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Monitoring the gravitational reflex of the ectoparasitic mite Varroa destructor: A novel bioassay for assessing toxic effects of acaricides
Pesticide Biochemistry and Physiology 101 (2011) 109–117
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Monitoring the gravitational reflex of the ectoparasitic mite Varroa destructor: A novel bioassay for assessing toxic effects of acaricides Alexandros Papachristoforou a,b,c,1, Chrisovalantis Papaefthimiou a, Georgia Zafeiridou a, Vasiliki Goundy a, Max Watkins d, George Theophilidis a,⇑ a
Laboratory of Animal Physiology, School of Biology, Aristotle University of Thessaloniki, 54 124 Thessaloniki, Greece Laboratoire Evolution, Génomes, Spéciation, CNRS UPR 9034, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France Department of Agricultural Sciences, Biotechnology and Food Science, Cyprus University of Technology, p.b. 50329, 3603 Limassol, Cyprus d Vita (Europe) Limited, 21/23 Wote Street, Basingstoke, Hampshire RG21 7NE, UK b c
a r t i c l e
i n f o
Article history: Received 26 April 2011 Accepted 23 August 2011 Available online 27 August 2011 Keywords: Varroa destructor Gravitational reflex Leg flexion Sternum expansion Acaricides Mortality assessment
a b s t r a c t We investigated the effect of several acaricides on Varroa destructor by monitoring the rhythmic expansion of the sternum, followed by a strong flexion of the legs, initiated when the mite was placed in a dorsal side-down position, as an indication of a mite’s vitality. The pulses generated by the force of the rhythmic expansions had an average duration of 3.11 s, force (amplitude) of 73 lN, and frequency of 0.228 Hz. These parameters remained constant for the first 10 h of recording, whilst significant changes occurred after 15 h. The rhythmic sternal expansion is an indication of a Varroa mite’s gravitational reflex, or attempt to return to an upright position; this reflex is observed in all invertebrates and vertebrates. The sternal expansion can be recorded for over 20 h, or for as long as the Varroa mite remains alive, and the expansion stops as soon as the mite is placed in a normal, upright position. Proper function of the chain of proprioceptors, interneurons, motorneurons, neuromuscular junctions, and muscles of Varroa is required for the initiation and maintenance of such a behavioural motor pattern. Any deleterious effect of synthetic chemicals or natural compounds (acaricides) may have a direct effect on one or more of these links, thereby disturbing or even inhibiting the reflex. Topical application of 1.81 103 mg/mite of amitraz completely inhibited the gravitational reflex within 60–70 min for all mites tested. The volatile acaricides formic acid (13.83 mg), thymol crystals (250 mg), and ApiguardÒ (1000 mg) eliminated the reflex within 10–35 min. This bioassay, based on the gravitational reflex, could be a useful tool for accurate assessment of the acaricidal action of numerous compounds under laboratory conditions, saving money and time necessary to conduct field trials. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Over the last four decades, the ectoparasitic mite, Varroa destructor [1], has become the most serious worldwide pest of the honeybee, Apis mellifera L [2,3]. Colony mortality caused by varroosis is close to 100% for untreated apiaries, and colonies might collapse within a few weeks or months if the infestation rate of adult honeybees reaches a level of 30% during the summer [4]. As a result, over the last forty years research has been focused on controlling Varroa infestation, and various products and methods have been tested and applied in laboratory and field trials. Synthetic pesticides acting on Varroa, such as tau-fluvalinate, coumaphos, bromopropylate, amitraz, and flumethrin in numerous products and formulations have been widely applied to beehives around ⇑ Corresponding author. Fax: +30 2310998269. E-mail address: [email protected] (G. Theophilidis). Present address: Department of Agricultural Sciences, Biotechnology and Food Science, Cyprus University of Technology, p.b. 50329, 3603 Limassol, Cyprus. 1
0048-3575/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2011.08.008
the globe [5–7]. Alternatively, many natural products have been used to control varroosis. Organic acids such as formic acid, lactic acid, and oxalic acid are among the most commonly used in applied apiculture [8,9]. In addition, some plant extracts, especially thymol, have demonstrated adequate acaricidal properties [10,11]. A lot of beekeepers currently tend to use thymol-based products in many countries. Research on the development of new acaricides requires a variety of tests, and some of these must be performed under laboratory conditions before field studies can be conducted. The efficacy of each compound tested is assessed in terms of the percent mite mortality following application. The susceptibility and possible development of resistance to current acaricides is also evaluated by estimating the percentage of dead mites after acaricide application in laboratory trials [12–15]. However, accurate assessment of mortality may include a number of challenges. In all cases, the definition of mite mortality is based on visual observation of their mobility [16–18]. However, visual observations carry a risk of inaccurate evaluation or of variability among methodologies. For example, De Guzman et al. [19] considered a mite that showed
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only uncoordinated leg movement dead, while Elzen et al. [20] considered such a mite alive. Moreover, results of studies in which mites are exposed to sublethal concentrations of acaricides appear to be greatly variable, because in some cases mites could be considered alive while in others the same mite would be considered ‘‘paralyzed’’ [16]. These inconsistencies indicate that more accurate methods are required to assess the effects of acaricides used for the control of Varroa. During our preliminary studies on the neuronal control of the legs of Varroa, we noticed that a rhythmic movement of the legs was initiated when a mite was fixed dorsal side-down on a substrate, and that this movement was well coordinated with expansion of the sternal shield. This rhythmic behavioural motor pattern, known as gravitational reflex in vertebrates and invertebrates [21], is completely different from the movement observed in other acarians and insects placed in the same position. Coordination of the central and peripheral nervous system in both invertebrates and vertebrates is required to initiate such a reflex [22–24,21]. The gravitational reflex can last for many hours and for as long as Varroa is alive and fixed in dorsal side-down position, and it was used in our study as an indication that a mite’s nervous system was functioning properly. The first objective of this study was to monitor and quantify the gravitation reflex – the rhythmic movement of legs and sternum of V. destructor – as an indication of the vitality of the mite under specific conditions (fixed in a dorsal side-down position). The second objective was to use this Varroa-preparation to evaluate the deleterious effects of several acaricides: amitraz, formic acid, thymol crystals, and ApiguardÒ – all widely applied for the control of varroosis.
