Lithium chloride treatments in free flying honey bee colonies: efficacy, brood survival and within colony distribution

doi:10.21203/rs.3.rs-3235022/v1

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Lithium chloride treatments in free flying honey bee colonies: efficacy, brood survival and within colony distribution

https://doi.org/10.21203/rs.3.rs-3235022/v1

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published 22 Dec, 2023

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The efficacy of various Lithium Chloride (LiCl) applications in eradicating the parasitic mite Varroa destructor in honey bee colonies was investigated, with a specific focus on its impact on brood development. In broodless colonies (3 weeks post queen caging), the highest mite mortality rate of 98% was achieved with a 9-day treatment of 2.5 kg of 50 mM LiCl fondant candy. A shorter 5-day treatment with 2 kg of 50 mM LiCl candy resulted in a mite mortality rate of 77.5%. In colonies with brood, a repeated short-term application of 4*0.5 kg 50 mM LiCl candy yielded an efficacy of 88%. LiCl led to a removal of the first reared brood after release of the queen. However, no long-term effects on colony growth were observed, and the colonies successfully overwintered. Additionally, the study demonstrated that Lithium is rapidly distributed among the bees of a colony within 2 days, yet only low concentrations were detected in stored food samples. This suggests that the bees efficiently absorb and distribute LiCl within the colony. The harvested honey in the following spring revealed a lithium concentration of 0.1–0.2 mg/kg, which is below naturally occurring lithium levels in honey. Based on these findings, LiCl can be considered an effective and easy-to-apply acaricide in broodless colonies, and even in colonies with brood, it had no long-term effects on colony survival. Further research may be necessary to determine the optimal treatment period for achieving an efficacy over 95%.

Varroa destructor

Apis mellifera

lithium chloride

brood survival

distribution

The negative impacts from globalisation are exemplified by the worldwide spread of the honey bee mite Varroa destructor, a striking example of the redistribution of diseases and parasites in wildlife and livestock (Travis et al. 2011). Varroa destructor is originally a parasite of the Eastern honey bee Apis cerana in Asia. Intensive trade of managed Apis mellifera colonies during the last century enabled the mite’s shift to this new host and promoted widespread distribution within a few decades (Chantawannakul et al. 2016). The mite arrived and established itself in Australia in 2022, hitherto the last continent free of V. destructor infested hives (Commonwealth of Australia 2022; Vince 2023).

V. destructor causes severe morbidity and mortality of entire A. mellifera colonies by feeding on host tissues and transmitting viruses (Rosenkranz et al. 2010; Traynor et al. 2020). Natural Varroa-resistance in the new host has - with few exceptions - only evolved in regions with huge feral honey bee populations, predominantly in tropical and subtropical regions (Le Conte et al. 2020; Locke 2016; Rosenkranz 1999; Seeley 2017). In the temperate climates of Europe and North America varroosis continues to be the major cause of colony losses despite decades of experience with and selection against this parasitic mite. Regular Varroa treatments are indispensable, for without beekeeper intervention, colonies dwindle (Genersch et al. 2010; Gray et al. 2019; Hernandez et al. 2022; Seitz et al. 2015; Stahlmann-Brown et al. 2022). Colony losses due to Varroa infestation continue to be high for two main reasons. To date we have had limited success in breeding viable Varroa-resistant honey bees (Büchler et al. 2010; Mondet et al. 2020a; Mondet et al. 2020b). Additionally, the biology of V. destructor hinders the effectiveness of treatments. The life cycle of V. destructor can be divided into a dispersal phase, where the mites are riding on adult bees consuming their haemolymph and fat body and a reproductive phase, which exclusively takes place inside the capped brood cells of the host (Traynor et al. 2020). During the season, when brood is present in the colonies, the majority of the mite population is located within these capped brood cells (Fuchs 1985, 1992; Ifantids 1988) and thus protected against the efficacy of nearly all common acaricides (Rosenkranz et al. 2010). A further challenge to controlling V. destructor under temperate climate conditions is that (i) chemical treatments should be avoided during nectar flows when bees collect nectar to make honey, yet (ii) a treatment must be performed in late summer before the bees rear their long-lived winter bees, so that their longevity is not compromised through high levels of parasitisation. High mite infestation levels in late summer and the subsequent damage to winter bees, as mite levels continue to be high in fall, are the main drivers of winter colony losses (Genersch et al. 2010; Gray et al. 2019; Seitz et al. 2015). The relatively small window of time for late summer treatment increases the need for highly effective varroacides in mite management strategies. Several varroacides are registered, however the majority are based on only a few active compounds such as formic and oxalic acid, thymol and a few synthetic substances from the group of pyrethroids and amidine (Mutinelli 2016; Qadir et al. 2021; Vilarem et al. 2021). None of these registered products fulfill all desired characteristics of an “ideal” veterinary drug: 1) high and reliable efficacy, 2) limited side effects, 3) no residues in bee products above critical thresholds, 4) low risk of mite resistance and 5) easy-to-apply. The widely used organic acids are poorly tolerated by bees or brood with significant impacts on colony size, especially in warm climates (Elzen et al. 2004; Satta et al. 2005). In particular formic acid, a common agent used in late summer as it penetrates into the brood nest, requires specific environmental conditions and its efficacy is strongly influenced by colony factors, such as colony size and amount of colony thermoregulation (Pietropaoli and Formato 2019; Steube et al. 2021; Underwood and Currie 2003). Repeated applications are necessary to achieve sufficient efficacy, which makes the use of organic acids time consuming and thus costly, especially in large beekeeping operations (Berry et al. 2022). Synthetic varroacides often result in accumulating levels of residues in beeswax, with potential impacts on later larval survival and queen quality (Albero et al. 2023; Haarmann et al. 2002; Kast et al. 2021), and frequent reuse leads to the development of mite resistance (Higes et al. 2020; Mitton et al. 2022). Due to these shortcomings of the currently options for chemical control of Varroa, there is still urgent demand for further varroacidal compounds.

