Pilot study: From RNAi to lithium chloride

In a pilot study, mite-infested honey bees were caged and fed sucrose syrup containing dsRNAs of potentially essential Varroa genes (Supplementary Table S1). Plain sucrose syrup (untreated) and syrup with dsRNA based on the coding sequence for the green fluorescent protein GFP (dsGFP ctrl) served as controls. GFP is expressed in the bioluminescent hydrozoan jellyfish Aequorea victoria. The GFP sequence was chosen as a control because no homologous gene exists in the genome of honey bees or the Varroa mite. In the untreated group mite mortality was <5%. In contrast, all mites on bees that received sucrose solution containing dsRNA targeting Varroa genes were effectively killed within three days. An identical effect on mites, however, was observed in a control experiment in which bees were fed GFP dsRNA (Supplementary Fig. S1). These results ruled out the proposed RNAi mediated mechanism but suggested either a yet unknown effect of RNA or the activity of other components in the test solution. As high concentrations of lithium chloride (LiCl) were used in the production of dsRNAs and therefore fed to the bees together with the dsRNA, we chose to feed LiCl in sucrose solution to caged bees to test its activity against Varroa mites. Strikingly, LiCl at concentrations of 25 mM, which corresponds to the calculated concentration in the dsRNA solution, killed the mites as effectively as the test substances containing dsRNA. Moreover, after LiCl was largely removed from dsRNA by extensive washing (washed dsGFP ctrl), the miticidal activity was substantially diminished as indicated by delayed onset and reduced activity (Supplementary Fig. S1). From these data, we concluded that LiCl, not RNA knockdown, mediated the observed activity on Varroa mites and that it would be worthwhile to analyse the potential of LiCl as a varroacide.

Effective concentration of lithium chloride

To corroborate the primary observations of our pilot study, we established cage experiments with different concentrations of LiCl for robust statistical analysis. In addition to the 25 mM concentration, which was found to be effective in the pilot study, we used concentrations of 2 mM, 4 mM and 10 mM in order to determine the lower threshold of efficacy. The results supported the findings of the previous study and demonstrated significant miticidal effects for LiCl concentrations as low as 2 mM at which a substantial increase in mite mortality (P < 0.001, log-rank test; Supplementary Table S2) was shown. Higher concentrations of 10 mM and 25 mM both significantly enhanced mite mortality starting at day two of treatment and achieved extermination of more than 96% of the treated mites at the end of the experiment (Fig. 1a, Supplementary Table S2). In the control experiments without LiCl in the feeding solution, mite mortality reached on average 9.3% and was therefore well within the range of mortality rates obtained for mites kept on untreated cage bees under different environmental conditions33. Based on these results, we confirmed a clear effect of LiCl on mite viability in a concentration range between 2 mM and 25 mM.

Figure 1 Mortality of phoretic Varroa mites and honey bees after feeding lithium chloride (LiCl) to caged bees. (a) Kaplan-Meier survival curve of female Varroa mites kept on caged bees fed with LiCl in concentrations between 2 mM–25 mM (n = 33, 9, 9, 12, and 9 cages for 0 mM (control), 2 mM, 4 mM, 10 mM and 25 mM, respectively). At all concentrations, the survival of mites in the treatment groups was significantly different from the control (P < 0.001, log-rank test with Bonferroni correction). (b) Kaplan-Meier survival curve of caged worker bees and female Varroa mites after 24 h LiCl exposure (n = 9 cages). The survival of mites in the treatment group was significantly different from the control group (P < 0.001, log-rank test) but there were no significant group differences in bee mortality. Full size image

In these experiments, the caged bees were fed with the respective concentration of LiCl over several days until all mites were killed by the treatment. However, for potential use in beekeeping practice, a shorter and defined treatment period would be preferable. We therefore performed an additional experiment, in which the most effective concentration of 25 mM LiCl (Fig. 1a) was administered for 24 h followed by feeding with sugar solution for additional six days. At the end of the observation period, 92.9% of the mites (n = 225 mites, P < 0.001, log-rank test) were killed without any significant effect on the treated bees (see next paragraph). This result clearly demonstrates that even a short-term feeding of 25 mM LiCl is sufficient to substantially diminish the mite population.

