Alkali metal salt for use in treatment of <i>Varroa destructor </i>mite infestation of honey bees

Abstract

The present disclosure relates to the use of alkali metal salts and preferably lithium chloride for treatment of Varroa destructor infestation of honey bees.

Claims

1. A method of prophylactic, therapeutic, or prophylactic and therapeutic treatment of Varroa destructor mite infestation of bees, the method comprising administering an organic or inorganic salt of lithium to said bees, wherein the lithium in the lithium salt is a miticidal agent, and wherein the organic or inorganic salt of lithium is administered in a concentration range which is equimolar to the concentration range of about 1 to about 150 mM LiCl anhydrous with respect to the lithium ion.

2. The method of claim 1, wherein said organic or inorganic salt of lithium is provided as a bee-ingestible composition or sprayable composition.

3. The method of claim 2, wherein said bee-ingestible composition is in liquid form or is a feedpaste.

4. The method of claim 3, wherein said bee-ingestible composition is a nutritional solution which optionally comprises sucrose, corn syrup, and/or sucrose syrup.

5. The method of claim 2, wherein said bee-ingestible composition comprises said organic or inorganic salt of lithium in a concentration range which is equimolar to the concentration range of about 5 to about 100 mM LiCl anhydrous.

6. The method of claim 2, wherein said bee-ingestible composition comprises said organic or inorganic salt of lithium in a concentration range which is equimolar to the concentration range of about 10 to about 75 mM LiCl anhydrous.

7. The method of claim 1, wherein said organic or inorganic salt of lithium is a water-soluble salt.

8. The method of claim 1, wherein said bees are of the genus Apis with species Apis mellifera or Apis cerana.

9. The method of claim 1, wherein said bees are honeybees, foragers, hive bees, or a combination thereof.

10. The method of claim 2, wherein said lithium salt is a water-soluble salt.

11. The method of claim 2, wherein said bees are of the genus Apis with species Apis mellifera or Apis cerana.

12. The method of claim 1, wherein said bees are honeybees, foragers, hive bees, or a combination thereof.

13. The method of claim 1, wherein said lithium salt is administered to said bees during a phase of decreased egg-laying.

14. The method of claim 1, wherein said lithium salt is administered to said bees in one or more of September, October, November, and December.

15. The method of claim 2, wherein said lithium salt is administered to said bees during a phase of decreased egg-laying.

16. The method of claim 2, wherein said lithium salt is administered to said bees in one or more of September, October, November, and December.

17. The method of claim 1, wherein said lithium salt is administered to said bees as a solid composition by surface contact.

Description

FIGURE LEGENDS

(1) FIG. 1: depicts cage in which 100 bees were kept with 30 Varroa destructor mites and feed with sucrose solution comprising either dsRNA or lithium chloride

(2) FIG. 2: depicts number of dead mites after treatment with Varroa destructor specific dsRNAs or non-specific control RNAs. 100 bees were placed with 30 Varroa mites in 1 cage and treated as indicated in Experiment 1. FIG. 2 depicts the combined results from 3 cages for either Varroa destructor specific dsRNAs or non-specific control RNAs.

(3) FIG. 3 depicts number of dead mites after treatment with 70% sucrose solution only, non-specific control dsRNAs or 25 mM LiCl in 70% sucrose solution. 100 bees were placed with 30 Varroa mites in 1 cage and treated as indicated in Experiment 2. FIG. 3 depicts the results for either sucrose only treatment, non-specific control dsRNAs or 25 mM LiCl.

(4) FIG. 4: Kaplan-Meyer Plot of bees and Varroa mites in percentages. Dashed lines indicate bees, straight line indicate mites. Numbers 0, 2, 10, 50, and 250 indicate concentrations of 0 mM, 2 mM, 10 mM, 50 mM and 250 mM LiCl in the sucrose solution. The Y-axis indicates percent of surviving bees and mites. The X-axis indicates days after starting feeding LiCl. “Biene” stands for bee and “Milbe” stands for mite.