2. Materials and methods 2.1. The Varroa-preparation Experiments were performed on 20 adult female V. destructor. The mites were collected from untreated colonies of A. mellifera macedonica located in the suburbs of city of Thessaloniki, Greece (40°380 30.71500 N, 22°570 8.2300 E). The mites were removed gently by the use of thick needles with retuse edges. None of the compounds used during our pharmacological studies have ever been applied to the sampled colonies for controlling varroosis since their initial establishment, 8 years ago. Honeybees bearing mites were hand-collected and transferred immediately to the laboratory. Mites were fixed dorsal side-down on the top of a plastic cone (pc in Fig. 1A), using non-toxic wax glue (Cenco Scientific Company, USA) and left to settle for over 1 h. The mites were handled using a stereomicroscope. The plastic cone was placed in the centre of a Petri dish (volume of 55 mL) (pd, Fig. 1A). Video recordings of the gravitational reflex were obtained using the stereoscope and a digital video camera (Euromex, DSP, Netherlands) in order to examine the movements of the legs and the sternal shield of a Varroa mite in detail (Movie 1 in the Supplementary Data). To monitor the force generated by either the rhythmic flexion of the legs or the movement of the sternal shield, a sensitive isometric force displacement transducer (FT-03C, Grass Instrument Company, USA) was used. The transducer was mounted on a micromanipulator, while its probe was extended to a micropin (t, in Fig. 1A). The transducer was gently attached either on the surface of the sternal shield or on the tips of the left (or right) legs, usually the 3rd and 4th pairs (Fig. 1B 1st diagram). Due to the very weak signal obtained from the sternal shield, the Varroa-preparation was placed in an anti-vibration table where the recording system was supported by air-strings. This precaution was necessary in order to minimize any background vibrations, which usually
appeared in the recordings as noise. Finally, the whole preparation was placed in a Faraday cage. The analog signal of the transducer was amplified 70,000-fold, using the proper preamplifier and an AC/DC Neurolog NL 106 amplifier (Digitimer, England, UK). Electromyograms (EMGs) from the leg muscles, converging under the sternal shield [25], were obtained using a tungsten metal microelectrode (Harvard Apparatus, Massachusetts USA). The tip of the microelectrode penetrated the thin cuticle of the ventral sternal shield of immobilised Varroa (Fig. 1B diagram 1), and was inserted among the fibres of the leg muscles. Also, the transducer (t, Fig. 1B, 1st diagram) was placed on the legs and was used as an indication of leg flexion. In this way it was possible to simultaneously record the EMGs and the contraction of the flexor muscles. The active input of the microelectrode was connected to an AC differentiated amplifier (Neurolog, NL 104, Digitimer, UK), while the ground electrode of the amplifier, a silver wire with a diameter of 0.010 mm, was placed in a small opening of the sternal shield. The amplified signal from either the transducer or the AC-amplifier was initially filtered (Neurolog NL115), then digitized (1000 Hz) using a data-acquisition card interface (Keithley KPCI-3102, Keithley Instruments, Cleveland, OH, USA) and analyzed using the proper software (Labview 5.1, National Instruments USA). A sample of such recordings is shown in Fig. 1C and D. The entire experimental procedure used to obtain recordings from the sternum via the transducer is presented in Movie 2 (in the Supplementary Data). 2.2. Acaricide application 2.2.1. Topical application Experiments were performed on 10 adult female V. destructor, (5 amitraz + solvent and 5 solvent-control). Amitraz (Sigma– Aldrich, Germany), a formamidine insecticide frequently applied against Varroa, was dissolved in dimethyl sulfoxide (DMSO) (Panreac, Spain), and freshly made stocks containing 0.02% amitraz (w/v) were prepared on a daily basis. Both DMSO and amitraz solution were applied topically to Varroa using a micropipette (mp, Fig. 1B 1st diagram). For this purpose, fine-tipped micropipettes were pulled from glass tubing (internal diameter 1.1–1.2 mm, external 1.5–1.6) (Marienfeld, 4360, Germany) using a Flaming/ Brown P-97 pipette puller (Sutter Instruments, Novato, CA, USA). The tips of the micropipettes were broken and backfilled with either pure DMSO, or amitraz 0.02%. Then, the micropipette was connected with the proper tubing to a pressure stimulus controller (Stim. Contr. CS-27, Syntech, Netherlands). Air pulses (100 ms in duration) were applied and fine spherical drops of the experimental solution with a volume 0.078 mm3 were generated at the tip of the micropipette. The entire system including the micropipette and the tubing was mounted on a micromanipulator, which permitted topical application of the drop containing the drug onto the abdomen of Varroa. Approximately 1.81 103 mg of amitraz was applied to each mite. 2.2.2. Volatile products Experiments were performed on 32 adult female V. destructor. Formic acid was applied to 10 Varroa mites while thymol crystals and ApiguardÒ were each applied to 11 mites. To test any possible sublethal effects, eight extra mites were used; four were treated with thymol crystals and four with ApiguardÒ gel. Before the acaricide application, the response and sensitivity of the Varroapreparation were tested on four other separate Varroa mites using CO2, a gas applied extensively as an anaesthetic in insect experiments [26]. CO2 was introduced through a small opening at the rear side of the Petri dish (Fig. 1A). Three volatile products were tested: thymol crystals (Merck Chemicals, USA), ApiguardÒ gel (Vita (Europe) Ltd., UK), and formic acid (Merck Chemicals, USA). The first two, thymol crystals and ApiguardÒ gel, were fixed on
