Changes in Lithium Levels in Bees and Their Products Following Anti-Varroa Treatment

SIMPLE SUMMARY: Varroosis caused by the ectoparasitic mite Varroa destructor has been the biggest threat to managed bee colonies over recent decades. Chemicals available to treat the disease imply problems of resistance, inconsistent efficacy, and residues in bee products. Recently, alongside novel compounds to defeat the pest, lithium chloride has been found to be effective. In this study, we found that lithium treatments leave beeswax residue-free. The possibility of decontamination in adult bees, bee bread, and uncapped honey was revealed. On the other hand, ripe honey was found to be affected by lithium administered via feeding. Case studies are necessary to uncover the level of exposition in harvested honey to estimate its potential risk once it becomes a registered veterinary medicine. ABSTRACT: The biggest threat to beekeeping is varroosis caused by the mite Varroa destructor. Chemicals available to treat this fatal disease may present problems of resistance or inconsistent efficacy. Recently, lithium chloride has appeared as a potential alternative. To date, the amount of residue lithium treatments may leave in honeybee products is poorly understood. Honeybees were fed with 25 mM lithiated sugar syrup, which was used in earlier studies. The accumulation and elimination of the lithium were monitored in bees and their products for 22 days. Lithium concentration increased in the entire body of the bees to day 4 post-treatment and then recovered rapidly to the control level. Lithium exposure was found to affect uncapped honey in the short term (<16 days), but ripe (capped) honey measured at the end of the trial remained affected. On the other hand, lithium treatment left beeswax lithium-free. Based on these data, we propose that comprehensive research on harvested honey is needed to decide on the veterinary use of lithium

Lithium Chloride Shows Effectiveness against the Poultry Red Mite (Dermanyssus gallinae)

The poultry red mite (Dermanyssus gallinae) is the main pest of poultry, causing severe problems by being a vector of several animal and human pathogens. The number of miticides is few, and their efficacy in practice implies problems of residues and resistance; therefore, the demand for a new and safe agent is constant. The present publication investigated the effectiveness of lithium chloride under in vitro conditions on poultry red mites. This chemical currently appears to be one of the most promising alternatives to study amongst potential applicants to treat varroosis, a fatal disease of honey bees. In Experiment I, the previously used experimental doses (5.52 M, 2.76 M, 1.38 M) on Varroa mites confirmed their in vitro activity on the poultry red mite. Three event times (uncontrolled movement, immobilisation and death) were recorded to base the response to treatment for each concentration. In Experiment II, the LD 50 value was calculated, i.e., the value at which 50% of the mites were killed by the treatment. This Experiment showed that the LD50 of lithium chloride = 0.265 M in the poultry red mite. It is to note that the study remained restricted to in vitro confirmation of lithium chloride&rsquo;s effectiveness on the parasite. Thus, further extensive studies are needed to decide whether it has any relevance in practice against D. gallinae, and also to assess potential residue problems that could affect poultry products

Efficacy of In Vitro Lithium Chloride Treatments on <i>Dermacentor reticulatus</i>

Dermacentor reticulatus (Fabr., 1794) (Acari: Ixodidae) is parasite that spreads many diseases which are dangerous to humans and animals. Microelement lithium was found to have promising potential against the detrimental bee pest Varroa destructor. Furthermore, its effectiveness was confirmed against Dermanyssus gallinae, a major parasite of poultry, in vitro. In the present study, we investigated whether the efficacy of lithium chloride extends to other parasitic species, such as D. reticulatus. Our results revealed, for the first time, that the effectiveness of lithium chloride extends to D. reticulatus, confirmed to have 100% mortality at a relatively high minimum concentration of 1.38 M in vitro. The 24 h and 48 h median lethal concentration (LC50) values proved to be 0.654 M and 0.481 M, respectively, for this species. Our pilot study may contribute to a better understanding of the properties of lithium ion. Furthermore, it may elicit further studies aiming to reveal whether the different environmental mineral conditions may influence the D. reticulatus population. Further studies might reveal whether lithium has any possible veterinary relevance

Efficacy of In Vitro Lithium Chloride Treatments on Dermacentor reticulatus

Dermacentor reticulatus (Fabr., 1794) (Acari: Ixodidae) is parasite that spreads many diseases which are dangerous to humans and animals. Microelement lithium was found to have promising potential against the detrimental bee pest Varroa destructor. Furthermore, its effectiveness was confirmed against Dermanyssus gallinae, a major parasite of poultry, in vitro. In the present study, we investigated whether the efficacy of lithium chloride extends to other parasitic species, such as D. reticulatus. Our results revealed, for the first time, that the effectiveness of lithium chloride extends to D. reticulatus, confirmed to have 100% mortality at a relatively high minimum concentration of 1.38 M in vitro. The 24 h and 48 h median lethal concentration (LC50) values proved to be 0.654 M and 0.481 M, respectively, for this species. Our pilot study may contribute to a better understanding of the properties of lithium ion. Furthermore, it may elicit further studies aiming to reveal whether the different environmental mineral conditions may influence the D. reticulatus population. Further studies might reveal whether lithium has any possible veterinary relevance

Thymic Atrophy and Apoptosis of CD4+CD8+ Thymocytes in the Cuprizone Model of Multiple Sclerosis.