(5) FIG. 5: Kaplan-Meyer Plot of bees and Varroa mites in percentages. Dashed lines indicate bees, straight line indicate mites. Numbers 0 and 100 indicate concentrations of 0 mM and 100 mM LiCl in the sucrose solution. The Y-axis indicates percent of surviving bees and mites. The X-axis indicates days after starting feeding LiCl. “Biene” stands for bee and “Milbe” stands for mite.

(6) FIG. 6: depicts mean number of dead mites with standard deviation after treatment with Varroa destructor specific dsRNAs (“ds RNA MW”) or non-specific control RNAs (“neg RNA MW”). A control with 70% sucrose solution only is also included (“ApiInvert MW”). Results represent mean values derived from three cages whereby 100 bees were placed with 30 Varroa mites in 1 cage and treated as indicated in Experiment 1.

(7) FIG. 7: depicts number of dead mites after treatment with 70% sucrose solution only (“Kontrolle”), non-specific control dsRNAs (“dsRNA [500 μg/Tag]”) or 25 mM LiCl in 70% sucrose solution (“25 mM LiCl”). 100 bees were placed with 30 Varroa mites in 1 cage and treated as indicated in Experiment 2.

(8) FIG. 8: depicts the mean number of dead mites upon treatment with 70% sucrose solution only (“Kon Avg”), 2 mM LiCl in 70% sucrose solution (“2 mM Avg”), 10 mM LiCl in 70% sucrose solution (“10 mM Avg”) or 25 mM LiCl in 70% sucrose solution (“25 mM Avg”) over a treatment period of 23 days.

DETAILED DESCRIPTION OF THE INVENTION

(9) The following examples illustrate specific embodiments of the aspects described above. They are not to be construed as limiting.

(10) Experiment 1—siRNA Approach to Fight Varroa destructor Infestation

(11) The publication of Garbian et al. (Garbian Y, Maori E, Kalev H, Shafir S, Sela I. Bidirectional Transfer of RNAi between Honey Bee and Varroa destructor: Varroa Gene Silencing Reduces Varroa Population. Schneider DS, ed. PLoS Pathogens. 2012; 8(12):e1003035. doi:10.1371/journal.ppat.1003035) showed that dsRNA complementary to essential genes in Varroa destructor could be used to combat varroosis. DsRNA was dissolved in glucose syrup and fed to bees and subsequently taken up by parasitizing mites via the bees' hemolymph. The publication suggested that ingested dsRNA could knock down the respective Varroa genes by a systemic RNAi based mechanism.

(12) In a first experiment a refined list of potentially essential Varroa genes was assembled and dsRNA molecules were synthesized. In a first experiment, a refined list of potentially essential Varroa genes was assembled and dsRNA molecules were synthesized. The refined list was generated by retrieving essential genes from various eukaryote species (fly, worm, human, zebrafish) (http://ogeedb.embl.de). Where possible, homologous genes were determined using NCBI HomoloGene DB. Then genes from Drosophila with homology to the Contig sequences published by Cornman et al were identified. (Cornman et al.: http://www.ncbi.nlm.nih.gov/pubmed/20973996). The identified genes were ranked according to the number of species where they were essential.

(13) To generate the respective dsRNA, Varroa RNA was extracted and used to generate cDNA. Specific primers were designed to synthesize dsRNA by in vitro transcription and subsequent hybridization. Primer sequences for the respective genes were based on the sequence of the fly database which can be found at http://flybase.org. The list comprised the following genes:

(14) TABLE-US-00001 TABLE 1 Flybase Fly_symbol accession no. human_description Human symbol RPN1* FBgn0028695 proteasome (prosome, PSMD2 macropain) 26S subunit RPN3* FBgn0261396 proteasome (prosome, PSMD3 macropain) 26S subunit NOI* FBgn0014366 splicing factor 3a, SF3A3 subunit 3, 60 kDa CG2807* FBgn0031266 splicing factor 3b, SF3B1 subunit 1, 155 kDa RPL7* FBgn0005593 ribosomal protein L7 RPL7 ATPsyn* FBgn0010217 ATP synthase, H+ ATP5B transporting, mitochondrial F1 complex, beta polypeptide Tbp1 FBgn0028684 proteasome (prosome, PSMC3 macropain) 26S subunit, ATPase, 3 AlphaTub* FBgn0003884 tubulin, alpha 1c TUBA1C His2B* FBgn0061209 histone cluster 1, H2bj HIST1H2BJ RPT4 FBgn0028685 proteasome (prosome, PSMC6 macropain) 26S subunit, ATPase, 6 Pros26.4 FBgn0015282 proteasome (prosome, PSMC1 macropain) 26S subunit, ATPase, 1 PP2A-B FBgn0042693 protein phosphatase 2, PPP2R5D regulatory subunit B′, delta RPL15* FBgn0028697 ribosomal protein L15 RPL15 RPN2 FBgn0028692 proteasome (prosome, PSMD1 macropain) 26S subunit, non-ATPase, 1 Blw* FBgn0011211 ATP synthase, H+ ATP5A1 transporting, mitochondrial F1 complex So* FBgn0003460 SIX homeobox 1 SIX1 Med* FBgn0011655 SMAD family member 4 SMAD4

(15) DsRNA was precipitated using standard procedures and lyophilized. RNA was provided at 1 μg/μl. As control dsRNA corresponding to human GAPDH was synthesized, hybridized and purified. Alternatively, a mix of uncleaved siPOOL dsRNA (as described in WO 2013/160393 A1) not targeting any gene in the human genome was used as negative control.

(16) 12 genes were selected for a first experiment (marked with an asterisk in Table 1).

(17) In accordance to Garbian et al. (vide supra), 100 bees were kept in a container and 30 mites were added to each container (see FIG. 1). Bees were fed 80 g per gene and day. In view of 12 genes this adds up to 960 μg of dsRNA per container and day dissolved in 2 ml of 70% sucrose solution. In the publication of Garbian et al. (vide supra), the experiments were carried out with 2 different dsRNA mixes, with each mix comprising 200 μg of either 5 or 14 different dsRNA species targeting different Varroa genes in 5 ml sucrose solution. As such, different total amounts of dsRNA were fed to each test hive. Moreover, control experiments were carried out with either sucrose solution only or unrelated dsRNA encoding GFP. The GFP control however was carried out with only 200 μg total dsRNA in 5 ml sucrose solution exposing the bees to less dsRNA irrespective of its nucleotide sequence.

(18) Other than Garbian et al. (vide supra) we choose to control our experiments with similar total amounts of control RNA to exclude a sequence unspecific effect of dsRNA on the mites.

(19) 3 cages with each cage having 100 bees/30 mites were treated with 12 dsRNA species, 80 μg per gene and day over a period of 6 days. Dead mites and bees were collected and counted. In parallel 3 cages were treated with unrelated control dsRNA at corresponding total amount of dsRNA. Additionally, 2 cages were treated with sucrose solution only.

(20) In cages treated with dsRNA targeting mite-specific genes, mites were killed effectively within the first 2 days. Complete loss of mites was observed after 3 days (day 1: 18 mites, day 2: 68 mites, day 3: 5 mites, see FIG. 2 for combined results or see FIG. 6 for mean numbers of dead mites per treatment day). Interestingly a similar response was found in mites fed with sucrose containing dsRNA not targeting any gene in Varroa (day 1: 10 mites, day 2: 64 mites, day 3: 13 mites, day 4: 2 mites, see FIG. 2 for combined results or see FIG. 6 for mean numbers of dead mites per treatment day).

(21) In summary, all mites in either treatment regimen were killed within the first 3 days of treatment and a difference in response to either treatment could not be observed. Cages treated with sucrose alone did not show any response. Of note, bees showed toxicity effects starting from day 3 and around 30% of the bees were lost within the first 6 days of treatment (data not shown). Bees and mites in cages that were fed sucrose without RNA did not exhibit any toxicity responses.