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Fig. 1. Diagrammatic representation of the Varroa-preparation used for recording the rhythmic movements of the legs and the sternum expansion. (A) Varroa (V) was fixed, ventral side–up, on a plastic cone (pc) inside a Petri dish (pd). A micropin attached on the isometric force displacement transducer (t) was placed either on the legs or on the sternal shield (the positioning of the transducer is indicated in B1). A small opening on the top of the Petri dish allowed for contact between the transducer and the mite. The Petri dish was further modified to allow exposure of Varroa to CO2, formic acid, thymol, and ApiguardÒ. (B) Diagrammatic representation of the placement of the transducers (t) on the legs and the sternum of Varroa. A micropipette (mp) was used for the topical application of amitraz. (C) Simultaneous recordings of the pulses generated by the expansion of the sternum (upper trace) and the flexion of legs (lower trace) using two transducers (the position of the transducers is indicated in B1). The massive contraction of leg muscles causes the immediate increase in the volume of the sternum exerting a force on the transducer (B2). As soon as the leg movement stops the abdominal cavity returns to its initial volume (B3). t: time between two sequential pulsed, d: duration of each pulse. (D) Expanded record (in terms of time and amplitude) of leg movement (lower trace) and volume of the sternum (upper trace). The flexion of the legs corresponds in time with the increase in the volume of the sternum, as indicated by the dotted lines. (E) Recordings of the force of the sternum expansion combined with EMGs obtained from the region of the leg muscles. The position of the microelectrode (me) is shown in B1. The expansion of the thorax corresponds with the EMGs recorded from the leg muscles.
the cover of the Petri dish just above the Varroa mite (Fig. 1A), since the thymol vapours are heavier than air. To fix thymol crystals and ApiguardÒ gel, we placed a layer of honeybee wax on the top of the Petri dish and the products were easily attached on the wax surface. In apicultural practice, thymol-based anti-Varroa formula-
tions are applied on the top of the frames inside a beehive. In contrast, formic acid was applied at the base of the cone where Varroa was attached and was allowed to evaporate freely (Fig. 1A). To maintain a saturated microenvironment around the mite in all cases, the Petri dish was closed, leaving only a small
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opening of 1–2 mm2 to allow the contact of the probe from the transducer with the restrained Varroa mite (t, in Fig. 1A). The rhythmic expansion of the sternal shield was monitored continuously for 1 h before the application of each compound (control), as described above, and then monitored continuously during and after acaricide application. Each acaricide was applied for 1 h before replacement with air. For the evaluation of possible sublethal effects, the same procedure was followed but the products were removed 20 min post-application. Varroa mites were exposed to 13.83 mg of formic acid (16.2 ll at a concentration of 70% v/v: a dilution commonly applied to colonies by beekeepers). Similarly, the quantity of thymol crystals used for each application was 250 mg, and the quantity of ApiguardÒ gel was 1000 mg (containing 250 mg of thymol), both corresponding to the equivalent dose regularly applied in colonies. 2.3. Data analysis statistics To quantify and validate the gravitational reflex of the Varroapreparation, the following parameters were calculated from the positive pulses of force generated by the rhythmic flexion of the legs (Fig. 1C and D, second trace): (1) the frequency of the pulses was estimated from the period of a full cycle (t, in Fig. 1C; (2) the duration of the pulses was estimated as the time difference between the beginning and the end (onset and offset) of the pulse (d, in Fig. 1C); and (3) the force of the pulse was estimated as the maximum amplitude of the pulse measured from the baseline to the peak (Fig. 1C, vertical calibration mark). The above parameters were measured 1 h after all Varroa-preparations were left to settle. These values served as a control prior to the application of acaricides. Mean values and standard deviations (sd) of frequency, duration, and force of the pulse of tension generated by the expansion of the sternum were then calculated from recordings lasting 100 s at 1 h after fixation (control) and 2 or 5 h time intervals following acaricide application. For vitality experiments, long-term recordings, continuing for more than 20 h, were obtained from five mites while 15 more mites were also recorded for 5 h. For the long-term experiments, frequency, duration and force of the pulses were calculated from recordings lasting 100 s at 1, 5, 10, 15, and 20 h postfixation. The statistical significance of the recordings before and after acaricide application was determined using the Mann–Whitney test for two independent samples. Despite the fact that each individual Varroa mite was considered as an independent experiment, we also compared all parameters between all control mites using non-parametric Kruskal–Wallis one-way analysis of variance. The criterion for all statistical significance was a P value less than 0.05. 