DESTRUCTOR MITES

[001] This invention relates to compositions comprising a hydroalcoholic solution comprising a Quassia amara extract, the solution comprising at least 80 %v/v of at least one alcohol, as well as to the use of such compositions in controlling Varroa destructor mites, and methods for preparing the solution.

[002] Background

[003] Varroa destructor mites (hereafter also referred to as Varroa mites) are parasitic mites that attack and feed on honey bees. The Varroa mite is considered the parasite with the most pronounced economic impact on the beekeeping industry. Varroa mites are commonly found in bee colonies globally, resulting in major economic losses for both beekeepers and farmers of crops, which rely on pollination. Detection often results in intense activities to control the spread of the mite and prevent the infestation of colonies and collapse of the honey industry.

[004] The current leading method of controlling Varroa mites in honey bee colonies is the application, usually as a spray, of oxalic acid. However, a problem with the preparation of the oxalic acid solution is that it is toxic, meaning that it has to be handled with extreme care.

[005] WO 2016/185379 A1 and WO 2016/185380 A1 relate the use of hydroxyapatite as a carrier of bioactive substances, including Quassia amara, for application to plants with vascular diseases. WO 2010/089567 A2 relates to herbal compositions comprising various components, including Quassia, for the control of hematophagous parasites. This document does not mention Varroa mites, and the compositions are prepared by cell culturing.

[006] Esquivel, F., Mejia, L., Flores, B., Duttmann, C., Castillo, G., Arguello, O., & Demedio, J. (2014). Evaluation of Quassia amara as a treatment against varroosis in three apiaries in the municipality of Leon, Nicaragua. Universitas (Leon): Scientific Journal of the UNAN Leon , 5(1), 100-106, describes a study which sought to evaluate the efficacy of an aqueous solution comprising a Quassia amara extract against Varroa mites.

[007] An improved composition for use in controlling Varroa mites in honey bee colonies has been sought.

[008] Statement of invention

[009] This invention relates to a composition comprising:

(a) a hydroalcoholic solution comprising a Quassia amara extract, wherein the solution comprises at least 80 %v/v of at least one alcohol, and

(b) an aqueous sucrose solution.

[0010] In particular, the hydroalcoholic solution may comprise at least 90 %v/v of the at least one alcohol, even more particularly at least 95 %v/v of the at least one alcohol. More particularly, the at least one alcohol may comprise ethanol and/or isopropyl alcohol, even more particularly ethanol.

[0011] In the context of the invention, the phrase "hydroalcoholic solution comprising a Quassia amara extract" is used to refer to the solution obtained when a solvent, for example a mixture of water and at least one alcohol, is contacted with Quassia amara plant material for a defined period of time in order to extract components from the Quassia amara plant material.

[0012] In particular, the composition may comprise 0.1-5 parts by volume of the hydroalcoholic solution as defined above, more particularly 0.5-2 parts by volume, even more particularly about 1 part by volume. In particular, the composition may comprise 15-25 parts by volume of the aqueous sucrose solution, more particularly 18-22 parts by volume, even more particularly about 19 parts by volume of the aqueous sucrose solution.

[0013] In particular, the aqueous sucrose solution may comprise 40-60 wt% sucrose, more particularly 45-55 wt% sucrose, even more particularly about 50 wt% sucrose. [0014] In addition, this invention relates to a spray comprising a spray device and a composition as defined above. In particular, the spray may be for use in controlling Varroa destructor mites. More particularly, the spray may comprise a chamber which contains the composition as defined above. In particular, the spray device may be a hand pressure spray device.

[0015] This invention also relates to a method of controlling Varroa destructor mites comprising applying to a bee colony a hydroalcoholic solution comprising a Quassia amara extract, wherein the solution comprises at least 80 %v/v of at least one alcohol. In addition, this invention relates to a method of controlling Varroa destructor mites comprising applying to a bee colony a composition as defined above. This invention also relates to a method of treating Varroa destructor mite infestation in a bee colony comprising applying to the bee colony a hydroalcoholic solution comprising a Quassia amara extract, wherein the solution comprises at least 80 %v/v of at least one alcohol. In addition, this invention relates to a method of treating Varroa destructor mite infestation in a bee colony comprising applying to the bee colony a composition as defined above. In particular, the applying may be by spraying.