(22) Experiment 2—Identification of Lithium Chloride as Miticide Against Varroa destructor

(23) Based on the observations in Experiment 1 we reasoned that effects of dsRNA did not relate to the sequence and maybe a non-specific response to dsRNA or agents copurifiying with dsRNA in the production process.

(24) To reproduce the observation, the experiment was repeated with the negative control dsRNAs and the results confirmed the findings of Experiment 1 lending support to an unspecific RNA effect.

(25) Additionally, we set out to pursue the question of potentially copurifying toxic agents. In the dsRNA production process dsRNA is precipitated with LiCl at 7.5M. We reasoned that even after extensive washing substantial amounts of lithium may be present in the RNA samples. In view of 2 washes with 70% EtOH after precipitation we considered 25 mM LiCl a reasonable concentration to integrate into the experiment.

(26) The next set of experiments was carried out in plastic container with 50 bees and 20 mites each. Bees were fed the same amount of unspecific dsRNA that has been used in previous assays in 70% sucrose solution, LiCl at a concentration of 25 mM in 70%, sucrose solution, and 70% sucrose solution only.

(27) Bees and mites in cages supplied with sucrose syrup only did not show an impact on viability of either mite or bee.

(28) Cages that were fed sucrose containing dsRNA that has been subjected to extensive washing displayed activity against Varroa destructor but was tolerated well by the bees. After 24 h only one mite was killed and a total of 4 mites were dead after 72 hours. After 4 days 75% of the mites were killed, indicating a decreased activity compared to the results from the previous experiments where identical concentrations of ds RNA was fed to the bees. No bees were affected.

(29) LiCl at a concentration of 25 mM however displayed high activity against Varroa destructor, with 4 mites dead after 24 h, 15 and 20 after 48 and 72 h of treatment, respectively. Bees however were not affected within the first 4 days of treatment with 25 mM LiCl.

(30) The results are summarized in FIG. 3 as a bar graph and in FIG. 7 as a line graph.

(31) Experiment 3—Dose Response Relation of Lithium Chloride as Miticide Against Varroa destructor

(32) To analyze the therapeutically active concentrations of LiCl in more detail we devised a series of experiments in cages of 50 bees to each of which 25 mites were added. LiCl was added to 70% sucrose solution to final concentrations of 16 μM, 80 μM, 400 μM, 2 mM, 10 mM, 50 Mm, and 250 mM, respectively. 2 ml of the respective solution was fed to the bees, if the sucrose/LiCl solution was entirely ingested additional sucrose syrup was offered ad libidum. Uptake of LiCl solution as well as survival rate of bees and mites was monitored 3 times daily (morning, noon, evening). Additionally, the healthiness of bees was scored based on characteristic behavioral features. LiCl was administered until all mites were dead, and the experiment continued with plain sucrose syrup to record the response of bees over the entire 2 weeks of the experiment. For control bees treated with sucrose syrup only, 9 mites died within the course of the experiment with even timely distribution of the events, indicating natural death of mites. 26 bees died in the same period of time. In view of the life span of a worker bee (4 weeks) this relates to the natural death rate that can be expected in a time span of 14 days. However, 12 bees were lost in a single day equaling almost 50% of the total bee loss in this control experiment. Moreover, bees treated with LiCl up to 50 mM did not exhibit comparable responses. For that reasons the sudden loss of bees on day 14 is considered to result from a mistake in bee treatment as feeding inappropriate amounts of sucrose syrup. (See FIG. 4, dotted red line)

(33) Cages that were fed sucrose with 250 mM LiCl lost all mites within the first 48 hours of the experiment, the majority of which (21/25) within the timespan between 30 h and 48 h. Bees were fed sucrose syrup only after all mites were dead and the viability of bees was monitored for the remaining experiment. 27 bees were lost in total with an average rate of 3 bees per day, indicating a toxic effect on bees even after treatment was terminated and bees were fed sucrose only (FIG. 4, yellow lines)

(34) A 5-fold decrease of LiCl to 50 mM LiCl in the sucrose syrup showed similar results with all mites dead after 48 h of treatment. Bee survival was similar with 20 bees lost in the timespan of the entire experiment. The average bee loss over time relates to the decay curve of controls until day 12 and thus indicates good tolerability by the bees (FIG. 4, purple lines).

