Hydrophobic protease activities were decreased and even not observed at all in relation to casein and gelatine (Table 1). Asparagine and serine proteases, but no thiolic proteases or metal-proteases, were detected on the apian cuticles because proteolytic activity was identified in the case of pepstatin‑A and phenylmethylsulfonyl fluoride (PMSF) but was not observed in relation to iodoacetamide or o-phenanthroline (diagnostic inhibitors of proteases).

Amitraz and oxalic acid treatments increased the number of acidic and alkaline protease (both hydrophilic and hydrophobic) and neutral hydrophobic protease bands, and reduced the number of bands associated with neutral hydrophilic proteases in electrophorograms (Table 2). Furthermore, the width of the bands of alkaline proteases increased, whereas that of neutral proteases diminished. Considering Table 1, it may be assumed that varroacide treatment may render cuticle proteins/peptides inactive.

Amitraz and oxalic acid caused a complete loss or considerable reduction (Figure 2) in the activities of natural inhibitors of acidic, neutral, and alkaline proteases (both hydrophilic and hydrophobic). In the electrophorograms of the control group, poorly pronounced narrow bands associated with both high- and low-molecular inhibitors of asparagine and serine proteases were observed (Figure 3, Table 3). After applying amitraz and oxalic acid, additional bands associated with high- and low-molecular protease inhibitors appeared. These were broader and well-pronounced at acidic and neutral but narrower and less prominent at alkaline pH. It may be assumed that amitraz and oxalic acid deactivated protease inhibitors or reacted with the proteases/substrates. Moreover, they may have destroyed the structure of one/multiple components of the protease/substrate/inhibitor complex. In all the cases more inactive cuticle proteins/peptides may appear.

Our results showed that both amitraz and oxalic acid reduced the activities of the cuticle proteolytic systems of worker bees, especially as regards hydrophobic proteases. This was particularly noticeable in the case of natural protease inhibitors. This suppressive effect may stem from the varroacids’ toxicity. Howis et al. [23] proved that organic acids damage anatomic and physiological structures of the alimentary and body cover, and can facilitate pathogen penetration by debilitating the proteolytic system. Additionally, organic acids create a favorable environment for fungal development. Moreover, oxalic acid probably has a negative effect on the Krebs cycle. Drugs to fight V. destructor used to be selected by their efficacy, ease of application, and affordability [25]. In this context, amitraz seems a good solution. Nevertheless, our results reveal that amitraz is not advantageous as regards the proteolytic barrier. A “vicious circle” is created, in which drug use reduces resistance, leading to weaker colonies, and thereby to more losses in the apiary, which then require more intensive drug application. This finding corresponds with feedback from beekeepers, who have observed the spreading of various diseases in their hives after intensive use of the above drugs [26]. Amitraz-containing drugs considerably reduced the amount of the main biochemical compounds in the hemolymph of adult workers from their emergence until foraging age, and to a lower extent, the amount of proteins in the body tissues of the workers [27]. Our results demonstrate that amitraz also lowers protein concentration and suppresses proteolytic system activity on the body surface of honeybees. Therefore, among the side effects of therapy against V. destructor, we should enumerate the extenuation of the proteolytic barrier.

Our previous experiments proved that some external factors could supress the proteolytic barrier on the apian cuticle. In a polluted environment, lower values of protein concentrations, protease activities, and natural protease inhibitor activities were observed in the worker cuticles as compared with the cuticles of bees from an unpolluted environment [11]. Bee diet may also affect the barrier efficiency [28]. The present study revealed that amitraz and oxalic acid theraphy may also reduce cuticular proteolytic system activity and therefore may be regarded as harmful external factors.

The rinsings from the body surface of workers in the control group showed activity in relation to all the microorganisms (Table 4). In bees treated with amitraz and oxalic acid, no activity towards A. niger or S. aureus was observed. Moreover, after applying oxalic acid, no activity was identified in relation to S. typhimurium or P. aeruginosa. This may correspond with a low in vitro activity of the proteolytic system, due to the influence of those varroacids, and confirms their suppressive effect on the proteolytic barrier especially in the case of fungal infection. On the other hand, amitraz increased the activity towards Bacillus subtilis, Salmonella typhimurium, Pseudomonas aeruginosa. Hence, suppression was not always observed, though mostly.