[0016] This invention also relates to the use of a hydroalcoholic solution comprising a Quassia amara extract, wherein the solution comprises at least 80 %v/v of at least one alcohol, in controlling Varroa destructor mites. In addition, this invention relates to a hydroalcoholic solution comprising a Quassia amara extract, wherein the solution comprises at least 80 %v/v of at least one alcohol, for use in controlling Varroa destructor mites. This invention also relates to the use of a hydroalcoholic solution comprising a Quassia amara extract, wherein the solution comprises at least 80 %v/v of at least one alcohol, in treating Varroa destructor mite infestation in a bee colony. In addition, this invention relates to a hydroalcoholic solution comprising a Quassia amara extract, wherein the solution comprises at least 80 %v/v of at least one alcohol, for use in treating Varroa destructor mite infestation in a bee colony.

[0017] This invention also relates to the use of a composition as defined above in controlling Varroa destructor mites. In addition, this invention relates to a composition as defined above for use in controlling Varroa destructor mites. This invention also relates to the use of a composition as defined above in treating Varroa destructor mite infestation in a bee colony. In addition, this invention relates to a composition as defined above for use in treating Varroa destructor mite infestation in a bee colony.

[0018] This invention also relates to a method for preparing a hydroalcoholic solution comprising a Quassia amara extract, wherein the solution comprises at least 80 %v/v of at least one alcohol, the method comprising the steps of:

(a) adding a mixture of water and at least one alcohol, the mixture comprising at least 80 %v/v of the at least one alcohol, to Quassia amara plant material,

(b) allowing the mixture of water and the at least one to alcohol contact the Quassia amara plant material for a defined period of time, and

(c) removing the Quassia amara plant material from the mixture to provide a hydroalcoholic solution comprising a Quassia amara extract.

[0019] In particular, the at least one alcohol may comprise ethanol and/or isopropyl alcohol, more particularly ethanol. In particular, the hydroalcoholic solution may comprise at least 90 %v/v of the at least one alcohol, more particularly at least 95 %v/v of the at least one alcohol. When the hydroalcoholic solution is to be used to prepare a composition comprising an aqueous sucrose solution (as described above), the amount of Quassia amara plant material to which the mixture of water and at least one alcohol was added may be 100-1000 mg of plant material per ml of the mixture, more particularly 250-750 mg/ml, even more particularly 400-600 mg/ml. When the hydroalcoholic solution is to be used to prepare a composition comprising a vegetable oil (as described below), the amount of Quassia amara plant material to which the mixture of water and at least one alcohol was added may be 50-300 mg of plant material per ml of the mixture, in particular 75-275 mg/ml, more particularly 150-250 mg/ml, even more particularly 175-225 mg/ml.

[0020] In particular, the defined period of time may be 6-48 hours, more particularly 12-36 hours, even more particularly 18-30 hours.

[0021] In addition, this invention relates to a hydroalcoholic solution comprising a Quassia amara extract, wherein the solution comprises at least 80 %v/v of at least one alcohol, obtainable by the method defined above. The invention also relates to the compositions, methods and uses defined above utilising such a hydroalcoholic solution.

[0022] This invention also relates to a method for preparing a composition as defined above, the method comprising the steps of:

(a) providing a hydroalcoholic solution comprising a Quassia amara extract, wherein the solution comprises at least 80 %v/v of at least one alcohol, and

(b) mixing 0.1-5 parts by volume of the hydroalcoholic solution with 15-25 parts by volume of an aqueous sucrose solution.

[0023] In particular, step (b) may comprise mixing 0.5-2 parts by volume, more particularly about 1 part by volume, of the hydroalcoholic solution. More particularly, step (b) may comprise mixing 18-22 parts by volume, more particularly about 19 parts by volume, of the aqueous sucrose solution. The composition of the aqueous sucrose solution may be as defined above.

[0024] In particular, step (a) may comprise preparing a hydroalcoholic solution by the method as defined above.

[0025] In addition, this invention relates to a composition obtainable by the method defined above. The invention also relates to the methods and uses defined above utilising such a composition.

[0026] In a further embodiment, this invention relates to a composition comprising:

(a) a hydroalcoholic solution comprising a Quassia amara extract, wherein the solution comprises at least 80 %v/v of at least one alcohol, and

(b) a vegetable oil.

[0027] Vegetable oils are defined as oils extracted from seeds or from other parts of fruits. Examples of seed oils include soybean oil and grape seed oil. Examples of oils from other parts of fruits include olive oil and palm oil. In some embodiments, the vegetable oil may be olive oil, although other vegetable oils can be used.

[0028] In particular, the hydroalcoholic solution may comprise at least 90 %v/v of the at least one alcohol, even more particularly at least 95 %v/v of the at least one alcohol. More particularly, the at least one alcohol may comprise ethanol and/or isopropyl alcohol, even more particularly ethanol.