The recently discovered lithium chloride (LiCl) meets many of the above mentioned requirements for a varroacide (Ziegelmann et al. 2018) and so we wanted to examine its efficacy and distribution in free flying colonies. LiCl is a widely distributed salt (Szklarska and Rzymski 2019), a natural component of honey (Bogdanov et al. 2008; Karabagias et al. 2017), and is used in therapeutic treatments of bipolar disorders and depression (Ferensztajn-Rochowiak et al. 2021; Gomes-da-Costa et al. 2022). LiCl is very effective at killing mites via contact and displays a systemic mode of action, while being well-tolerated by adult honey bees (Kolics et al. 2020a; Kolics et al. 2020b; Ziegelmann et al. 2018). A limitation for a broad applicability of LiCl is currently the low tolerability by honey bee brood (Rein et al. 2022).

Changing climate is altering the way beekeepers manage colonies, as often colonies no longer have a natural brood break which enables an effective winter treatment. In southern Europe it has therefore become common to cage queens after the summer honey harvest and then treat the mites in the dispersal phase on adult bees when colonies are broodless (Büchler et al. 2020; Lodesani et al. 2014). LiCl is an ideal compound under these circumstances, due to its high efficacy and good tolerability by adult bees (Stanimirovic et al. 2021; Ziegelmann et al. 2018). Some field tests with lithium salts have already been performed, however most of them used a repeated dribbling method similar to the application of oxalic acid (Jovanovic et al. 2022; Kolics et al. 2020b; Kolics et al. 2022).

In our approach we wanted to capitalize on the systemic mode of action of LiCl, i.e. the administration of the active substance via food to the colonies. This new application method would enable a quick and “easy-to-apply” treatment. We present the efficacy and distribution of LiCl after different food applications in broodless colonies under realistic field conditions with a special focus on the subsequent development of the brood. In one of these studies, we compared the broodless application with repeated short-term treatments in brood rearing colonies. We hypothesized that such short-term treatments would reduce the exposure time and risk of honey bee larvae to LiCl and therefore potentially reduce the loss of brood as described in Rein et al. (2022).

2.1. Experimental Setup

Field trials were performed in two apiaries. The preliminary experiment in 2018 took place at the field station Heidfeldhof of the University of Hohenheim (48°42'55.3"N 9°10'49.2"E), whereas the main experiments in 2021 and 2022 were performed in the local apiary of the State Institute of Bee Research at the University of Hohenheim (48°42'32.7"N 9°12'38.9"E). Each colony consisted of a total of 20 frames in two Zander size brood boxes with similar colony strength and headed by healthy sister queens.

The setups of the different experiments are shown in Table 1. Our main objective was to investigate different applications of LiCl in broodless colonies during summer, so to achieve broodless colonies we first had to cage the queen. Each queen was placed in a small plastic cage (Varroa control box, Rubee®) on a frame in the middle of the brood nest in the bottom hive body at the start of each trial (Fig. 1). The cages are designed with queen-excluder sized material, allowing worker bees to pass through while confining the queen. After 3 weeks, when the last of the brood emerged, the queen was released and LiCl food was applied.

In the preliminary experiment we tested two different LiCl applications with 25 mM LiCl syrup and 50 mM LiCl candy and compared the efficacy to control colonies treated with 250 ml formic acid (60%, Nassenheider® evaporator) (Table 1).

