Contact Effect Contribution to the High Efficiency ofLithium Chloride Against the Mite Parasite of theHoney Bee

Éva Kolics¹, Kinga Mátyás¹, János Taller¹, András Specziár²  and Balázs Kolics¹,^*
¹ Department of Plant Sciences and Biotechnology, Georgikon Faculty, University of Pannonia,
H-8360 Keszthely, Hungary; kolicseva@gmail.com (É.K.); petrovicsnemkk@gmail.com (K.M.);
taller@georgikon.hu (J.T.)
² Balaton Limnological Institute, Centre for Ecological Research, H-8237 Tihany, Hungary;
specziar.andras@okologia.mta.hu
^* Correspondence: bkolics@gmail.com

Abstract: Lithium chemicals have been proven to be very effective in eradicating Varroa destructor,
the detrimental parasite of the honey bee; however, little is known about the side effects on brood and
long term consequences on the colony. Earlier, it was proposed that the action mechanisms of lithium
chloride do not include the contact mode. Here, we investigate this question using a paper strip test
to demonstrate the concentration-dependent effectiveness of lithium in the contact mode of action,
confirming that it is also a contact agent against the Varroa mite. According to our knowledge, this is
the first report on the high varroicidal effect of lithium in the contact mode of action. Our findings
may open up possibilities for novel ways of treatmen

Keywords: lithium chloride; contact mode of action; Apis mellifera; Varroa

1. Introduction

Maintenance of commercial honey bee (Apis mellifera) colonies is highly dependent upon the
successful control of the parasitic mite Varroa destructor, recognized as the biggest threat to the western
honey bee worldwide. Left untreated, mites can kill an entire colony within one or two years [¹,²];
however, in areas of high bee density, it may occur within an apicultural season. Controls can be
effective and of low risk of building resistance [3–6], but in some countries, they are restricted mainly to
only a few chemicals, implying the potential development of acaricide resistance [7,8] and reducing the
possibility of mite eradication in the foreseeable future. Parallel to this, there is an increasing demand
to avoid the build-up of miticide residues or their metabolites in honey and wax. Alongside novel
RNAi-based approaches [9], it was observed that lithium salts may offer promising and easy-to-use
chemicals for effectively treating Varroa infestation. Furthermore, treatments have been published
where 100% mite mortality was found in the brood-free period with minor or no mortality of adult
bees, with certain concentrations of lithium-containing chemicals [10,11]. Lithium chloride has been
described as a varroicide that acts in a systemic mode of action in a wide range of concentrations [10].
High miticidal activity was exerted in artificial swarms applying 25 and 50 mM lithium chloride in
sugar syrup and patties, respectively [12,13].
Based on earlier unpublished attempts where lithium chloride showed high effectiveness at very
low concentrations, we supposed that it might have an additional effect in a contact mode of action.
The aim of this study was to test this hypothesis with insights from in situ application to enable
demonstration in commercial bee colonies.

2. Materials and Methods

Adult mites were freshly obtained from sealed brood cells and collected using a powder sugar
test of heavily infested Apis mellifera carnica colonies. Mites were placed onto a vertical paper towel
using a fine brush. To preselect vital individuals, mites that were unable to grasp strongly for about
30 min were discarded (6%). The remaining mites were kept at 25 ◦C for a maximum of 120 min in
order to prevent a decrease in vitality and mobility. Subsequently, mites were placed on experimental
paper strips one at a time with the help of sterile syringe needles. While transferring specimens from
the paper towel, an additional pre-selection was implemented, as only mites willing to climb onto
the needle by themselves were used (96%). Experimental paperboard strips (1.5 mm in thickness,
3 cm × 20 cm in area) were evenly impregnated with 2 mL lithium chloride solution (LiCl 1H2O) of one
of the 11 tested concentrations ranging from 10.78 mM to 11.04 M (10.78, 21.55, 43.11, and 86.22 mM,
and 0.17, 0.34, 0.69, 1.38, 2.76, 5.52, and 11.04 M). The strips were fixed on a flat glass surface at a
45◦ angle. Control strips were impregnated with deionized water from the same stock that we used
for preparing the lithiated solutions. One mite was placed onto each lithiated strip and another mite
onto the control strip at the same time (the number of individuals used for each exposure varied
between three and 11; the total number of mites used was 71 each for the treated and control groups).
After initiation, the first event recorded was the onset of tremorous movements accompanied by
uncontrollable movements. The second recorded event was when the mite fell off the strip, which we
considered to be the miticidal threshold. For overview of the in vitro experimental design, see Figure 1.
The concentration-dependent contact effect of LiCl on the log10 transformed time of the first
tremorous movement and of the drop of the mites was evaluated by analysis of variance (ANOVA)
followed by Tukey HSD post hoc tests. Levene’s test for the homogeneity of variance of data
(F10;60 = 1.45, p = 0.180 for time to first tremorous movement and F10;60 = 1.65, p = 0.114 for time to
drop) and the Kolmogorov–Smirnov test of normality on residuals (N = 71, D = 0.103, p = 0.405 for
time to first tremorous movement and N = 71, D = 0.109, p = 0.345 for time to drop) proved that
the assumptions of the ANOVA were met. Since none of the control mites showed any tremorous
movements or dropped down from the experimental paperboard during the 120 min of the observation
period, the control groups could not be included in the ANOVA test. Therefore, the Z-test was used
to compare the proportion of mites that responded (i.e., showed tremorous movement and dropped)
between the control and LiCl treatments.
In order to demonstrate that lithiated strips show in situ effectiveness, strips were prepared by
impregnating each strip with the amount used for a trickling dose for one hive (2.28 mL 5.52 M LiCl 1
H2O = 0.76 g LiCl 1 H2O). This demonstration was carried out in three broodless commercial bee
colonies, in the pre-wintering period in November 2019, at Keszthely, Hungary (GPS: 46◦45055.6” N,
17◦14052.6” E), registering the number of mites counted on the sticky board. Commercial colonies
were selected according to their previous mite fall; we picked these colonies from the most infected
ones of an apiary of 120 colonies, based on the mite fall carried out using Apivar strips at the end of
September in the broodright stage. At that time, mite fall was 116, 102 and 67 for hives No. 1, 2 and 3,
respectively. Additionally, in all of these colonies, bees with mites on the thorax could be observed in
October, suggesting an elevated level of infestation. The colonies had not been treated from September
until the start of the experiment in order to preserve the mites. Experimental hive No. 1, used as
control, was left untreated until the end of the trial. In hives No. 2 and 3, treatment was started with
one lithiated strip placed in the middle of the nest. Colonies were broodless in order to assure that the
varroicide effect was not influenced by the addition of hive-born mites. After tracking the effect for
five days by counting mites, five additional strips were inserted and the recording of mite fall was
continued. Finally, all three hives were controlled by trickling lithiated syrup (40 mL, 250 mM) at the
same time.

1. Spivak, M.; Reuter, G. A Sustainable Approach to Controlling Honey Bee Diseases and Varroa Mites; USDA:
Washington, DC, USA, 2005; pp. 1–6. ↩︎
2. Barlow, V.M.; Fell, R.D. Sampling Methods for Varroa Mites on the Domesticated Honeybee; Virginia Cooperative
Extension: Virginia, VA, USA, 2006; pp. 1–3. ↩︎