of food is given in Fig. 6.
  • Example 2 Toxicity of BPQ A in an aqueous solution when sprayed onto honeybees
  • Hoarding cages were prepared as described in example 1 .
  • BPQ A and solvent were made up at similar concentrations as described in example 1 , but in an aqueous solution, rather than a sugar /syrup solution. The latter was used to prevent bees becoming sticky when the solution is sprayed directly onto bees.
  • Three replicates were analysed at each concentration of BPQ A and solvent, together with a control group which were only sprayed with water.
  • Bees in all cages were initially fed with l OmL of suganwater syrup using a gravity feeder and placed in an incubator at 32°C. Bees were fed further syrup when required and the percentage survival of bees in each treatment group were monitored on a daily basis over a 7 day period.
  • Solvent in suganwater feed (equivalent solvent concentration to that in BPQ A -sugar/water solutions)
  • the present cage experiment indicates that spraying the bees with BPQ A in an aqueous solution at the stated concentrations has no negative effects on survival rate (Figs. 7 and 8) or feeding behaviour of honeybees.
  • Example 3 Toxicity of BPQ A in an aqueous solution when sprayed on honeybee larvae
  • Results Results indicate that spraying BPQ A or solvent had no negative effects on honeybee larvae survival.
  • the mean survival rate ( ⁇ se) for the BPQ A, the solvent and the control were 86.1 ⁇ 1.19, 87.7 ⁇ 1.02 and 86.0 ⁇ 3.55 respectively.
  • Example 4 Toxicity of BPQ B to honeybee larvae when fed treated syrup
  • BPQ B BPQ B, dissolved in the solvent (N-methyl pyrollidone) and diluted in an aqueous solution.
  • Apidae boxes were used to raise young queens and when virgin queens started to lay an area 6cm x 3cm were marked and missing cells were noted.
  • the miniature colony was fed treated syrup, which was made by dissolving BPQ B powder in solvent, followed by serial dilutions using a sucrose solution (2 parts sugar: 1 part water). The concentrations administered are given below. Two replicates were carried out for the product, while 1 colony (apidae) was used for each of the solvent concentrations.
  • Solvent in suganwater feed (equivalent solvent concentration to that in BPQ B -sugar water solutions)
  • Treating developing larvae with BPQ showed no negative effects on their development and the queen continued to lay during the treatment period.
  • the percentage emergence in the solvent treated colonies was lower than BPQ B, but the number of missing cells in this group was also higher, thus indicating a possible problem with the fertility of the eggs in these colonies.
  • Varroa mites only feed on haemolymph of larvae and adult bees and thus would not ingest BPQ in a syrup/water solution. Therefore, it was necessary to identify a volume sufficient to permeate through the cuticle, but not drown the mites. After preliminary investigation it was concluded that 3 ⁇ /10 mites/eppendorf tube gave the maximum viability and the latter was used for this trial.
  • BPQ A/solvent A total of 3 ⁇ of BPQ A/solvent was pipetted into each eppendorf tube. Ten mites taken from purple eye drone pupae were submerged in the liquid by tapping the tube gently and a fresh pupa was placed in each tube. 20 pinholes were made in each of the tubes and all samples were placed in the incubator at 30°C. Three replicates were made for each concentration and water was used in the control group. Concentrations of BPQ A and solvent were as described above (Section 1 .1 ). The number of damaged or dead mites was counted on a daily basis. Damaged mites were mites that would not move in a coordinated fashion when touched gently 3 times with a fine brush.
  • the survival curves indicate that at higher concentrations of BPQ, Varroa mite mortality is higher and more rapid than at lower concentrations (Fig. 9).
  • the mean number of live mites on day 3 was 3.33 ⁇ 2.4 (mean ⁇ 2.4) while at concentrations 0.0005 ⁇ g/mL (f) and 0.00005 ⁇ g/mL (g) the mean number of live mites on the same day was 7.66 ⁇ 1.33 and 8.66 ⁇ 1.73 respectively. It should be noted that not all mites were dead, but were damaged and immobile and consequently not able to stay attached to bees. This is typical of many treatments.
  • Example 6 Toxicity of BPQ B to Varroa mites on honeybees when fed treated sucrose solution
  • a total of 20 newly emerged Varroa infested bees were placed in hoarding cages.
  • the cages were prepared as described in Section 1 .0, except that the air vents were reduced in size using parafilm to prevent the Varroa mites escaping during the test period.
  • the treatment solution was made by dissolving BPQ B in solvent, followed by two aqueous serial dilutions producing a concentration of 50C ⁇ g/mL and 5 ⁇ g/mL. From the latter, further serial dilutions were carried with sucrose solution producing the following concentrations:
  • % efficacy mites knocked from treatment/ total mites (treatment+Apistan)* 100
  • Example 7 Toxicity of BPQ B to Varroa mites on honeybees when sprayed with an aqueous solution of the active ingredient
  • % efficacy mites knocked from treatment/ total mites (treatment+Apistan)* 100 Results
  • Solutions tested contain Buparvaquone dispersed in a 66% sucrose solution.
  • the mean particle size of a 0.5 mcg/mL dispersion of Buparvaquone in 66% w/v sucrose is approximately 6 micron, while all particles are below 20 micron
  • sucrose solution 660 g of sucrose are dissolved in approximately 500 mL of purified water (heating at 40-50°C if necessary to facilitate dissolution). This is then brought up to 1 L with purified water.
  • Solution 0.05 mcg/mL 200 ⁇ of BPQ concentrate solution is added to 500 mL of purified water with gentle and continuous stirring to maintain insoluble BPQ in suspension. 660 grams of sucrose is added, and the solution is warmed to 40-50°C until all sucrose is dissolved. The final solution is then brought to 1 L with purified water (0.05 ⁇ g/mL)
  • Solution 0.005 mcg/mL 100 mL of Solution 0.05 mcg/mL is added to 900 mL of 66% w/v sucrose solution and mixed thoroughly to ensure homogenization.
  • Solution 0.0005 mcg/mL 100 mL of Solution 0.005 mcg/mL is added to 900 mL of 66% w/v sucrose solution and mixed thoroughly to ensure homogenization. Hoarding cages:
  • Hoarding cages consisted of plastic tumblers (600ml) with an opening diameter of 84mm. The tumblers were paired with standard plastic petri-dishes and glass scintillation vials. To allow sufficient ventilation, 33 holes, approximately 3mm in diameter were made in the sides of each tumbler, in addition to two 30mm x 30mm holes fitted with nylon mesh. A circular hole was cut out of the bottom of the cup, which produced an opening large enough to allow a lidded scintillation vial to pass through the hole. A piece of disposable incontinence under-pad was cut to be slightly larger than the petri-dish so that drips from the scintillation vial would be absorbed. Fresh water was made available to bees throughout the trial using a gravity feeder attached to the cage. Food was administered through a gravity feeder placed on top of the hoarding cage.
  • the product is highly toxic to free living Varroa mites
  • Varroa mites attached to bees can be attacked via a bee feed route or throu: topical application
  • Topical application is -40% effective
  • Mites detached through treatment with BPQ may be incapable of reattachment contrast to mites detached in the caged control study