In the main experiments we used 50 mM LiCl candy, as it was the more promising approach due to ease of application combined with a slower, more consistent consumption of the LiCl food. In 2021 we tested a single application (A1) of 2.5 kg 50 mM LiCl (fed via a plastic bowl protected by an empty super) and compared it with the control group, treated with 250 ml formic acid (60%, Nassenheider® evaporator) (Table 1). In 2022, we modified the LiCl application and offered the candy via small Ziplock plastic bags (1L SafeLoc^®, Toppits^®) put directly on top of the frames (A2) (Fig. 2), due to ease of application and to provide ready access for the bees. We also shortened the treatment period from 9 to 5 days to eliminate exposure of the newly developed larvae with LiCl. A flipped top feeder (Nicot^®) provided enough space for the bees to access the treatment food.

To test whether a repeated short-term application (A3) can prevent brood removal with the same Varroa control efficacy, we applied 0.5 kg LiCl candy four times in 7-day intervals without caging the queen, meaning that all brood stages were present during the treatment. The plastic bags with these 0.5 kg candy were emptied within 2 days of application, therefore the treatment period was defined as 4 x 2 days.

Data were collected on Varroa mite mortality, brood development, and distribution of lithium in sampled bees and stored food as described below.

Table 1

Specifications of the tested treatment methods with acronym, LiCl concentration used, treatment period, number of tested colonies and intervals of data collection for the preliminary and full experiments
Treatment Method		LiCl Concentration	Treatment Period [days]	Number of Colonies	Start of Brood Assessment	Sampling Interval of Bees and Food
Preliminary Experiment
Control:	250 ml formic acid; Nassenheider®	-	8	4	-	-
P1:	7 litres syrup; 1 day after ROQ*	25 mM	8	4	-	-
P2:	4.5 kg candy; 1 day after ROQ*	50 mM	8	5	-	-
Experiment I − 2021
Control:	250 ml formic acid; Nassenheider®	-	9	9	-	-
A1:	2.5 kg candy; on the day of ROQ*	50 mM	9	6	5 days after application	4 days
Experiment II − 2022
A2:	2 kg candy; on the day of ROQ*	50 mM	5	10	3 + 19 days after application	2 days
A3:	4*0.5 kg in 7 day intervals; breeding colonies	50 mM	4 x 2	10	0 + 16 days after 1st application	2 days
*ROQ = release of queen

2.2. LiCl food production

For the syrup we used Apiinvert® (saccharose, fructose and glucose, Suedzucker Group©) and mixed it with the respective amount of lithium chloride (> 99.9%, p.a., ultra-quality, Roth®) to reach a 25 mM concentration (1.06 g LiCl per litre). We added food colouring (“Tannengrün”, Städter®) to the syrup, which dyed it green, so we could identify where the syrup was stored in the combs. Such food colouring has no negative impact on honey bees or honey bee brood (Ehrenberg et al. 2019).

The candy was made from powdered sugar (Suedzucker Group©) and honey produced from summer nectar, mixed in the ratio of 2:1. Additionally a concentration of 50 mM LiCl and red food colouring (Allura Red AC, Sigma-Aldrich®) was added into the mixture. For 1 kg of candy we used 1.57 g LiCl dissolved in water. This was mixed until the food colouring was evenly distributed using a commercial dough mixer (ITR50 2V “Evo”, Prismafood DE).

2.3. Varroa mite mortality and calculation of efficacy

The mite mortality was recorded by counting the mites on the sticky board inserted under the screened bottom board of the hive. The sticky board was prepared with a single layer of kitchen paper towel, moistened with oil, which prevents other insects from removing the dead mites. The natural mite fall was recorded two weeks prior to the treatments. Dead mites found on the sticky board during LiCl food application and the consecutive 7 days where considered killed by LiCl. For the control colonies treated with formic acid we considered dead mites on the sticky board from 1st till 20th day after treatment administration. Formic acid penetrates the sealed brood cells to kill mites inside capped cells, which is why we extended the monitoring period to cover an entire brood cycle.

Each colony received a follow-up treatment for four to six weeks with either Bayvarol® (2018 and 2022) or Apivar® (2021). We alternated the compound for the follow-up treatments to avoid development of mite resistance. The efficacy in % of the main treatment was calculated as follows:

$$\frac{number of mites killed by main treatment}{number of mites killed by main treatment+follow-up treatment}\times 100$$

2.4. Honey bee brood survival

To evaluate the effects of the different LiCl treatments on brood, we conducted a brood assessment of newly laid eggs in the main experiment. We marked the position of eggs on a transparent acetate sheet (= brood area fixing day (BFD)), according to the method described by Schur et al. (2003). We inspected the viability of the brood every 4 days and marked cells with viable larvae as well as cleared out cells on a new transparent sheet. As eggs don’t hatch for three days, the exact age of the larvae varied between 1 and 72h. To keep the error rate low and to guarantee that empty cells were cleared out and the bees hadn’t emerged, we terminated the assessment on day 16 (BFD + 16). From these data we calculated the brood survival rate for each assessment.