3. Results 3.1. The Varroa-preparation Rhythmic movement of the legs began immediately when a Varroa was fixed dorsal side-down (Fig. 1B, 1st diagram). Visual inspection of the Varroa-preparation (Movie 1 in the Supplementary Data) revealed that the leg movement was not random, as it is in insects and arachnids placed in a similar position. There was a simultaneous and coordinated flexion of the legs for a few seconds, followed by a relaxation. The force generated by the flexion of the legs appeared as positive pulses in the recordings shown in Fig. 1C and D (second trace). Detailed analysis of these records revealed the development of small peaks that build on the main pulse (asterisks in Fig. 1D, second trace), which represent extra, short flexions of the leg that are also followed by a short relaxation.
This rhythmic flexion of the legs could be observed only in the last three pairs of legs. The first pair of legs, though also activated during flexion, appeared incapable of either flexion or extension, producing a random movement that, however, was always synchronised in time with the movement of the rest of the legs (Movie 1 in the Supplementary Data). Furthermore, we observed a rhythmic movement of the cuticle of the sternal shield (or sternum) and the abdomen along with the rhythmic flexion of the legs (Movie 1 in the Supplementary Data). Using two tension transducers (Fig. 1B, 1st diagram), it was possible to simultaneously monitor both the pulse of force of the flexion of the legs (Fig. 1C, lower trace) and the pulse of the force generated by the rhythmic expansion of the sternum (Fig. 1C, upper trace). Comparison of the two pulses showed that there was an exact one-to-one correspondence between them. Further detailed analysis of the expanded pulses (Fig. 1D), indicated that even the fine flexions of the legs that built on the main pulse of the flexion (asterisks in the second trace of Fig. 1D) were synchronised, and were well coordinated with the fine expansion of the sternum (dotted lines in Fig. 1D). Furthermore, the EMG recordings from the leg muscles (Fig. 1E, lower trace) indicated that pulse generated by the flexion of the leg muscles also corresponded directly with the pulses of the force generated by the expansion of the sternum (dotted lines, in Fig. 1E). This evidence shows that the rhythmic expansion of the sternum is caused by contraction of the muscles that control flexion of the legs. These muscles converge from the four pair of legs to the region exactly under the mite’s sternum. All of the flexor muscles are confined within the small volume of the sternum cavity: their simultaneous contraction pushes the sternal shield and increases the sternal volume, as shown in the 2nd diagram of Fig. 1B. When the flexor muscles are relaxed, the sternum returns to its initial volume (Fig. 1B, 3rd diagram). Comparison of the recordings shown in Fig. 1C reveals that the pulses of the force recorded from the transducer attached on the sternum (upper trace) were more stable than those obtained from the transducer placed on the legs (lower trace). This is because the sternal shield has a stable surface to which the probe of the transducer can be firmly attached, but the tips of the legs can flex with a phase difference, distorting the final force output of the transducer and therefore producing less stable and less accurate recordings. Therefore, in order to monitor the rhythmic contraction of a mite’s leg muscles – a behavioural motor pattern generated by the central nervous system (CNS) – we chose to record the stable pulses generated by the expansion of the sternal shield, rather than the unstable force of the flexion of the legs, even though the force of the latter is about 10 times larger than that of the former. The rhythmic expansion of the sternal shield is a behavioural motor pattern that persists for hours – as long as the Varroa mite remains in the dorsal side-down position. Since the recording technique was non-invasive and caused no apparent injury, it was possible to obtain measurements over a prolonged period of time from the same Varroa mite (see the sample recordings of the force of the sternum expansion in Fig. 2A obtained at time t1 = 1 h, t2 = 5 h, t3 = 10 h, t4 = 15 h and t5 = 20 h). The parameters of the pulses were stable for each individual Varroa during the first 10 h (frequency, P = 0.3672; duration, P = 0.9653; force, P = 0.1283). Surprisingly, the rhythmic expansions of the sternum of an immobilised Varroa mite fixed dorsal side-down were maintained for 20–24 h (recordings overnight). Comparison of parameters after more than 10 h showed a slight but non-significant decrease in frequency (P = 0.1659) and force (P = 0.2545); this decrease was still not significant after 20 h of recording. However, the pulses were significantly shorter (P < 0.0001) after the 15th hour of continuous recording (Fig. 2A, t4).