[0029] In particular, the composition may comprise 80-90 % by volume of the hydroalcoholic solution as defined above, more particularly 82-88 % by volume, even more particularly 84-85 % by volume. In particular, the composition may comprise IQ- 20 % by volume of the vegetable oil, more particularly 12-18 % by volume, even more particularly 14-15 % by volume of the vegetable oil.

[0030] In particular, the composition may be in the form of an emulsion. In some embodiments, the composition may additionally comprise an emulsifier. In particular, the emulsifier may comprise castor oil. Suitable emulsifiers include Rimulgan® and LV42®. In particular, the composition may comprise 0.2-1.1 % by volume of the emulsifier, more particularly 0.4-0.9 % by volume, even more particularly 0.6-0.7 % by volume of the emulsifier.

[0031] In addition, this invention relates to a porous material, in particular a sponge, impregnated with a composition as defined above.

[0032] This invention also relates to a method of controlling Varroa destructor mites comprising contacting a bee colony with the composition of the further embodiment as defined above. This invention also relates to a method of treating Varroa destructor mite infestation in a bee colony comprising contacting the bee colony with the composition of the further embodiment as defined above. In particular, the contacting may be by evaporation.

[0033] This invention also relates to the use of the composition of the further embodiment as defined above in controlling Varroa destructor mites. In addition, this invention relates to the composition of the further embodiment as defined above for use in controlling Varroa destructor mites. This invention also relates to the use of the composition of the further embodiment as defined above in treating Varroa destructor mite infestation in a bee colony. In addition, this invention relates to the composition of the further embodiment as defined above for use in treating Varroa destructor mite infestation in a bee colony. [0034] This invention also relates to a method for preparing a composition (i.e. that of the further embodiment) as defined above, the method comprising the steps of:

(a) providing a hydroalcoholic solution comprising a Quassia amara extract, wherein the solution comprises at least 80 %v/v of at least one alcohol, and

(b) mixing 80-90 % by volume of the hydroalcoholic solution with 10-20 % by volume of a vegetable oil.

[0035] In particular, step (b) may comprise mixing 82-88 % by volume, more particularly 84-85 % by volume, of the hydroalcoholic solution. More particularly, step (b) may comprise mixing 12-18 % by volume, more particularly 14-15 % by volume, of the vegetable oil. The vegetable oil may be as defined above.

[0036] In particular, step (a) may comprise preparing a hydroalcoholic solution by the method as defined above.

[0037] In addition, this invention relates to a composition obtainable by the method defined above. The invention also relates to the methods and uses defined above utilising such a composition.

[0038] Brief description of the drawings

[0039] This invention will be further described by reference to the following Figures which are not intended to limit the scope of the invention claimed, in which:

Figure 1 shows a graph of Varroa mite Infestation Rate (IR %) in adult bees before and after the application of a Quassia amara aqueous solution,

Figure 2 shows a graph of Varroa mite Infestation Rate (IR %) in adult bees before and after the application of a Quassia amara hydroalcoholic solution, Figure 3 shows a graph of Varroa mite Extension of Invasion (El %) in brood cells before and after the application of a Quassia amara aqueous solution, Figure 4 shows a graph of Varroa mite Extension of Invasion (El %) in brood cells before and after the application of a Quassia amara hydroalcoholic solution,

Figure 5 shows a graph comparing Varroa mite Infestation Rate (IR %) in adult bees for the aqueous and hydroalcoholic solutions, both before and after the application of the Quassia amara solution, Figure 6 shows a graph depicting the correlation between beehive condition and reduction in Infestation Rate (IR),

Figure 7 shows a graph comparing the average number of dead Varroa mites between the different experimental groups during the 8 hours post-exposure to the evaporation formulation,

Figure 8 shows a line graph comparing the average number of dead Varroa mites between the different experimental groups during the 8 hours postexposure to the evaporation formulation,

Figure 9 shows a line graph comparing the percentage of dead Varroa mites between the different experimental groups during the 8 hours post-exposure to the evaporation formulation, and

Figure 10 shows comparing Varroa mite Infestation Rate (IR %) in adult bees for the evaporation formulation (referred to as "oasis"), both before and after application.

[0040] Detailed description

[0041] Spray formulation

[0042] Preparation for In-vivo testing

[0043] Study site: In Nicaragua, the dry season starts at the end of November and the beginning of December. This is the time when flowering starts and so it is the beginning of the productive period for beekeepers. Testing was carried out before the start of this productive period, i.e. between May and November. Several beekeepers were contacted in order to find one with a small apiary with Varroa infested beehives which was not yet ready to enter the productive period. After checking the conditions of the apiary, it was decided to work with seven beehives that consisted of two boxes each. Before the application of the compositions, the strength of each beehive was estimated for later analysis.