To precisely determine the ingested amount of LiCl by the bees that is necessary to kill parasitizing mites, 12 newly hatched bees were artificially fed 10 µl of LiCl solutions of 4 mM to 100 mM and kept individually with a phoretic mite for five days within cages. With the 4 mM and 10 mM solutions, which corresponded to an uptake of 1.7 µg and 4.2 µg of LiCl, respectively, the effect was not significantly different from the untreated control (n = 12 mites, P = 1.000, log-rank test, Supplementary Table S3). However, a single dose of 25 mM, corresponding to 10.6 µg of LiCl consumed by the bee was sufficient to kill 100% of the phoretic mites within 48 hours (Fig. 2).

Figure 2 Mortality of phoretic Varroa mites kept on bees that were individually fed 10 µl of a lithium chloride solution at concentrations ranging from 4 mM to 100 mM. The bees were fed LiCl only once at the beginning of the experiment, then they received sucrose syrup over five days. For each concentration, 12 cages with one bee and one Varroa mite each were analysed. The survival of mites was significantly reduced compared to the control group when concentrations of 25 mM and higher were fed to the bees (P < 0.001, log-rank test). Full size image

Effect on worker bees

For the analysis of the tolerability of LiCl to worker bees, the test cages that were used to analyse mite mortality (Fig. 1a) were additionally recorded for the mortality of the worker bees. After exposure to 2 mM, 10 mM and 25 mM LiCl which have been shown to exert miticidal activity, the treated worker bee mortality ranged on average from 3 to 7% within the different feeding groups. With the exception of the 10 mM LiCl group (n = 12 cages, P = 0.015, log-rank test; Supplementary Table S4), the values were not significantly different from the 4% mortality in the untreated control group. Furthermore, the mortality rates of our controls were well within the range of the mortality of non-treated cage bees required as a control in toxicology tests34, therefore confirming the validity of our test system. Also the 24 h treatment with LiCl did not affect the mortality of worker bees (Fig. 1b; n = 9 cages, P = 0.308, log-rank test). A good tolerability of LiCl to bees was also confirmed by the feeding of a single dose (for mite mortality see Fig. 2) that did not elicit a significant increase in worker bee mortality (P = 1.000, log-rank test; Supplementary Table S5).

Next, different concentrations of LiCl were continuously fed until the last caged bee died to investigate response to long-term exposure. Here, the treatment significantly reduced the average lifespan of freshly hatched worker bees from 26 days in the untreated control cages to 23 and 22 days for 2 mM and 10 mM LiCl, respectively (n = 60 bees, P = 0.024, log-rank test; Supplementary Table S6). In bees that received the highest concentration of 25 mM LiCl the lifespan was significantly reduced to 19 days on average (Fig. 3a).

Figure 3 Mortality of honey bees after feeding lithium chloride to caged bees. (a) Kaplan-Meier survival curve of caged worker bees during chronic LiCl exposure. LiCl diets at concentrations of 2 mM, 10 mM and 25 mM were fed ad libitum till the death of the last bee (n = 6 cages with 10 bees each). The survival of all treated groups was significantly different from the sugar syrup control (P < 0.01, log-rank test with Bonferroni correction). (b) Kaplan-Meier survival curve of caged worker bees after a 24 h LiCl exposure. LiCl diets at concentrations of 2 mM, 10 mM and 25 mM were fed ad libitum for the first 24 h after hatching and then replaced by sucrose syrup (n = 12 cages with 10 bees each). The survival of all treated groups was not significantly different from the sugar syrup control (P > 0.1, log-rank test with Bonferroni correction). Full size image

However, LiCl appears to impede bee viability only if administered over an extended period of time as indicated by an additional experiment in which LiCl was fed for the first 24 h after hatching and was then replaced by sucrose syrup until the last caged bee died (Fig. 3b). Here, the average lifespan of freshly hatched worker bees ranged from 22 days (10 mM) to 24 days (control) without significant differences between the treatments (n = 120 bees per treatment, P ≥ 0.126, log-rank test; Supplementary Table S7). Based on this data from caged bees, we conclude that even a short-term LiCl treatment is sufficient to completely eradicate Varroa mite infestation with little or no impact on the viability of worker bees. These results obtained in cage tests under controlled conditions represent a successful and promising first step towards a new approach to Varroa treatment. However, efficiency and side effects must be confirmed under field conditions.