After being caged for 3 weeks, the queen needs a few days to start laying eggs again, therefore we started the brood assessment in treatment group A1 5 days after the release of the queen and the start of the treatment application. In treatment group A2 we were already able to find enough eggs 3 days after the release. In 2022, we not only examined the development of the first brood cycle, but also a second brood cycle 16 days later to evaluate possible long-term effects (Table 1) for both the A2 and A3 treatment groups.

2.5. Distribution of lithium among worker bees, stored food, and residue in honey

An even distribution of lithium among the worker bees is important for a sufficient treatment efficacy. In 2021. we took bee samples (approx. 30 bees) from each treated colony at regular intervals during and post treatment (as shown in Online Resource 1). In 2022, we took bee samples from the central frames of the hives every other day for 3 weeks in trial A2 and for 4 weeks in trial A3, as well as collecting additional bees 39 days post-treatment for both groups. Stored food samples were taken once a week until day 39 post-treatment from 4 combs containing food stores in the top hive body.

To investigate whether the LiCl treatments had an effect on the lithium levels in food and honey of the following year, we took a sample from the brood chamber of each colony before installing the honey supers on 19th of April 2023. Three weeks later, we took samples from the honey super of each colony that already collected nectar. On the 9th of June the honey was harvested for each treatment group individually (A2 and A3) and samples were analysed.

After sampling, the bees were frozen (-20°C) until dissection of the honey crop. Bees were thawed and we gently pulled on the abdomen with forceps, which exposes the crop. The crops of 20 bees per sample were pooled, homogenized and analyzed with ICP-MS (Inductively Coupled Mass Spectrometry) for the concentration of lithium. The samples of stored food from the colony were also frozen (-20°C) until analysis.

For ICP-MS analysis about 500 mg of honey or crop samples, respectively were weighed into glass tubes. Two ml of HNO₃ were added and tubes were filled up with double distilled water to a final volume of 10 ml and vortexed to ensure sample homogeneity. Following this, microwave digestion was done with an Ultra Clave III (MLS Mikrowellen-Labor-Systeme GmbH, Leutkirch, Germany) where temperature was gradually increased from 80°C to 200°C at 900 W and 100 bar. Samples were cooled down and diluted adequately for ICP-MS analysis (NexION 300 X, PerkinElmer, Waltham, MA, USA). A ICP multi-element standard solution (Merck KGaA, Darmstadt, Germany) was used for calibration at concentrations of 0.1 µg/l, 0.2 µg/l, 1 µg/l, 10 µg/l, 20 µg/l prepared with double distilled water and HNO₃. A CertiPUR Rhodium ICP-standard solution (c = 1000 mg/l Rh, Merck KGaA, Darmstadt, Germany) served as internal standard (LOQ < 0.025 mg/kg).

2.6. Statistics

The data were statistically analysed using JMP Pro 16. Data on efficacy were checked with a Shapiro-Wilk test for normal distribution and evaluated with ANOVA followed by the Wilcoxon Rank Sum-Test. For comparison of the 1st and 2nd brood assessment we used the Chi-Square-Test.

3.1. Efficacy of different treatments

In the preliminary experiment in 2018, the broodless colonies treated with LiCl demonstrated an efficacy of 92.0 ± 4.5% for the syrup application (P1) where each colony received 7 litres of 25 mM LiCl spiked Apiinvert®. For the candy application (P2) we applied 4.5 kg 50 mM LiCl which resulted in a mean food uptake of 2,040 ± 786 g per colony in 8 days (additional data are given in Online Resource 2) and an efficacy of 89.8 ± 5.6%, with no significant difference between the treatments (Fig. 3p = 0.54, Wilcoxon Rank Sum Test, Online Resource 3). Treatment with LiCl outperformed the control treatment with formic acid, where only 59.5 ± 8.5% of the mites were killed. The formic acid treated colonies did not have caged queens, as the treatment penetrates into the brood nest and we wanted to compare LiCl results with the most commonly used method of Varroa control in Germany.

In the years 2021 and 2022, LiCl was exclusively applied via candy (Fig. 4). For treatment A1 each colony received 2.5 kg 50 mM LiCl candy, which resulted in a mean food uptake of 1,739 ± 167 g in 9 days and the highest mite mortality with 98.1 ± 0.7%, followed by A3 with 87.9 ± 10.5% and A2 with 77.5 ± 12.8%. For treatment A2 and A3 each colony consumed a total of 2 kg of 50 mM LiCl candy, which incorporates to 3.1 g LiCl-salt (additional data are given in Online Resource 2). Control treatment with formic acid was the least effective (69.8 ± 25.2%) and showed the highest variation between colonies, ranging in efficacy from 22.4–96.9%. Due to higher variance in A2 and A3, they did not differ significantly from the control treatment (p = 0.71 and p = 0.07 respectively, Wilcoxon Rank Sum Test, Online Resource 4) (Fig. 4).