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Fig. 2. (A) Monitoring the force generated by the rhythmic expansion of the sternum of an immobilized Varroa. Recordings were obtained at time t1 = 1 h, t2 = 5 h, t3 = 10 h, t4 = 15 h, and t5 = 20 h. (B) Monitoring the rhythmic expansion of the sternum of Varroa before, during, and after application of CO2. The first arrow indicates the application of CO2 and the second the replacement with air.
For the 20 mites examined at time t = 1 h, the mean force was 73 lN (sd = 1.041), the mean frequency was 0.228 Hz (sd = 1.424), and the average duration was 3.112 s (sd = 0.972). There were no significant differences between individual mites recorded at time t = 1 h and those recorded at time t = 10 h (force, 0.921 < P < 0.111; frequency, 0.933 < P < 0.082; duration, 0.889 < P < 0.075). When all mites (n = 20) were considered as a group, comparison of parameters of the pulses at time t = 1 h showed that there were significant differences between mites in pulse frequency (P < 0.0001, Nonparametric ANOVA – Kruskal–Wallis Test, KW = 48.003), duration (P < 0.0001, Nonparametric ANOVA – Kruskal– Wallis Test, KW = 57.024) and force (P < 0.0001, Nonparametric ANOVA – Kruskal–Wallis Test, KW = 59.089). 3.2. Acaricide application The pulses of the rhythmic expansions of the sternum can be recorded for many hours and for as long the mite is left in dorsal side-down position. However, recordings obtained after 15–20 h, though similar to the initial recordings at time t = 1 h and t = 5 h,
presented significant differences in some of the main parameters. Therefore, recordings after 15 h can be used to verify the vitality of a Varroa mite, but cannot be used to quantify the effects of acaricides. Recordings obtained within the first 3 h following application were used to assess acaricide toxicity. Before application of any acaricide, rhythmic sternum expansions were recorded for 1 h as a control (Fig. 2B), after the Varroa mite had been left for 1 h to settle. 3.2.1. Topical application (amitraz) Amitraz, 1.81 103 mg/mite, was applied to the surface of the abdomen or sternum (Fig. 3B, first arrow). The force of the pulses of sternal expansion gradually decreased. The frequency of these pulses, however, drastically increased, resulting in a decrease in their duration (see Fig. 3B, the comparison of the expanded records at t = 3 min [control] and at time t = 60 min after exposure to amitraz). After 120 min of continuous exposure to amitraz, the pulses of the sternal expansion of all mites (n = 5) ceased (expanded record at time t = 120 min in Fig. 3B). There was no recovery after 10 h of recordings.
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Fig. 3. (A) Monitoring the force generated by the rhythmic expansion of the sternum of an immobilized Varroa starting 20 min before and continuing after topical application of DMSO using the micropipette. DMSO had no effect on the rhythmic expansion of the sternum even after 180 min. The parallel lines on the record indicate a time gap of 120 min. (B) Monitoring the force generated by the rhythmic expansion of the sternum of an immobilized Varroa starting 20 min before and continuing after topical application of 0.02% (w/v) amitraz on the cuticle of Varroa using a micropipette. The topical application of amitraz completely eliminated the rhythmic expansion of the sternum 60–70 min post-application. The region of the record marked with boxes indicates the time when the expanded records were extracted from the main record. The parallel lines on the record indicate a time gap of 120 min.