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
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  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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  • Tropical Medicine & Parasitology (AREA)
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  • Agricultural Chemicals And Associated Chemicals (AREA)

Abstract

L'invention concerne un procédé de lutte contre les acariens Varroa qui parasitent les abeilles, qui comprend l'administration aux abeilles d'une quantité miticide efficace et non toxique de buparvaquone. Le procédé peut comprendre l'alimentation des abeilles avec de la buparvaquone dans une solution sucrée.
PCT/IE2011/000049 2010-09-23 2011-09-09 Lutte contre les acariens WO2012038945A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
GB1302471.6A GB2495683A (en) 2010-09-23 2011-09-09 Control of mites

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US34472910P 2010-09-23 2010-09-23
US61/344,729 2010-09-23

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Publication Number Publication Date
WO2012038945A2 true WO2012038945A2 (fr) 2012-03-29
WO2012038945A3 WO2012038945A3 (fr) 2012-07-05

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104535680A (zh) * 2014-12-25 2015-04-22 河北科星药业有限公司 测定布帕伐醌注射液中布帕伐醌含量的方法

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Publication number Priority date Publication date Assignee Title
US2572946A (en) * 1949-11-19 1951-10-30 Du Pont Composition comprising 2-aliphatic-3-hydroxy-1, 4 napthoquinone and method for controlling mites and aphids
DE3801743A1 (de) * 1987-07-03 1989-01-19 Bayer Ag Schaedlingsbekaempfungsmittel auf basis von substituierten 1,4-naphthochinonen und neue substituierte 1,4-naphthochinone
GB9612403D0 (en) * 1996-06-13 1996-08-14 Sandoz Ltd Organic compounds
US7767234B2 (en) * 2006-03-31 2010-08-03 John I. Haas, Inc. Compositions and methods for controlling a honey bee parasitic mite
ITMI20070941A1 (it) * 2007-05-09 2008-11-10 Chimico Internazi0Nale S P A L Procedimento per la preparazione di naftochinoni trans2,3-disostituiti
EP2433624B1 (fr) * 2010-09-23 2013-08-21 Bimeda International Pharmaceuticals Limited Utilisation du buparvaquone pour contrôler les acariens parasitisants les abeilles

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Title
None

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104535680A (zh) * 2014-12-25 2015-04-22 河北科星药业有限公司 测定布帕伐醌注射液中布帕伐醌含量的方法

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GB2495683A (en) 2013-04-17
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