The total counted mites in 2021 showed an average mite load per colony of 3,087 ± 915 for control colonies and 2,129 ± 967 for A1 colonies, whereas in 2022 the mite loads were on average 1,429 ± 1,155 mites for the A3 colonies and substantially lower in the A2 colonies with 456 ± 296 mites (Table 2).

Table 2

Total number of dead mites counted during the observation period (main treatment and follow-up treatment) and mean mites per colony ± standard deviation. Statistical different mite counts are indicated by different letters (Wilcoxon Rank Sum Test p < 0.05). Control colonies were treated with 250 ml 60% formic acid. For A1 we applied 2.5 kg 50 mM LiCl over period of 9 days; A2: 2 kg in 5 days; A3: 4*0.5 kg 50 mM LiCl candy in seven-day intervals
Treatment	Year	Total Mites	Number of Colonies	Mean Mites per Colony
Control	2021	27,783	9	3,087 ± 915 a
A1	2021	12,776	6	2,129 ± 96 ab
A2	2022	4,558	10	456 ± 296 c
A3	2022	14,291	10	1,429 ± 1,155 bc

3.2. Honey bee brood survival

In total we assessed 9,463 brood cells from the freshly laid egg stage in 6 colonies of A1 and 10 colonies for both brood assessments of A3. Due to absence of eggs in two colonies on the assessment day, we could only examine the brood development for 8 of 10 colonies in A2.

Treatment A1 with 9 days of LiCl treatment led to the highest brood removal rates and only 4.7% of the brood survived until BFD + 16 (Fig. 5). The first assessment of A2 (A2-1) showed a high variability in the survival probability of brood from the 8 treated colonies with a mean brood survival rate of 45%. The 2nd assessment (A2-2), which started 19 days after the LiCl application, had the highest brood survival rates with 82%, which was statistically different to the first assessment (p = 0.02, Chi²-Test). The mean brood survival rate of the first assessment in A3 was 52% and thus similar to the first assessment of A2. However, in the 2nd brood assessment (A3-2), which started 2 days after the 3rd LiCl application, the brood survival rate decreased to only 8%, which was significantly lower than A3-1 (p = 0.004, Chi²-Test).

Colony winter survival

In winter 2021, we lost one colony from the control group due to Nosema disease. In the 2022 experiments, we found one colony dead in the following spring probably due to queen loss in fall. However, the queen was still present during the first brood assessment (A2-1), however, during the second assessment (A2-2), we were not able to find eggs. All the other colonies overwintered well.

3.3. Distribution and detection of LiCl

In colonies treated over a period of 9 days (A1), we took 66 stored food and 61 adult bee samples over a period of 21 days. The 1st sampling was conducted before LiCl application, showing no lithium contamination in bees nor in food (Online Resource 5). Within 5 days of commencing the treatment, the concentration of lithium in the honey crop of bees sampled from the central area of the lower hive body rose to 69 mg/kg lithium and doubled four days later to reach the maximum of 131 mg/kg. After the remaining LiCl food was removed on day 9, the lithium concentration quickly dropped through day 17; thereafter it increased slightly during the last sampling 21 days post treatment. On no sampling day was there a significant difference between the lithium concentration from bees sampled from top or bottom hive body in our two brood chamber set-up, indicating an equal distribution within the colony. We also saw similar distribution patterns among the sampled colonies, indicated by the low standard error. The increase in the lithium concentration in the honey crop was mirrored by the number of fallen mites. The mite drop reached a maximum on the fifth day, when a mean of 983 mites dropped, decreasing thereafter as shown in the figure (Online Resource 5).

In the stored food samples, we first detected lithium in uncapped cells on day 9 after commencing the LiCl treatment (25 mg/kg), whereas the first detection in capped cells was on day 13 (4.4 mg/kg). The lithium concentration was always lower in samples taken from capped cells and was significantly lower on day 17 with 5.2 mg/kg lithium compared to 36 mg/kg in open cells (p = 0.013, Tukey-Kramer HSD) (Online Resource 5).

In 2022 when colonies were treated for 5 days (A2), 130 bee samples were taken from the treated hives. From the colonies repeatedly treated for 4 x 2 days (A3) 170 bee samples were taken. Lithium concentration in the honey crop of bees from the A2 treatment displayed a similar distribution as in A1 group with a peak of 104 mg/kg lithium on day 3, decreasing after termination of treatment (Fig. 6). Maximum lithium concentration in stored food occurred 13 days after the start of the treatment (18 mg/kg).

For the repeated short-term treatments used in A3, we detected several short peaks after each application of the 0.5 kg LiCl candy (Fig. 7). The highest concentration in the honey crop was found on day 16 after the 3rd application with 98 mg/kg lithium. The concentration of lithium in the sampled food reached a maximum on day 28 with 20 mg/kg. For both treatments, A2 and A3, we took the last samples 39 days after the last application of LiCl and measured 15.4 mg/kg lithium in the A2 treated colonies and 13.2 mg/kg in the A3 treated colonies.