The solvent (DMSO), when tested alone, had a minor effect on the rhythmic expansion of the sternal shield over the first 60 min of continuous exposure (t = 60 min, Fig. 3A). However, there were no significant differences in any of the parameters measured before and after 180 min (Fig. 3A, insert record at t = 180 min) of topical application of DMSO: force (0.5763 < P < 0.9216); frequency (0.7391 < P < 0.9104); duration (0.6239 < P < 0.9216) of the movements for all mites exposed to DMSO (n = 5). 3.2.2. Application of volatile products (formic acid, crystal thymol, and ApiguardÒ) The response of the Varroa-preparation to volatile products was tested using CO2. When Varroa mites were exposed to CO2 for 40 s, the rhythmic pulses of the sternal shield stopped completely (Fig. 2B, first arrow), and negative pressure developed in the sternal shield – an indication of complete relaxation of the leg muscles. When CO2 was removed (Fig. 2 B, second arrow), there was gradual recovery of the rhythmic expansions of the sternum within 3–5 min for all mites tested (n = 4). When Varroa mites were exposed to formic acid (Fig. 4A, first arrow), the pulses of the rhythmic sternal expansion, or the rhythmic contraction of the flexor muscles, gradually decreased. At around 6–8 min there was a massive tetanic contraction of the leg muscles causing drastic expansions of the sternal shield over a few seconds (Fig. 4A). As soon as the expansion reached its maximum, activity of the flexor muscles ceased completely and the muscles relaxed completely, causing a vacuum in the body cavity (indicated by the asterisks in Fig. 4A). The rhythmic pulses ceased completely after 8.74–11.37 min (n = 10, mean = 9.63 min, sd = 1.07) of exposure. When formic acid was removed from the chamber and replaced with air (Fig. 4A, second arrow) there was
no recovery of sternal movement recorded, even after 10 h, indicating that all mites were dead (n = 10). Application of thymol crystals drastically increased the frequency of the pulses of sternal expansion within 3–5 min (Fig. 4B, first arrow). The rhythmic pulses were eventually eliminated completely after 18.37–51.08 min (n = 11, mean = 33.43 min, sd = 10.87) of exposure (see the expanded record: Fig. 4B, t2). When the thymol crystal vapours were replaced with air (Fig. 4B, second arrow), there was no recovery of the pulses (see the expanded record: Fig. 4B, t3). Inspection of all mites 10 h post-application confirmed that all mites were dead (n = 11). During the extra tests for the evaluation of possible sublethal effects, three out of the eight mites (37.5%) showed no recovery after the removal of thymol crystals (20 min post application), while the rest demonstrated sternal movements that lasted for four up to more than 20 h, signalling their recovering. However, in all cases the parameters of the contraction (frequency, force and duration) differed significantly from the recordings before application (P < 0.001 for frequency, force and duration), indicating some possible sublethal effects of the application. Before application (control) the average force was 70 lN (sd = 1.186), the average frequency was 0.203 Hz (sd = 1.610) and the average duration was 3.301 s (sd = 1.522). Two hours after the removal of thymol crystals the average force was 48 lN (sd = 4.339), the average frequency was 0.019 Hz (sd = 4.558) and the average duration was 1.674 s (sd = 6.237). When the ApiguardÒ gel was applied (Fig. 4C, first arrow), there was a temporal excitation of Varroa with an increase in both force and frequency of the pulses of rhythmic sternal expansions. This excitation lasted for about 10 min and was followed by a drastic decrease in both force and frequency of the pulses (see the expanded record in Fig. 4C, t2). The pulses of sternal expansions
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Fig. 4. (A) Monitoring the force generated by the rhythmic expansions of the sternum from an immobilized Varroa starting 20 min before evaporation of formic acid, 20 min during, and continuing immediately after replacement with air. (B and C) Application of thymol crystals (B), and ApiguardÒ (C) starting 20 min before evaporation of the acaricides, 1 h during, and continuing immediately after replacement of each acaricide with air. In all records, the first arrow indicates application of each acaricide while the second arrow indicates replacement with air. The expanded record for (A) was obtained at time t1 = 15 min, t2 = 15 min post-application of formic acid, and t3 = 10 min in air. The expanded record for (B) was obtained at time t1 = 19 min, t2 = 2 min post-application of thymol crystals, and t3 = 2 min in air. The expanded record for (C) was obtained at time t1 = 18 min, t2 = 8 min post-application of ApiguardÒ, and t3 = 2 min in air. Horizontal scale bar at (A) corresponds to 2 min for upper trace and 20 s for lower trace. Horizontal scale bar at (B) and (C) corresponds to 1 min for upper trace and 10 s for lower trace. The parallel lines on the record indicate a time gap of 30 min.
were eliminated completely within 12.33–15.45 min postapplication (n = 11, mean = 14.01 min, sd = 1.11) and there was no recovery when this acaricide was replaced by air (Fig. 4C, second arrow). Inspection of all mites 10 h post-application confirmed that all mites were dead (n = 11). During the tests for the evaluation of possible sublethal effects five out of the eight mites (54.55%) showed no recovery after the removal of ApiguardÒ, while the rest demonstrated sternal movements that lasted for two up to more than 18 h. Similarly to crystal thymol, the parameters of the contraction differed from the recordings before the application of ApiguardÒ (P < 0.001 for frequency, force and duration). Before application (control) the average force was 75 lN (sd = 0.933), the average frequency was 0.261 Hz (sd = 1.171) and the average duration was 3.465 s (sd = 1.772). Two hours after the removal of ApiguardÒ the average force was 39 lN (sd = 3.018), the average frequency was 0.025 Hz (sd = 3.211) and the average duration was 1.504 s (sd = 4.673).