[0044] A hydroalcoholic solution comprising an extract of Quassia amara was prepared as follows. 2 kg of branches of the Quassia amara tree were weighed. The dry branches were cut into pieces approximately 10 cm long and 2 cm wide and placed in a container. 4 litres of 95 %v/v ethanol were added (i.e. 500 mg of plant material to 1 ml of liquid, 500 mg/ml). After 30 hours resting at room temperature, the resulting hydroalcoholic solution comprising a Quassia amara extract was filtered and stored in the dark. A hydroalcoholic solution using isopropyl alcohol instead of ethanol has also been produced and successfully tested.

[0045] The hydroalcoholic solution comprising a Quassia amara extract was then diluted for testing. An aqueous sugar solution was prepared by mixing 1 kg of sugar per 1 litre of warm water, and then allowing the solution to cool. 50 ml of the hydroalcoholic solution comprising a Quassia amara extract was then mixed with 950 ml of the sugar solution to get a volume of 1000 ml (i.e. a dilution to 25 mg/ml).

[0046] Estimation of the strength of the beehives for analysis

[0047] The strength of a beehive can be estimated by a subjective method that relies on visual estimates of various parameters. This method is less accurate than a quantitative mode, but is far less disruptive to the bees. It allows the investigator to preserve the social cohesion and health of tested colonies, and even collect data on variables that determine the status quo of the colony.

[0048] The beehive was opened and the combs evaluated sequentially, working with both sides of each comb. It was not possible to proceed in each hive the same way, because there were some hives with an extreme reaction resulting in the majority of the bees leaving the hive at the moment of revision. If a large number of bees left the hive, we "resorted" the bees by imaginatively moving them into a contiguous mass on the comb surface. The aim of this was to approximate the density of the bee-covered frame. We worked with the estimates of bee densities described by Delaplane et al. 2013. Table 1 below shows bee capacity per frame in different regions in the world compared to Nicaragua. In addition, we visually estimated the bee movement at the hive entrance, the quantity of bees on combs, the presence of queen cells and drone brood, as well as the number of combs with brood, the percentage of the comb surface covered by brood cells, the brood pattern and the quantity of honey and pollen next to the brood. Table 2 below shows the method used in this study for scoring the strength of each beehive.

Own measurement during the study

Source: Delaplane et al. 2013 Standard methods for estimating strength parameters of Apis mellifera colonies

Table 1

Data of adult bees Characteristics Evaluation

Movement at the hive entrance Little (1) Regular (2) Plenty (3)

Quantity of bees on combs Few (1) Regular (2) Plenty (3)

Production of queen cells Yes (1) No (0)

Drone brood Few (1) Regular (2) Plenty (3)

Data of bee brood Characteristics Evaluation

# Number of brood combs 1-2 (1) 3-4 (2) 5-6 (3) 7-8 (4)

Area covered with brood (%) 0-25 (1) 26-50 (2) 51-75 (3) >75 (4)

Brood pattern Solidity (1) Contiguity (0)

Quantity of stored honey & Few (1) Regular Plenty (3) pollen near brood (2)

The numbers in brackets are the assigned code for strength estimation

Table 2 [0049] Application of the composition

[0050] To apply the composition, the hive was opened and the hydroalcoholic dilution of Quassia amara mixed with the sugar solution was sprayed directly onto the bees moving in the spaces between the frames. [0051] The strength of the hive was taken into account in applying the composition. If the hive was weak, the amount of composition used was reduced. The composition was applied to all frames with bees, using between 60 and 80 ml of the composition per hive. Given that any Varroa mites in the capped brood cells of the hive are not affected by the composition sprayed into the spaces between the frames, the procedure was repeated 4 times at intervals of 4 days in order to kill any hatched mites. Thus, the complete process per hive consisted of four applications.

[0052] The sampling to determine the Infestation Rate (IR) of varroosis was carried out on 13th November. The first application of the composition was on 16th November, the second on 20th November, the third on 24th November and the fourth on 29th November. On 3rd December, the final sampling of the adult bees and brood in all of the hives to which the composition was applied was carried out in order to determine the effectiveness of the hydroalcoholic solution comprising an extract of Quassia amara.

[0053] Analysis

[0054] It was decided to sample twice to determine the IR in the adult bees and brood cells. The first sample was prior to the first application of the composition, and this second was four days after the final application. The reason for minimising the number of sample points was to reduce disturbance during the In-vivo test. This was because it was observed in previous In-vivo testing that the sampling process resulted in the bee population becoming very stressed.