The stored food sampled from brood chambers in Spring 2023 revealed low concentrations of 5.4 ± 2.4 mg/kg lithium in A2 treated colonies and an even lower concentration of 3.1 ± 2.4 mg/kg in the A3 treatment group (Table 3). In the freshly collected nectar from honey supers, we measured 0.8 ± 0.7 mg/kg lithium from A2 colonies, which was further reduced to 0.1 mg/kg in the harvested honey. For A3 we measured 0.2 ± 0.2 mg/kg and 0.2 ± 0.2 mg/kg, respectively (Table 3).

Table 3

Lithium concentration in spring 2023 of samples from stored food of brood chambers, freshly collected nectar in honey supers and harvested honey from colonies treated in summer 2022. Each given as mean ± standard deviation for the treatment groups. Number of analysed samples are given in brackets
		Lithium [mg/kg]
Treatment Method	Brood Chamber	Honey Super	Honey
A2	5.4 ± 2.4 (n = 9)	0.8 ± 0.7 (n = 6)	0.1 ± 0 (n = 4)
A3	3.1 ± 2.4 (n = 10)	0.2 ± 0.2 (n = 5)	0.2 ± 0.2 (n = 4)

Our experimental results demonstrated high efficacy for lithium chloride applications in colonies with and without brood when applied by feeding in syrup or candy. In the preliminary experiments in the year 2018 with broodless colonies, we already reached a mean efficacy of more than 90% with both the syrup and the candy application. We then decided to continue with the candy treatment, because of the slower consumption by the bees which enables a continuous flow of LiCl into the colony for more than one week through a single feeding. The field test with broodless colonies in the year 2021 revealed an even higher efficacy of 98%, which was significantly different from colonies treated with the standard formic acid Varroa control in the same apiary. This confirmed that the high efficacy of LiCl, which had previously been demonstrated by Kolics et al. (2020b) with the dribbling method, can also be achieved using a single application of 2.5 kg candy spiked with 50 mM LiCl. The next field test with broodless colonies in the year 2022 had only a 78% efficacy. Two reasons could potentially have caused this significantly lower efficacy compared to the 2021 study. First, we reduced the duration of the treatment from 9 to 5 days to reduce the impact on brood loss in the first subsequent brood cycle seen in the 2021 experiment (see below). But this shortened period was obviously not sufficient to achieve a high efficacy. Second, the mean infestation per colony was 456 mites compared to 2,129 in the year before. In colonies with relatively low mite numbers, the reinvasion of mites in late summer and early fall (Frey and Rosenkranz 2014), a common occurrence in apiaries in this part of Germany, can have a greater impact on the calculation of the efficacy. Our experimental apiaries were not in the direct vicinity of colonies managed by other beekeepers, however even over a distance of 1.5 km an invasion of more than 100 mites per colony is possible (Frey et al. 2011).

Our repeated short-term LiCl treatments of colonies with brood also revealed a good efficacy of 88% and confirmed the results of former field tests with lithium salts in breeding colonies (Stanimirovic et al. 2021), however at the cost of high brood removal rates (see below).

Our analysis of honey bee crops and stored food for traces of lithium demonstrated an even distribution of the active ingredient lithium within the colony, despite food uptake differing somewhat between colonies. This low variance between colonies in the distribution of the active ingredient Lithium and the efficacy of treatment results in high levels of treatment success throughout an apiary, which cuts down on varroa reinvasion and potential transfer of mites between strong and weak colonies (Giacobino et al. 2023).

In contrast to currently available varroacides, our application method of lithium chloride takes advantage of the social behaviour of food sharing in bees, called trophallaxis (LeBoeuf 2017). This leads to a rapid distribution of the treatment food throughout the whole colony (Crailsheim 1998; Nixon and Ribbands 1952) with sufficient amounts of lithium to kill Varroa mites in sampled bees within 48 hours of application, as demonstrated by the spike in mite fall within two days of LiCl administration (see Fig. 6). We always detected substantially lower levels of lithium in the food samples than in the crop of the sampled bees. In 2021, when about 1.7 kg of a 50 mM candy was applied per colony, the maximum lithium content found in open food cells was 36 mg/kg, whereas in the crop it rose to 131 mg/kg. In 2022 when the feeding period was reduced from 9 to 5 days, the maximum lithium levels were somewhat lower, with similar low levels in food compared to bee crops as seen in 2021. To produce honey from nectar or stored food from a beekeeper applied food source like syrup or candy, honey bees rework the food source multiple times, before permanently storing it in a cell (Park 1925). During this food processing, the bee’s ventriculus removes contaminations and excess water. Honey bees also have the capacity to remove heavy metals from collected nectar in the process of producing honey (Borsuk et al. 2021), which could help explain why we always detected substantially lower lithium concentrations in stored food compared to crop samples.