4. Discussion Our assessment of the toxicity of certain acaricides to Varroa, and our attempt to locate any rhythmic neural response as an indication of vitality, involved recording rhythmic movement of the four pairs of legs. This movement was only initiated when the mite was placed in a dorsal side-down position. It is worth noting that while the last three pairs of legs created a well- coordinated rhythmic flexion, the movement of the first pair was synchronised but never identical to the others, and instead moved randomly during the period of flexion. This independent action of the first pair of legs was previously described by Rickli et al. [27]. Rhythmic flexion of the legs appears to be an attempt by the Varroa mite to recover its normal, dorsal side-up, position – a behavioural motor pattern observed in almost all insects and arachnids. However, for Varroa this particular motor activity has a rhythmic and continuous pattern that is maintained for as long
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as Varroa was alive in this position – in some cases for over 20 h. The synchronized contraction of the flexor muscles, converging under the sternal shield, generated an expansion of the flexile sternum that also was monitored as a pulse. It is worth noting that during the flexion of the legs both expansion of the sternum and electrical activity of the flexor muscles were recorded, indicating strong motor activity; no equivalent activity was ever recorded during the extension of the legs. We assume that this extension is caused by a hydraulic pressure build-up inside the body, as has been observed in other arachnids [28]. This rhythmic pulse may play in important role in physiology of Varroa, a very specialized parasite with very little profound results on various aspects of physiology. For example, the movements could aid respiration, since the main tracheal system of Varroa is located directly under the sternum [29]. In addition, many arachnids lack extensor muscles at the femoropatellar (knee) joint of their legs and they extend this joint with hydraulic pressure during locomotion; a certain level a continuous pumping is required for the maintenance of the prosomal pressure. In Varroa, it is possible that this regulation in pressure could be achieved through rhythmic movement of the sternum and the abdomen. However, the rhythmic pulse ceases completely as soon as Varroa is placed in a normal, dorsal side-up position; the legs instantly engage in another behavioural motor activity, i.e. walking. This is an indication that the specific motor pattern is not related to either respiration or prosomal pressure, since neither system can be turned off under any circumstances. The only behavioural motor pattern that can be initiated and maintained continuously by placing an animal in a dorsal side-down position is the gravitational reflex in both invertebrates and vertebrates [21–24]. This is probably the first time that such a powerful gravitation reflex has been reported in such a small organism. The parameters characterizing the recordings of this gravitational reflex (frequency, force and duration) remained stable for periods reaching 20 h of continuous recordings in some cases. The high variability amongst the total of tested mites could be a result of the sampling method. Since we collected mites from adult honeybees, we could either pick a female which has been mated and laid within a brood cell or we could pick a virgin female Varroa which has exited the brood cell, before its re-entrance to a new cell for reproduction. During additional experiments we tested the gravitational reflex using Varroa mites obtained from sealed brood cells just before the emerging of honeybees (Data not shown). The gravitational reflex of the mated female differed significantly from its first offspring though there were no differences between all mated mites and all offspring tested. This area is a field of future research and application of this methodology. Additionally, the impact of other factors like the nutritional conditions of mites could not be excluded as a source of extra variation. Nevertheless, what is most important during the application of this method is the recording of each individual and the changes presented before and after the application of any tested compound. Whether the Varroa-preparation was suitable for assessing the toxic effects of acaricides was initially tested using CO2 – a gas that probably remains the most popular anaesthetic in entomological research. There was strong and instant inhibition on the gravitational reflex by CO2. The effect of CO2 on the nervous system of invertebrates has been elucidated elsewhere [26–30]. The instant response of the Varroa-preparation to CO2 indicates that the same preparation could be vulnerable to other toxic compounds, like acaricides. For example, topical application of 1.81 103 mg amitraz/mite caused a drastic decrease in the force of the pulses generated by the rhythmic expansions of the sternum, but a drastic simultaneous increase in the frequency of the pulses. Then, within 180 min, all Varroa activity was eliminated completely. The main target of this acaricide in Varroa is unknown,
but the mode of action of formamidine pesticides like amitraz in insects is believed to be via the toxic effects on a G protein-coupled receptor for a neuromodulator, octopamine [31]. It seems that each acaricide has its own pattern of action on the gravitational reflex (or on the vitality of mites); Varroa responded in a completely different manner when exposed to each acaricide. Vapours of formic acid led to massive contraction of the flexor muscles, followed by an intense relaxation that culminated as a permanent negative phase. The effect was so strong that less than 10 min of continuous exposure was enough to kill Varroa mites. The effects of thymol crystals and ApiguardÒ (a thymol-based product) vapours were milder, but both completely inhibited the gravitational reflex within 15–35 min. In insects and mammals, thymol acts on the nervous system by binding to c-aminobutyric acid (GABA) receptors and potentiating their response [32]. The action of thymol crystals and ApiguardÒ was relatively slow. Initially, an increase in pulse force and frequency could