(35) Cages that were fed sucrose syrup with 10 mM LiCl showed delayed response compared to higher concentrated dosage. All mites were killed within the first 48 h of treatment (day 1: 3 mites, day 2: 10 mites, day 3: 7 mites, day 4: 3 mites, additionally 2 mites were found dead at the end of the experiment). The survival of bees improved over high concentrated LiCl to 34 bees alive at the end of the experiment (FIG. 4, green lines).

(36) 2 mM LiCl failed to kill all mites over the time span of the experiment but showed increased loss of mites compared to the control. 11 mites survived the treatment as well as 42 bees (FIG. 4, blue lines)

(37) Lower concentrations only marginally impacted the viability of mites (data not shown). As such active concentrations of LiCl in cage experiments range between 2 mM und 250 mM. Bees tolerate a dosage of 2 mM LiCl over a period of 2 weeks without pronounced decrease in viability. Concentrations of 10 mM and higher exhibit a clear acaricidal effect and eliminate all mites within maximally 4 days.

(38) These data are summarized for 0, 2, 10, 50 and 250 mM LiCl in FIG. 4.

(39) Additionally, we set out to test activity of a single dose on mite viability. Sucrose with 25 mM LiCl was fed to a cage of 50 bees infested with 25 mites and monitored as described above. Mites were effectively killed within 72 h (3 mites were lost) and no negative impact on bee health could be observed over the course of the entire experiment. As such, a single dose of LiCl can be used to kill mites under the described conditions.

(40) To explore the upper limit of dosage and limits of tolerability of bees we fed sucrose containing 100 mM of LiCl as only food source over 14 days. Again mites were effectively killed, however a time delayed continuous loss of bees showed toxicity of LiCl at high doses over a prolonged time of administration. In view of the latter results the effective window for concentration and administration and regimen of LiCl is sufficiently large to elevate low but effective concentrations (10 mM) more than 10 fold or prolong the time of administration substantially without negative impact on bee health. Taken together LiCl has been shown to exhibit strong acaricidal effects on Varroa mites without harming bees that can be administered systemically and such opens new opportunities to combat varroosis. The data is shown in FIG. 5.

(41) Experiment 4—Test of Acaricidal Effect of MgCl2

(42) To test the activity of bivalent ions we dissolved MgCl.sub.2 in sucrose syrup at a final concentration of 100 mM. 2 cages of 50 bees to which 20 mites were added were fed with sucrose syrup only and syrup containing 100 mM MgCl.sub.2. No impact on viability was seen for the control cage, however MgCl.sub.2 appeared to impair bee health substantially. Moreover, only reduced amounts of sucrose syrup with MgCl.sub.2 was taken up by the bees.

(43) Experiment 5—Comparison of Acaricidal Effects of LICl, NaCl and MgCl.sub.2

(44) In a further experiment LiCl, NaCl and MgCl.sub.2 were selected and their acaricidal activity compared. The experiment was performed in cages of 50 bees, to each of which 25 mites were added. Bees in control cages received sucrose only. LiCl, NaCl or MgCl.sub.2 were added to 70% sucrose solutions at a final concentration of 25 mM each and fed to the bees for seven days. Survival rate of bees and mites were monitored daily. Dead bees and mites were removed from the cages.

(45) As can be seen from Table 2, column “Sum”, only 3 of 50 bees died during treatment with LiCl while all mites were killed inferring a strong acaricidal effect. The numbers represent mean values derived from 6 cages. In contrast, during treatment with NaCl, 2 bees died while also only 2 mites died. The number of dead mites upon treatment with NaCl corresponds to the number of dead mites for the control which implies that NaCl fails to exhibit an acaricidal effect. As regards MgCl.sub.2, all bees were dead after 5 days of treatment. Hence, MgCl.sub.2 strongly impacts the viability of bees. Numbers for NaCl, MgCl.sub.2 and control represent mean values derived from 3 separate cages.