As shown in previous studies, exposure to lithium chloride during larval development can result in high brood mortality (Rein et al. 2022). We thus paid particular attention to the survival of the first batch of brood reared after release of the queen. In 2021, the long treatment period of 9 days resulted in low brood survival, most likely due to an overlap of applied LiCl food and the presence of the first larvae. To avoid this loss of brood, we shortened the treatment period to 5 days in 2022, reducing the exposure of the sensitive early larval stages to LiCl. Brood survival increased, but with high variance in survival rates among the colonies. We assessed a second brood cycle, which showed that there are no long-term effects to brood survival from LiCl as was also observed in 2021. This is also supported by the fact that 34 colonies treated with LiCl (from all experiments) overwintered well and only one colony died due to queen loss in fall 2022. Thus although we see a short time frame of brood loss immediately after treatment, the colony then successfully rears brood and does not suffer any population loss from this brief interruption.

Other studies tested different lithium chloride applications in broodless colonies with even higher concentrations, but failed to evaluate the aftereffects on brood reared post treatment (Kolics et al. 2022). Stanimirovic et al.(2021) carried out lithium citrate treatments in colonies with brood and stated that there were no side effects observed during the year, which is too long an observation window to catch any carryover effects of lithium citrate on brood post treatment. Our experiments with repeated short-term treatments in colonies with a free-roaming queen and brood (A3) clearly confirm the low tolerability of honey bee larvae for LiCl. The goal with this new application method was to limit the contact of larvae to LiCl for a maximum of 1–2 days by feeding the treatment repeatedly in small amounts. However, this did not provide the desired reduction in brood loss. Both the first and second brood assessment during application resulted in low survival rates and so clearly a treatment with lithium chloride in colonies with brood is only possible at the cost of lost brood during the application period. As seen in our studies, the side effects on brood only occur during and shortly after the period of the treatment when lithium contaminated food is still circulating among the nurse bees.

From our comprehensive studies on LiCl, we can conclude the following regarding efficacy, distribution of the compound within the colony and side effects on brood for the treatment of broodless colonies:

A single feeding of 2 kg of LiCl candy kills more than 95% of Varroa mites under field conditions when exposure lasts 9 days. It could potentially be shortened, but 5 days was too short.

Side effects on the honey bee brood occurred only in the first brood cycle laid directly after the end of the application. If queens were released one week after treatment cessation, these side effects could largely be prevented.

Our data on the concentration and distribution of lithium within the worker bees of a treated colony indicate that within 48 hours lithium levels reach their maximum level within the bee´s crop and remain elevated for the duration of the treatment. Even some days after the end of the application the lithium concentrations within the crops of the bees should still be high enough to kill the parasitizing mites.

These results suggest that a promising strategy for future applications would be to start the treatment via candy one week before the release of the queen, when the colony only contains capped brood. The lithium concentration in the bees at the time of queen release should be sufficient to kill the few mites emerging with the last brood cells, yet subside to a harmless level by the time the first larvae hatch from queen laid eggs.

The long-term circulation of lithium in colonies post treatment and thus the potential risk of residue accumulation in the honey must be investigated before lithium-salts can be authorized as a varroacide. Lithium occurs naturally in some honeys (2.25–15.6 mg/kg) (Bogdanov et al. 2008), mineral water (1.7µg/l – 1,725µg/l) (Seidel et al. 2019), and even other beverages like wine and soft drinks (Seidel et al. 2020). We even found lithium at a concentration of 0.27 mg/kg in the APIINVERT® syrup sold as feed on the German market. It is thus hard to define an acceptable residue level, though the naturally occurring range in honey suggests a higher limit than with other varroacide residues is warranted. What is considered acceptable is currently debated; Kolics et al. (2021) found an increase in lithium in uncapped honey directly after application but claimed a “full recovery” by day 22 with a concentration below 0.25 mg/kg. Yet they found a concentration of 22.4 mg/kg in the ripe honey on day 28. In contrast, Stanimirovic et al. (2021) described an increase in lithium from honey supers from 0.018 mg/kg to 0.035 mg/kg seven days post treatment with lithium citrate as significant. Another study detected lithium concentrations seven days post-treatment between 0.05 and 0.9 mg/kg (Prešern et al. 2020). Our residue analysis of honey and stored bee food was conducted in the spring of the following year, which was almost ten months post treatment. We found lithium at rates of 3.1 to 5.4 mg/kg in the stored food of the brood chamber. Colonies were fed an additional 15–20 kg of sugar syrup post treatment to provide them with enough winter food stores, which must have diluted the lithium concentrations. In freshly made spring honey we harvested, we only found 0.1–0.2 mg/kg lithium which is far below the natural occurring concentrations. Our treatment method of feeding LiCl in candy does not lead to undesirable residues in honey. From a human nutrition perspective, lithium compounds are considered valuable micronutrients and a daily intake of 1 mg/day for a 70-kg adult is recommended (Schrauzer 2002; Szklarska and Rzymski 2019).