Ants have protective microbial biofilms on their body surfaces that are affected by various chemical substances [29]. Grzywnowicz et al. [9] suspected that bees might also have such biofilms. If they do, amitraz and oxalic acid could suppress the biofilm activity (anti-pathogen cuticle barrier) conditioned by the proteolytic system. Recent research [30] shows that the V. destructor parasite is equipped with a set of enzymes that belong to the category of asparagine and cysteine proteases, as well as metal‑proteases with a pH range of only 2.0–6.5. In turn, as evidenced in this research, A. mellifera have asparginic and serine proteases on their body surface whose pH range is much broader (2.4–11.2) than that of the acarids, which could help bees block “foreign” proteins.

Three experimental groups with 5 colonies of Buckfast bees in each (headed by sister-queens) were monitored (15 colonies). In the first group bees were sprayed with a 3.2% oxalic acid solution, 50 mL per colony. The solution was made using 15 g oxalic acid dihydrate, 200 g sucrose, and 200 mL water [24]. Each colony in the second group was fumigated with one tablet of Apiowarol AS^® (12.5 mg amitraz) single-time according to the producer’s recommendations. The treatments were repeated in 2009 and 2010 at the end of July. In the third, control group, neither amitraz nor oxalic acid treatments were performed.

Three samples comprising 10 nest worker bees were collected from each colony just before and two weeks after the treatments consecutively in 2009 and 2010 (together 6 samples). Totally the procedure produced 5 colonies × 6 samples × 2 years = 60 samples = 600 bees for each of the three group. The material was frozen in sterile bags at −8°C and stored for 1–2 months. Then, the samples were refrozen and rinsed in 10 ml distilled water for 20 seconds to remove impurities. Proteins were not found in the rinsings using the Lowry method as modified by Schacterle and Pollack [31]. Therefore, the rinsings were discarded. Subsequently, the samples were shaken/rinsed for 4 min at 3,400 rpm in 10 mL distilled water, and finally, after filtering through Miracloth, a solution was obtained that mostly contained hydrophilic proteins. The solution obtained from each sample was then divided into 4 portions, poured into four Eppendorf tubes, and frozen again at −40°C. The procedure produced:

Altogether, we obtained 60 samples × 4 portions = 240 portions per group.

Then, the solid samples that remained on the Miracloth were again shaken/rinsed (4 min at 3,400 rpm) in a 1% detergent solution (Triton X-100) in distilled water (10 mL). Similarly to the first rinsing, 4 portions (2 mL) were created from each sample, now containing mostly hydrophobic proteins.

The entire procedure resulted in a total of 1440 portions (3 groups × 60 samples × 2 rinsings × 4 portions).

For each of the three groups, 62 µL of the solution were sampled from each “portion a”, and mixed to create a one pooled group-sample (3 pooled group-samples, each consisting of 60 samples) to determine optimal pH values, at which protein activities were high. The values amounted to 2.2–3.4, 6.4–7.6, and 8.2–11.6 in the pooled samples. Therefore, the activities of acid, neutral and alkaline proteases/protease inhibitors were decided to be determinate at pH 2.4, 7.0 and 11.2, respectively.

Next, the remainder (1.938 mL) of each “portion a” was analyzed (portions were not pooled in this case; n = 60) as follows:

Polyacrylamide gel electrophoresis was performed using “portions b”, which had been previously lyophilized and then combined with 100 µL distilled water. Each portion (n = 60) was analyzed separately in this case. The following analyses were conducted:

“Portions c” from each group were pooled separately (3 pooled group-samples; each consisting of 60 portions), and then lyophilized to determine antifungal and antibacterial activities. To this end, the lyophilizates were combined with 200 µL distilled water. Subsequently, 10 µL of the mixtures were spackled on culture media using the double application method, with:

In all microbiological tests, each dish was photographed (SONY α100) to determine the area on which there was no microorganism growth, using the MultiScanBase [39].

To statistically verify an influence of amitraz and oxalic acid (experimental factors) on the protein contents as well as on the protease and protease inhibitor activities, one-way ANOVAs and the Duncan multiple range tests were performed using the SAS [40].