Previous studies on the degenerative animal model of multiple sclerosis suggested that the copper-chelator cuprizone might directly suppress T-cell functions. Peripheral T-cell function in the cuprizone model has already been explored; therefore, in the present study, we investigated, for the first time, how cuprizone feeding affects the thymus, the organ of T-cell maturation and selection. We found that even one week of cuprizone treatment induced significant thymic atrophy, affecting the cortex over the medulla. Fluorescent microscopy and flow-cytometric analyses of thymi from cuprizone- and vehicle-treated mice indicated that eradication of the cluster of the differentiation-4 (CD4)-CD8 double-positive T-cell subset was behind the substantial cell loss. This result was confirmed with CD3-CD4-CD8 triple-staining experiments. Ultrastructurally, we observed degraded as well as enlarged mitochondria, myelin-bodies, large lipid droplets, and large lysosomes in the thymi of cuprizone-treated mice. Some of these features were similar to those in physiological and steroid-induced accelerated aging. According to our results, apoptosis was mainly of mitochondrial origin mediated by both caspase-3- and apoptosis inducing factor-mediated mechanisms. Additionally, mitogen activated protein kinase activation and increased pro-apoptotic B cell lymphoma-2 family protein expression were the major underlying processes. Our results do not indicate a functional relationship between cuprizone-induced thymus involution and the absence of inflammatory responses or the selective demyelination observed in the cuprizone model. On the other hand, due to the reversible nature of cuprizone's deleterious effects, the cuprizone model could be valuable in studying thymus regeneration as well as remyelination processes

Effect of cuprizone on thymic epithelial cells.

<p>Four week-old male mice were treated with cuprizone for one week, then immune-staining (A) with FITC-labelled anti-EpCAM1 (green) and PE-labelled anti-Ly-51 (red) antibodies was performed on thymic sections of untreated (Control) and cuprizone-treated (CPZ) mice. Representative images (A) are presented of the green channel (top panels), the red channel (middle panels) and the merged channels (bottom panels) of three independent experiments, including at least three animals in each group for each experiment. Fluorescent photographs were taken using a 10x objective. The scale bar indicates 200 μm. In a parallel experiment, thymic MHC II and AIRE mRNA expression (B) was determined by using qPCR analysis in untreated (grey bars) and cuprizone-treated (black bars) mice. Results are presented as fold change, mean + SEM (n≥9). Significant difference from control; *p<0.05.</p

Macroscopic changes of the thymi upon cuprizone treatment.

<p>Four week-old male mice were treated with cuprizone for one week. Representative photographs (A) of the open chest of a control (Cont.) and a cuprizone-treated (CPZ) animal are demonstrated. Arrows point to the thymi of the mice. The scale bar indicates 5 mm. Thymus mass (B), body mass (C) and relative thymus mass (thymus tissue mass/ body mass) (E) of control (grey bars) and cuprizone-treated (black bars) animals are presented as bar diagrams, mean + SEM (n≥9). * denotes a significant difference from control p<0.05.</p

Characterisation of cuprizone-induced cell death.

<p>Four week-old male mice were treated with cuprizone for one week. Type of cell death was determined using flow cytometry following double staining with FITC-labelled AnnexinV and propidium iodide thymus suspensions of untreated (Control, grey bars) and cuprizone-treated (CPZ, black bars) mice. Results are presented as representative dot-plots (A) and bar diagrams (B), mean + SEM (n≥9). Significant difference from control; **p<0.01, *** p<0.001. DN: live cells (lower left quadrant); AnnexinV: early apoptotic cells (lower right quadrant); PI: necrotic cells (upper left quadrant); DP: late apoptotic cells (upper right quadrant).</p

Effect of cuprizone treatment on subcellular morphology.

<p>Four week-old male mice were treated with cuprizone for one week. Subcellular morphology was assessed in ultrathin thymic sections of control (A) and cuprizone-treated (B-E) mice. Representative images are presented of three independent experiments including at least three animals in each group for each experiment. Arrows indicate normal (A) and enlarged (B) mitochondria, a large lipid droplet (D), and large lysosomes packed with darkly stained material (E). Horizontal thin and vertical thick arrows in (C) point to myelin body and a degraded mitochondrion, respectively. Scale bars indicate 200 nm.</p

Effect of cuprizone treatment on death pathway and signalling proteins in the thymus.

<p>Four week-old male mice were treated with cuprizone for three days. Steady-state cytoplasmic levels of cytochrome C (Cyt C) (A), caspase 3 (casp 3) (A), cleaved caspase 3 (cleav. casp 3) (A) and nuclear apoptosis inducing factor (AIF) content (A), as well as cellular levels of Bad (B), BimEL (B), BimL (B) and Bax (B) were assessed in the thymi of untreated (Cont., grey bars) and cuprizone-treated (CPZ, black bars) mice by using specific primary antibodies and immunoblotting. The activation state of Bad (p-Bad) (B), JNK (p-JNK) (C), ERK (p-ERK) (C) and p38 MAPK (p-p38) (C) was also determined by using phosphorylation-specific primary antibodies and immunoblotting. GAPDH (A-C) and histone H1 (His H1) (A) were used as loading controls for cytoplasmic/cellular and nuclear fractions, respectively. Results are presented as representative immunoblots and bar diagrams, mean + SEM (n≥9). Significant difference from control; *p<0.05. Please note that the y-axis in B is broken to accommodate the very high Bax value.</p