(46) Overall, the results of this experiment substantiate that lithium is the causative acaricidal agent.

(47) TABLE-US-00002 TABLE 2 Day 1 2 3 4 5 6 7 Sum LiCl Number of dead bees 0.2 0.2 0.3 0.8 0.5 1.0 0.2 3.2 Number of dead mites 0.3 2.2 19.2 3.0 0.3 0.0 0.0 25.0 NaCl Number of dead bees 0.0 0.0 0.0 0.3 0.0 0.0 1.3 1.7 Number of dead mites 0.0 0.3 0.0 0.0 0.3 0.7 1.0 2.3 MgCl.sub.2 Number of dead bees 0.0 18.0 18.3 9.3 4.3 0.0 0.0 50.0 Number of dead mites 0.3 1.0 1.0 3.7 0.7 0.0 0.0 6.7 Control Number of dead bees 0.0 0.0 0.0 0.0 0.7 1.0 0.3 2.0 Number of dead mites 0.3 0.3 0.0 0.0 0.0 1.0 0.0 1.7
Experiment 6—Test of Acaricidal Effect of Various Lithium Salts

(48) To test the acaricidal activity of further lithium salts, Li-citrate, Li-sulfate, Li-acetate, Li-lactate and Li-carbonate were dissolved in sucrose syrup at a final concentration of 25 mM. Each Li-salt was tested in cages of 50 bees to which 25 mites were added and replicated three times (results in Table 3 represent mean values). The bees were fed with Li-salt/sucrose solutions for 7 days. Survival rate of bees and mites were monitored daily and dead bees or mites were removed.

(49) As can be seen from Table 3, bees treated with 25 mM Li-citrate, Li-sulfate, Li-acetate, Li-lactate or Li-carbonate lose all mites during the course of 7 days. The majority of mites is killed within the first 72 hours implying a high activity of said Li-salts against Varroa destructor.

(50) TABLE-US-00003 TABLE 3 Li-salt (25 mM) Day 1 2 3 4 5 6 7 Sum Li-sulfate Number of dead bees 0.0 0.0 0.0 1.0 3.7 1.7 0.0 6.3 Number of dead mites 0.7 18.0 5.7 0.7 0.0 0.0 0.0 25.0 Li-citrate Number of dead bees 0.0 0.0 0.3 0.0 0.7 1.7 0.3 3.0 Number of dead mites 0.3 10.0 14.7 0.0 0.0 0.0 0.0 25.0 Li-acetate Number of dead bees 0.0 0.0 0.3 0.3 0.3 0.7 1.0 2.7 Number of dead mites 0.7 14.0 10.3 0.0 0.0 0.0 0.0 25.0 Li-lactate Number of dead bees 0.3 0.0 0.0 0.7 2.0 0.3 1.7 5.0 Number of dead mites 0.7 14.0 6.3 4.0 0.0 0.0 0.0 25.0 Li-carbonate Number of dead bees 0.7 0.0 0.0 0.0 0.0 0.3 0.7 1.7 Number of dead mites 0.3 3.0 20.0 1.7 0.0 0.0 0.0 25.0

(51) Interestingly, even lower concentrations of lithium salts (data not shown) effectively killed mites. 4 mM of Li-sulfate, Li-citrate, Li-acetate, Li-lactate and Li-carbonate killed all mites during the 7-day treatment period without a significant negative impact on bee viability.

(52) Taken together, anorganic as well as organic lithium salts appear to exhibit a strong acaricidal effect on Varroa mites and might be valuable alternatives in the treatment of varroosis in bees.

(53) Experiment 7—Acaricidal Effect of LiCl in Mini-Hives

(54) In order to investigate if the miticidal effect of LiCl can also be detected under semi-field conditions, so-called “mini-hives” consisting of 2000 to 3000 bees each were treated with LiCl.