Lithium provides many advantages compared to current varroacides on the market. The most frequently used non-synthetic varroacides in Middle and Northern Europe are currently formic acid in the summer and oxalic acid in the winter, both of which require specific environmental or colony conditions for high efficacy and good tolerability (Adjlane et al. 2016; Steube et al. 2021). Our new application method, unlike these organic acids, allows the beekeeper to be independent of environmental factors. Even though caging the queen, in order to create a broodless period requires additional effort and time, especially in large beekeeping operations, such caging is becoming more standard as a method of integrated Varroa control (Büchler et al. 2020; Gregorc et al. 2017; van der Steen and Vejsnæs 2021). A brood interruption not only has the advantage that all mites are forced to reside on the adult bees, which makes them vulnerable to varroacides, it also interrupts the mite’s reproductive cycle which slows down this parasite’s population growth (Gabel et al. 2023; Jack and Ellis 2021; Rosenkranz et al. 2010). In the colonies with the repeated treatments where we allowed bees to keep rearing brood, three times as many mites fell (compare A3 with brood to A2 without brood in Table 2), even though the natural mite fall before the treatment was similar in both groups. Giacomelli et al. (2016) showed that queen caging alone resulted in a 40% reduction of Varroa populations, but should always be used in combination with a varroacide to achieve sufficient mite mortalities. As LiCl is a natural salt, the combination of queen caging and LiCl application could be a treatment option for organic beekeepers, too. A comparison of the additional workload generated by different summer brood interruption methods showed that queen caging was one of the least labor intensive ones with no impact on colony strength between the different methods (Büchler et al. 2020). A broodless period during late summer allows beekeepers to remove old combs and replace them with new ones, reducing pesticide residues in the lipophilic beeswax, which has been shown to have a beneficial effect on the productivity of the colony (Berry and Delaplane 2001; Taha et al. 2021).

When caging the queen is integrated into a varroa treatment strategy, the application of LiCl via candy feeding represents an effective and “easy to apply” treatment due to its rapid action and even distribution within a colony, regardless of external weather conditions. The systemic mode of action makes lithium an attractive new varroacide that deserves more attention and a communal effort to bring this new Varroa control tool to the market, where it can aid beekeepers in treating infested colonies sustainably and keeping their bees healthy.

Author Contribution Statement

CR: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Data Curation, Writing - Original Draft, Writing – Review & Editing, Visualization, Project administration, Funding acquisition

MB: Conceptualization, Methodology, Investigation

KT: Writing – Review & Editing

PR: Conceptualization, Methodology, Validation, Writing – Review & Editing, Supervision, Project administration, Funding acquisition

Acknowledgements

Our thanks to Dr. Sandra Renz and Andrea Ruf from the Core Facility Hohenheim for the analysis of lithium in samples. We also express our gratitude to Julia Renz and Markus Grünke for dissection of the bee samples in 2021. We appreciate the help of Nick Baumann and Adrian Preusch with the experiments in 2022, including colony management and sampling.

Funding

The project was supported by funds of the Federal Ministry of Food and Agriculture (BMEL) based on a decision of the Parliament of the Federal Republic of Germany via the Federal Office for Agriculture and Food (BLE) under the innovation support programme (Grant number 281C301A19).

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Data Availability

The datasets generated during and analysed during the current study are available from the corresponding author on reasonable request.

Ethics approval ‘Not applicable’

Consent to participate ‘Not applicable’

Consent to publish ‘Not applicable’

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Ziegelmann B, Abele E, Hannus S, Beitzinger M, Berg S, Rosenkranz P (2018) Lithium chloride effectively kills the honey bee parasite Varroa destructor by a systemic mode of action. Sci Rep 8:683. https://doi.org/10.1038/s41598-017-19137-5

No competing interests reported.

ReinLiCltreatmentsinfreeflyingcoloniesSupplementalInformation.docx

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published 22 Dec, 2023

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Lithium chloride treatments in free flying honey bee colonies: efficacy, brood survival and within colony distribution

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Journal Publication

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Abstract

Figures

1. Introduction

2. Materials and Methods

2.1. Experimental Setup

2.2. LiCl food production

2.3. Varroa mite mortality and calculation of efficacy

2.4. Honey bee brood survival

2.5. Distribution of lithium among worker bees, stored food, and residue in honey

2.6. Statistics

3. Results

3.1. Efficacy of different treatments

3.2. Honey bee brood survival

3.3. Distribution and detection of LiCl

4. Discussion

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1