[0055] The adult worker bee samples were placed in containers with 95% alcohol. To calculate the IR of varroosis, the content of the container with adult bees in alcohol was poured into a white plastic dish. The bees were counted and revised for detached mites, then the mites in the sample were counted and the IR was calculated using the following formula: 100 [0056] We included the revision of the brood (i.e. the cutting of a slice, 10 cm x 5 cm, of the comb) in order to study the reproduction activity of the Varroa mite within the cells and to obtain another way to define effectiveness of the acaricides. The IR in worker brood cells is also called the Extension (or Extent) of the Infestation (El). To determine the El, 100 cells with bee pupae were opened and examined for presence of mites. The El in the brood cells was then calculated using the following formula: number of mites

Extension of Inf estation (EE) = - - - - — — x 100 number of ceiis

[0057] The infestation of varroosis in adult bees and brood cells were measured twice across seven hives, three days before the first application and 4 days after the last application.

[0058] Results

[0059] The observed characteristics of strength estimation for the seven beehives tested (numbered 1-7) are shown in Table 3 below. The observed characteristics were codified using the Likert scale (assigned values in brackets in Table 2). The codification permits a correlation analysis with the results of varroosis. The strength values for the hives varied from 13 to 21 , 21 being the strongest hive.

Table 3

[0060] Table 4 below shows the estimated quantity of bees on the frames according to parameters from Delaplane et al (2013).

Table 4

[0061] Table 5 below then shows the Infestation Rate (IR %) of adult bees in the seven beehives before and after application of the composition (i.e. comprising the hydroalcoholic solution comprising a Quassia amara extract and the sugar solution).

Table 5

[0062] Beehives 1 , 3 and 7 showed a significant decrease in the infestation of mites in adult bees. Beehives 2, 5 and 6 maintained a low IR (we observed a difference of only one or two mites in the samples). The only beehive that demonstrated a higher IR after applying the composition was beehive number 4. In this beehive, we found a serious infestation with Aethina tumida, the Small Hive Beetle, another beekeeping pest. In addition, the conditions of the wooden material were severe, so the increase in this beehive with Varroa mites is believed to have been influenced by these factors.

[0063] Comparison of hydroalcoholic solution with aqueous solution in adult bees [0064] Previously, an identical study had been carried out using an aqueous solution comprising an extract of Quassia amara instead of a hydroalcoholic one. The aqueous solution comprising an extract of Quassia amara was prepared as follows. 100 g of branches of the Quassia amara tree were weighed. The dry branches were cut into

2.5 cm pieces and placed in a container. 1 litre of water was then added and the mixture was left to soak overnight (at least 16 hours). The mixture was then boiled for

1.5 hours, with water being added to keep the total amount of liquid at 1 litre. The mixture was then allowed to cool to room temperature, and the resulting aqueous solution comprising an extract of Quassia amara was filtered.

[0065] The aqueous solution comprising a Quassia amara extract was then diluted for testing. An aqueous sugar solution was prepared by mixing 1 kg of sugar per 1 litre of warm water, and then allowing the solution to cool. 1 litre of the aqueous solution comprising an extract of Quassia amara was then mixed with 3 litres of the sugar solution to get a volume of 4 litres.

[0066] The Varroa infestation results for the two studies are shown in Figure 1 (aqueous solution) and Figure 2 (hydroalcoholic solution). The "before" results are the front row of bar graphs, and the "after" results are the back row of bar graphs. The aqueous and hydroalcoholic solutions gave a similar pattern of reduced IR, but the hydroalcoholic solution was a more effective acaricide (see Figure 5) The hydroalcoholic solution is also easier for the beekeeper to use because, unlike the aqueous solution, it does not need to be refrigerated.

[0067] Comparison of hydroalcoholic solution with aqueous solution in brood cells

[0068] The effectiveness of the composition being tested is determined by the absolute decrease of Varroa mites in the brood cells of the worker bees. This is shown for the composition comprising the hydroalcoholic solution for the seven hives tested in Table 6 below. It is particularly notable that the extent of Varroa invasion in hive number 7 decreased to zero. This means that the reproductive phase of the Varroa mite was significantly interrupted.