Field tests with lithium chloride in artificial swarms

To approximate field conditions, we tested 25 mM and 50 mM LiCl in nine brood-free artificial swarms consisting of a queen and approximately 20,000 bees each. These concentrations were chosen based on previous experiments with caged bees using the highest dose that was still tolerated by bees in short application times (25 mM). Because an even distribution of sucrose syrup throughout the entire artificial swarm of approximately 20,000 bees might have been difficult to achieve, we additionally tested a 50 mM concentration of LiCl to ensure that each bee was exposed to sufficient amounts of lithium. Accordingly, the swarms were fed ad libitum with sucrose syrup containing 25 mM LiCl (n = 6) or 50 mM (n = 3) for a period of three days, followed by a topical application of Perizin®. Perizin® containing the organophosphate coumaphos as active ingredient, is a highly effective varroacide that is commonly used as a control treatment35. Mite mortality was monitored over a period of five days. Prior to the control treatment, 25 mM LiCl killed approximately 90% of the mites present within the artificial swarms (Table 1). The higher concentrated solution (50 mM), however, did not increase this effect (χ2 test, P = 0.953). Altogether, the efficacy was somewhat lower compared to the cage tests. One explanation might be that the distribution of LiCl within a cluster of thousands of bees requires more time until the last individual bee consumes a sufficient dosage to kill the respective parasitizing mite. The necessary feeding time of such huge entities of 20,000 bees and more must be analysed in further experiments.

Table 1 Comparison of the varroacidal action of two lithium chloride diets administered to artificial swarms for five days. Full size table

Efficacy of other lithium compounds and non-lithium salts

To confirm lithium as the active component for the effect on Varroa mites we tested a series of lithium compounds and compared the miticidal effects with non-lithium salts. Of particular interest were lithium citrate, a compound with three lithium ions, lithium sulphate and lithium carbonate, which have two lithium ions compared to only one lithium ion in LiCl. Additional compounds with one lithium ion (lithium lactate, lithium acetate), but different solubility, chemical reactivity and price were included to analyse efficacy and tolerability in comparison to LiCl. In cage experiments all compounds eliminated 100% of the mites at 25 mM within three (lithium citrate and lithium acetate) to four days (lithium sulphate, lithium lactate and lithium carbonate). Also, the 4-mM test solutions, entirely killed phoretic mites within five (lithium citrate, lithium sulphate, and lithium acetate) to seven days (lithium lactate) except for lithium carbonate (Table 2; Supplementary Table S8).

Table 2 Mortality of phoretic Varroa mites and worker bees after feeding two concentrations of different lithium compounds over a maximum feeding period of seven days. Full size table

Worker bee mortality was not significantly increased at either concentration compared to the untreated control bees, except for 25 mM lithium sulphate and 25 mM lithium lactate (Supplementary Table S9). With these experiments we could confirm that other lithium compounds have a similar potential for the use as a systemic acaricide. This might increase the flexibility for the possible design of a veterinary product. Considering the price, lithium chloride and lithium citrate are the cheapest compounds. Lithium sulphate is less suitable due to the lower bee tolerability and lithium carbonate due to a relatively low water solubility.

To investigate the concentration-dependent efficacy of lithium compounds in more detail, we compared LiCl with lithium citrate (Li 3 C 6 H 5 O 7 ), which had the greatest difference in the number of lithium ions per molecule, at five different concentrations in a range of 1 mM–25 mM. All concentrations of lithium citrate exhibited significantly higher acaricidal activity compared to LiCl, but there was no difference in the mortality of the bees (Table 3, Supplementary Tables S9 and S11). Therefore, lithium citrate might represent an even better active ingredient.