(55) LiCl was dissolved in 70% sucrose to final concentrations of 2 mM, 10 mM and 25 mM. Each concentration was replicated 6 times and fed to the bees for 23 days. Dead mites were documented daily. After 23 days of LiCl treatment, remaining mites were killed with Perizin® (commercial product containing the active ingredient coumaphos) and counted (see Table 4 “Remaining mites”).

(56) As can be taken from FIG. 8 and Table 4, a majority of mites was killed for the 25 mM treatment group with only 24 mites left after LiCl treatment. Regarding a treatment with 10 mM LiCl, more mites are killed as compared to the control with only 54 mites remaining after LiCl treatment. A treatment with 2 mM LiCl appeared to have a similar mite fall as the control.

(57) Overall, it can be inferred that a treatment with LiCl is effective against Varroa destructor infestation of bees under semi-field conditions and may be extended to natural hives.

(58) TABLE-US-00004 TABLE 4 Remaining mites Control 234  2 mM LiCl 234 10 mM LiCl 54 25 mM LiCl 24
Experiment 8—the Effect of LiCl on Larval Development In Vitro

(59) In order to assess the effect of LiCl on larval development in vitro, larvae were obtained by caging the queen for 24 h on broodless combs in the colony. After 5 to 6 days when the larvae reached the age of about 48 h they were transferred to petri dishes filled with larval food. The petri dishes were placed in plastic boxed filled with 8% sulfuric acid in order to prevent fungi infection and kept in a chamber at 34° C. and 95% relative humidity. The larvae were fed as needed with a mixture consisting of 53% royal jelly, 4% glucose, 8% fructose, 1% yeast extract and water (control). For samples treated with LiCl, 10 mM, 25 mM or 50 mM LiCl were added to the mixture. Prior to the pupa phase, the larvae were placed on a tissue for defecating which occurred approximately on day 9. Afterwards larvae were placed in well plates until hatching. Larvae mortality was monitored on a daily basis.

(60) Table 5 summarizes the results expressed as survival rate of larvae or pupae. All larvae were lost within 72 hours implicating a strong lethal effect of LiCl on larvae. Since 25 mM LiCl is tolerated by bees very well but larvae viability is crucially impacted already at 10 mM LiCl, it follows that LiCl should ideally be applied when egg laying is decreased. Such a phase naturally occurs between the calender start of summer and the overwintering period.

(61) TABLE-US-00005 TABLE 5 Survival rate (%) Control 51.2 10 mM LiCl 0 25 mM LiCl 0 50 mM LiCl 0
Experiment 9—Artificial Swarms

(62) Six artificial swarms were fed with sucrose/LiCl after a starving phase of several hours. LiCl was dissolved in sucrose at final concentrations of 25 mM and 50 mM. Each artificial swarm received 500 ml of the sucrose/LiCl mixture in a feeding balloon. The total volume of sucrose/LiCl solution was consumed by the bees. Mite fall was monitored for 7 days. Afterwards, bees were treated with Perizin® to assess the number of remaining mites.

(63) As can be seen from Table 6, mites were effectively killed upon treatment of the bees with either 25 mM LiCl or 50 mM LiCl. A minority of mites was killed using Perizin® after the seventh day of LiCl treatment resulting in acaricidal efficacies of above 80% for all six swarms. Hence, it can be inferred that a treatment with LiCl will be effective against Varroa destructor infestation of natural bee hives under field conditions.

(64) TABLE-US-00006 TABLE 6 Swarm Mite fall upon LiCl treatment Remaining Efficacy no. LiCl Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Sum mites (%) 1 50 1 7 49 89 36 9 16 207 19 92 2 mM 1 0 8 35 48 7 22 121 23 84 3 0 5 25 87 35 23 32 207 25 89 4 25 3 6 82 242 108 60 82 583 70 89 5 mM 7 5 12 165 155 79 163 586 55 91 6 3 3 9 86 183 162 199 645 104 86