Table 6

[0069] The results presenting the Extension of the Invasion of the Varroa mites for the two studies are shown in Figure 3 (aqueous solution) and Figure 4 (hydroalcoholic solution). The "before" results are the front row of bar graphs, and the "after" results are the back row of bar graphs. The compositions comprising the aqueous and hydroalcoholic solutions gave a similar pattern of reduced El. However, again higher effectiveness was seen with the composition comprising the hydroalcoholic solution. There was a decline in the presence of Varroa mites within the brood cells. In addition, dead mites were found in and on the combs. The applied composition interfere with mite population growth by decreasing the rate at which mites successfully invade brood cells.

[0070] Analysis of the effectiveness of Quassia amara for the control of varroosis

[0071] In adult bees, the IR declined for both compositions, i.e. those comprising the aqueous and hydroalcoholic solutions comprising an extract of Quassia amara. Figure 5 compares the effectiveness of the aqueous and hydroalcoholic solutions comprising a Quassia amara extract. Both In-vivo tests demonstrated a reduction of IR % of varroosis, but the effect more noticeable and more homogeneous with the composition comprising the hydroalcoholic solution. The only outlier was beehive number 4, which was due to the severe conditions of the beehive.

[0072] Figure 6 shows the correlation between beehive strength and the reduction of IR of varroosis. A means of assessing the strength of each beehive was utilised as described above. The lowest possible result using this method was 8 points and maximum was 22 points. In the In-vivo testing the range was from 13 to 21 points. A higher reduction of IR was observed in the weaker hives (numbers 1 and 3) and in the hive with the higher IR at the beginning of the survey (beehive number 7). This is a positive feature of the product, because high infested and weak colonies need the most help to control varroosis. On the other hand, the hives with a low level of infestation maintained their IR in a range of +/-0.5%. This clearly demonstrates the effectiveness of Quassia amara in controlling varroosis in bees, and additionally shows that the composition comprising the hydroalcoholic solution is more effective than the composition comprising the aqueous solution.

[0073] Evaporation formulation

[0074] As well as a formulation which is applied by spraying, also disclosed herein is a formulation which is applied to the hive by evaporation. For example, a sponge is soaked with the formulation, which is then placed in the hive and the formulation is allowed to evaporate and thereby disperse through the hive. One difference between the spray formulation and the evaporation formulation is that the evaporation formulation comprises more alcohol. In addition, the evaporation formulation comprises a vegetable oil (e.g. olive oil), generally instead of the aqueous sucrose solution.

[0075] Preparation of the emulsion: hydroalcoholic extract of Quassia amara with olive oil

[0076] Before testing the effectiveness of the evaporation method, several tests were carried out to prepare a suitable emulsion consisting of ethanol 96% v/v and olive oil. Two different emulsifiers based on castor oil, Rimulgan® and LV41®, were tested. Emulsions comprising various amounts of olive oil in ethanol 96% were prepared. The test series showed that both emulsifiers were able to mix oil with ethanol to a 10% v/v emulsion. An emulsion of 15% olive oil by volume in ethanol 96% v/v was also prepared. It was decided to work with this concentration and the emulsifier Rimulgan®.

[0077] In-vitro test - laboratory work and diagnosis

[0078] Complete frames with sealed brood cells were examined in order to collect live mites. The sealed brood was capped one by one and the adult female mite gently removed from the Apis mellifera pupae or from the brood cell wall. Each recovered mite was placed immediately at the bottom of a Petri dish (5.5 x 1 .5 cm) and the dish was covered with tightly fixed gauze. This procedure was repeated until five mites were on the Petri dish. For efficacy testing, the vitality of the mites was checked. This means that the mites had to be active, moving firmly on the bottom of the Petri dish. Multiple Petri dishes were prepared in this way, as described below.

[0079] Efficacy testing of Quassia amara to control varroosis by determining the mortality after exposure to different compositions with the evaporation method

[0080] The acaricidal effectiveness of the evaporation method using Quassia amara extract was determined by In-vitro testing as follows. Small sponges with different concentrations of the composition were put on the gauzes of the prepared Petri dishes (each containing five mites). The prepared dishes were kept in the dark at an ambient temperature of about 32 °C without ventilation. The test emulsion (~50ml) was a 15% by volume emulsion of a hydroalcoholic extract of Quassia amara (prepared substantially as described below, but to provide two different concentrations, 100 mg/ml and 200 mg/ml) with olive oil: 42.5 ml extract - 7.5ml olive oil - 15 drops emulsifier.

[0081] Three groups were tested. Two of the groups tested Quassia amara compositions at different concentrations. In addition, there was one control group where either no composition or solvent was applied, or where ethanol only was applied, in order to define the mortality of the mites when exposed in the Petri dishes as a test location.