Table 3 Comparison of the efficacy and side effects of LiCl and lithium citrate using concentrations from 1 mM to 25 mM over a maximum feeding period of seven days. Full size table

As a lithium-free control and to rule out chloride as an active agent we also tested the alkaline salts sodium chloride (NaCl) and potassium chloride (KCl) and also magnesium chloride (MgCl) at 25 mM. We did not observe a varroacidal effect for NaCl or KCl (n = 3 cages, P = 1.000, log-rank test, Supplementary Table S12). In tests with MgCl 100% of the caged bees died within five days (P < 0.001, log-rank test), and according to the declining number of bees, the experiment was terminated before the effect on mites could be analysed. Based on these experiments, we concluded that lithium indeed mediates acaricidal activity in a dose-dependent manner and that lithium citrate exhibits the most favourable properties of all compounds tested so far.

Potential of lithium compounds as new varroacide

We have shown that not the initially hypothesized double stranded RNAs against essential Varroa genes but surprisingly lithium salts mediate a strong acaricidal effect on Varroa mites on caged bees and in artificial swarms. Thus, these results show that lithium compounds represent a new class of acaricidal agents with an outstanding potential and remarkably good tolerability by bees. The different susceptibility of mites and bees to LiCl is even more remarkable considering that due to dilution effects, the concentration of LiCl in the haemolymph of the bees will probably be substantially lower than the concentration fed to the bees.

Importantly, our findings do not imply that acaricidal effects of RNAi-based approaches as published by Garbian et al.32 are generally mediated by LiCl. After feeding of a mixture of dsRNA to honey bees over a period of 60 days, Garbian et al.32 recorded a slow increase in mite mortality with a final treatment efficacy of only 60%. In view of the quick and highly effective response of our artificial swarms to treatments with LiCl, different modes of action are likely: while RNAi mediated effects appear to exert long-term effects, lithium compounds represent an independent mechanism with fast onset and high efficacy.

As a varroacide, LiCl displays some features that are unique in this combination: (i) LiCl acts systemically via honey bee feeding (“easy-to-apply”), (ii) it is water soluble and will therefore not accumulate in beeswax which is a crucial problem for long-term treatment concepts using synthetic varroacides with lipophilic properties36,37 (iii) the oral toxicity of most lithium compounds to mammals is relatively low38 (iv) it has no repellent effect on the feeding solution within the relevant concentration range of 2–25 mM39 and (v) it is available at moderate prices. Highly promising is the fact that a single application of only 10 µl of LiCl in a 25 mM solution (corresponding to a dosage of 10.6 µg of LiCl) per individual bee is sufficient to kill phoretic mites. A challenge for further research will be the development of a smart application technique for full-sized swarms and colonies to ensure that all bees receive the critical amount of the active compound.

Currently, we do not know how LiCl is killing the phoretic Varroa mites, and there are few publications on the effect of LiCl in insects40. In human medicine, lithium has been used since the 1870s and is a mood-stabilizing agent indicated for the treatment of manic episodes and as maintenance treatment for bipolar disorder41. In view of their therapeutic use, lithium compounds and their toxicity profile have been carefully investigated. Thus far, a number of enzymes acting on metabolism, development, haematopoiesis and other processes have been proposed as potential targets42,43. These enzymes require metal ions and lithium exerts its activity in an uncompetitive manner, which most likely occurs by displacing a divalent cation. Admittedly, we have currently no indication that the observed miticidal effect of lithium compounds relies on a comparable mode of action.

We are also aware of the fact that our results represent only the first step towards the development of a new veterinary product. Field tests in free-flying colonies are just as necessary as analysis of sublethal and long-term side effects on adult bees and honey bee brood and possible residue problems in honey.

However, the results presented here already indicate that LiCl has potential as an effective and easy-to-apply treatment for artificial and natural swarms and particularly for the huge number of package bees used for pollination in the United States11,44. Furthermore, elucidation of the mechanism of action might open new avenues for the targeted development of veterinary products to combat Varroa mites.