[0082] Group 1 : Hydroalcoholic extract (100 mg/ml)

[0083] 2 ml of the emulsion was applied to each sponge. A sponge was then placed upon the gauze that covered each of the Petri dishes (each dish containing five Varroa destructor mites). This was repeated five times such that a total of 25 mites was exposed.

[0084] Group 2: Hydroalcoholic extract (200 mg/ml)

[0085] 2 ml of the emulsion was applied to each sponge. A sponge was then placed upon the gauze that covered each of the Petri dishes (each dish containing five Varroa destructor mites). This was repeated five times such that a total of 25 mites was exposed.

[0086] Group 3: Control group

[0087] The control group comprises five Petri dishes each containing five Varroa destructor mites without any added substance. In addition, two Petri dishes were prepared with pure ethanol applied to a sponge placed upon the gauze that covered each of the Petri dishes (each dish containing five Varroa destructor mites). Thus, there was a total of 35 control mites.

[0088] Results of the In-vitro testing to control the Varroa mite with Quassia amara using the evaporation method

[0089] The aim of this testing is to determine the effectiveness of Quassia amara formulation as a bio-acaricide to control varroosis in which the formulation does not have direct contact with the bees (as would be the case if using a spraying or dribbling method). The results set out the number of the dead mites, as well as the percentage of the mortality of the mites, during the evaluation of the different concentrations used in the emulsion of the hydroalcoholic extract of Quassia amara with olive oil. The results are set out in graphical form. The absolute number of dead Varroa mites is shown in Figures 7 and 8. The relative mortality of Varroa destructor mites is shown in Figure 9. Post-hoc testing by HSD Tukey test for pairwise comparison is shown in Table 7 below.

Table 7

[0090] The mean number of dead Varroa destructor mites after one hour was 1.4 in the group to which Quassia amara extract at a concentration of 200 mg/ml had been applied. There were no dead mites in the control group, or the group to which Quassia amara extract at 100 mg/ml had been applied. This is calculated to be a significant difference according to the ANOVA test (p=0.020). [0091] After 2 hours, the mean number of dead mites in the group to which Quassia extract at 200 mgl/ml had been applied was 2. This value is significantly higher (ANOVA: p=0.003) than the mean of 0.6 dead mites in the group to which Quassia extract at 100 mg/ml had been applied and in the control group. [0092] After 3 hours of exposure, the mean number of deaths in the group to which

Quassia extract at 200 mg/ml had been applied reached 2.6. This was significantly (ANOVA: p<0.05) higher than the mean number of deaths of 0.29 in the control group, but similar to the mean of 1.6 observed in the group to which Quassia 100 mg/ml (ANOVA: p>0.05) was applied. After 8 hours of follow-up, the average number of deaths in those to which Quassia at 200 mg/ml was applied was 3.80, in those to which Quassia 100 mg/ml was applied it was 3.00 and 1.71 in the control group (Figures 7 and 8).

[0093] In the group to which Quassia extract at a concentration of 200 mg/ml was applied, the percentage of mortality of Varroa destructor mites after one hour was 28%. In the control group and the group to which Quassia extract at 100 mg/ml was applied there were no dead mites. This is calculated to be a significant difference according to the ANOVA test (p=0.020).

[0094] After 2 hours, the mortality was 40% in the group to which Quassia extract at 200 mg/ml was applied. This value is significantly higher (ANOVA, p=0.003) than the group to which Quassia extract at 100 mg/ml (mortality 12%) was applied, as well as that observed in the control group (no mortality).

[0095] After 3 hours of exposure, the mortality in the group to which Quassia extract at 200 mg/ml was applied reached 52%. This was significantly higher than the 5.7% mortality in the control group (ANOVA: p<0.05), but similar to the 32 % mortality observed in the group to which Quassia 100 mg/ml (ANOVA: p>0.05) was applied. Mortality after 8 hours was 76% in the mite group to which Quassia 200 mg/ml was applied, 60% in those to which Quassia 100 mg/ml was applied and 34% in the control group (Figure 9).

[0096] The results of Tukey's Test on comparisons between the individual mean number of dead mites of the experimental groups generally indicated that the composition with the hydroalcoholic extract at a concentration of 200mg/ml was more efficient than a concentration of 100mg/ml. This was also confirmed by the results of the control group.

[0097] At one hour post use, ANOVA revealed that there was a statistically significant difference in mean exam score between at least two groups F<2, 14> = [5.263], p = 0.20. Tukey’s HSD Test for multiple comparisons confirmed that the efficacy of the group with the hydroalcoholic extract at the concentration of 200 mg/ml was significantly different to that of the group with the hydroalcoholic extract at the concentration of 100 mg/ml (p=0.016) and the control group (p=0.000). There was a similar result two hours post use ANOVA F<2, 14) = [9.023], p = 0.03; Tukey's Test indicated differences between the composition with a concentration of 200 mg/ml compared to the composition with a concentration of 100 mg/ml (p=0.040), and the control group (p=0.002).

[0098] At three hours post use, the mean number of dead mites in the experimental groups only differed between the composition with a concentration of 200 mg/ml and the control group (p=0.023), while ANOVA revealed F<2, 14) = [4.716], p = 0.027.

[0099] At four hours post use, the experimental groups that were previously different showed no significant statistical difference in mean exam scores, even though the number of the dead mites at every point in the testing was highest with the evaporation method using the hydroalcoholic extract at a concentration of 200mg/ml.

[00100] Preparation In-vivo test

[00101] To proceed with the In-vivo test, i.e. the use of the evaporation method in hives, as a first step sponges were impregnated with the emulsion of the hydroalcoholic extract of Quassia amara. The sponges (made of 70 wt% cellulose and 30 wt% cotton) were divided into two parts of the same size. These parts were then moistened with water and squeezed to remove excess water.

[00102] The preparation of the emulsion was based on the results of the In-vitro testing. Thus, a hydroalcoholic extract was prepared from 200 g of ground Quassia amara wood and 1000 ml of ethanol. This mixture was left to extract for 30 hours at room temperature. Of the 1000 ml of liquid (ethanol), 640 ml was recovered after extraction. To this extract was added 112 ml of olive oil and 100 drops of emulsifier (20 drops = 1 ml) in order to obtain 757 ml of the final 15% oil-emulsion to apply onto the sponges. In this case, Rimulgan was used as the emulsifier, although an alternative emulsifier may be used.

[00103] The In-vivo test was carried out with 16 hives, using the composition on eight of them and leaving another eight hives as a control group (i.e. without use of the composition). For each hive, two sponges were used (one sponge divided in 2 parts); the prepared emulsion (400 ml) was poured onto the 16 cut sponges. The In- vivo testing in the beehives was then carried out using these sponges.

[00104] In-vivo test - apiaries testing

[00105] The In-vivo test started with the sampling of adult bees to determine the infestation rate (IR) of varroosis in the 16 selected beehives located in two apiaries. At the same time, two impregnated sponges were placed into the brood chamber of eight of the 16 beehives. This was repeated for two further weeks, with an interval of one week between each use of the composition. One week after the last use, another sampling was carried out to determine IR post-use. After a further week, the hives were checked and their behaviour analysed to determine the tolerability to the composition.

[00106] Results of the In-vivo testing

[00107] The aim of the testing is to determine the effectiveness of Quassia amara as a bio-acaricide to control varroosis utilising an application method that is minimally invasive for the bees and is less time-consuming for the beekeeper. As set out above, the procedure involves placing sponges impregnated with an emulsion of the hydroalcoholic extract of Quassia amara with olive oil in the hive. This was repeated once a week for three weeks. The infestation rate of varroosis before and after the use of the composition was determined in order to demonstrate the efficacy of this method. The results are shown in a box-plot graph, indicating the median as the Central Tendency; range and guartiles as the variation indicator (Figure 10) and analysed by Student’s T-test for related samples (Tables 8(a) and (b)).

[00108] Analysing the results of the infestation rate (IR) of varroosis in the hives by a box-plot graph shows that the median increased in the control groups during the study. After use, the central tendency in the groups to which the composition was applied showed a slight decrease (see Figure 10).

[00109] At the beginning of the study, the infestation rate (IR) of varroosis in the hives of the control group was 0.90 %. At the end of the study the IR of the control group was 2.24 %. This was a significant increase according to the Student's T-test for related samples (p= 0.002). On the other hand, the hives to which the emulsion of the hydroalcoholic extract of Quassia amara (200 mg/ml) was applied showed an initial I R with Varroa destructor mites of 1 .65%. At the end of the study, there was a slightly increased IR of 1.76%. However, this increase was not significant according to the Student's T test tor related samples (p=0.876) (see Tables 8(a) and (b)).

Table 8(a)

Table 8(b)

[00110] In conclusion, the IR of varroosis increased when the composition of the invention was not used. Utilising an evaporation method with the emulsion of the hydroalcoholic extract of Quassia amara (200mg/ml), it was possible to maintain the I R. Given that the study was carried out during the production cycle with a high amount of brood cells, this was considered to be a positive result which confirms the efficacy of the evaporation method.