be observed as an indication of the withdrawal of any inhibitory input from the neural network. Then, almost all activity ceased, an effect that could either be related to inhibitory synapses in the CNS or in the muscles. The application of ApiguardÒ gel on Varroa mites resulted in faster mortality than the application of thymol crystals. This difference may be related to more controlled release of thymol from the ApiguardÒ matrix-gel. From the three volatile compounds examined using the novel method, it seems that formic acid is the most potent, inducing faster the death of Varroa at the lowest quantity (13.8 mg). Indeed, the time of death induced by Apiguard and thymol crystals (250–1000 mg) was 1.45–3 times higher compared to formic acid. These clear differences in time of death in relation to quantity applied are strong indications that the method presented here can successfully detect the potency of different compounds examined. However, for more accurate assessment of the toxicity of the compounds examined here or during future experiments, detailed dose–response curves (time of death vs. concentration) should be prepared. Results obtained from this bioassay showed that the inconsistency in mite death measured using visual observations no longer exists when using recordings of the gravitational reflex. Recordings from the toxicological tests showed that by using the Varroapreparation we can define the exact time of death caused by the compounds tested. A mite can be considered dead once no sternal movement is recorded. This bioassay could be used as a rapid, low cost, and accurate screening test for new substances that possess possible acaricidal properties. The application of these laboratory methods can provide some indication of the acaricide action of a compound, and this could save time, money and effort required for complicated field studies. An unlimited range of acaricide’s quantity, administered per mite, could be tested either topically, through feeding, or through fumigation or evaporation. Furthermore, honeybees could easily be tested for tolerance to the same quantities of acaricides by applying a similar, non-invasive in vivo method using respiratory rhythm as an indication of the honeybee’s vitality [33]. As demonstrated by some preliminary experimental results, one of the advantages of the Varroa-preparation is that it not only allows for precise measurement of the time required to ensure Varroa death, but also for detection of sublethal effects of low concentration of acaricides, provided that future studies incorporate more accurate recordings that use extremely sensitive force transducers and more complex mathematical analysis of the sternum pulses. The study of sublethal effects on mites could be significant because many compounds, especially natural acaricides, may also cause irreversible but non-lethal damage to Varroa [10,34]. Furthermore, this type of detailed analysis of the pulses of the sternum expansion due to the contraction of the flexor
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muscles controlled by the CNS could be used to assess possible acaricide resistance by comparing the response of resistant and non-resistant mites. Development of resistance is a major problem that has already been detected for many anti-Varroa products, such as tau-fluvalinate, flumethrin, and amitraz [13,16,35,36]. Acknowledgments We thank Tasos Gavriilidis for providing honeybees and Varroa mites from his apiary during experiments. We are grateful to Gerard Arnold for reviewing the manuscript. We are indebted to Lev Paraskevopoulos from L-Studio (www.l-studio.gr) who created the 3D animations. We dedicate this research to the memory of Jérôme Trouiller, who continuously supported our research on Varroa.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.pestbp.2011.08.008. Reference [1] D.L. Anderson, J.W.H. Trueman, Varroa jacobsoni (Acari:Varroidae) is more than one species, Exp. Appl. Acarol. 24 (2000) 165–189. [2] M. Spivak, Dynamics and control of Varroa parasitism on Apis, Apidologie 30 (1999) 81–83. [3] P. Rosenkranz, P. Aumeier, B. Ziegelmann, Biology and control of Varroa destructor, J. Invertbr. Pathol. 103 (2010) S69–S119. [4] I. Fries, H. Hansen, A. Imdorf, P. Rosenkranz, Swarming in honey bees (Apis mellifera) and Varroa destructor population development in Sweden, Apidologie 34 (2003) 389–398. [5] W. Ritter, Medications registered in Western Europe for varroatosis control, Apidologie 19 (1988) 113–116. [6] N. Koeniger, S. Fuchs, Control of Varroa jacobsoni Oud In honey bee colonies containing sealed brood cells, Apidologie 19 (1988) 117–130. [7] R. Borneck, B. Merle, New tests for Varroa control with Apistan (Fluvalinate), in: R. Cavalloro (Ed.), Present Status of Varroatosis in Europe and Progress in the Varroa Mite Control, ECSC, Luxembourg, 1989, pp. 311–314. [8] B. Kraus, S. Berg, Effect of a lactic acid treatment during winter in temperate climate upon Varroa jacobsoni Oud. and the bee (Apis mellifera L.) colony, Exp. Appl. Acarol. 18 (1994) 459–468. [9] A. Imdorf, V. Kilchenmann, S. Bogdanov, B. Bachofen, C. Beretta, Toxizitaet von thymol campher, menthol und eucalyptol auf Varroa jacobsoni und Apis mellifera im labortest, Apidologie 26 (1995) 27–31. [10] M.E. Colin, Essential oils for controlling honey bee varroosis, J. Appl. Ent. 110 (1990) 19–25. [11] F. Chiesa, Effective control of varroatosis using powdered thymol, Apidologie 22 (1991) 135–145. [12] W. Ritter, H. Roth, Experiments with mite resistance to varroacidal substances in the laboratory, European research on varroatosis control. In: Cavalloro, R. (ed.), Proceeding of a Meeting of the EC Experts’ Group, Bad Homburg. A.A. Balkema, Rotterdam 1988 pp. 157–160. [13] N. Milani, The resistance of Varroa jacobsoni Oud to pyrethroids: a laboratory assay, Apidologie 26 (1995) 41–54.
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Pesticide Biochemistry and Physiology journal homepage: www.elsevier.com/locate/pest
Monitoring the gravitational reflex of the ectoparasitic mite Varroa destructor: A novel bioassay for assessing toxic effects of acaricides Alexandros Papachristoforou a,b,c,1, Chrisovalantis Papaefthimiou a, Georgia Zafeiridou a, Vasiliki Goundy a, Max Watkins d, George Theophilidis a,⇑ a
Laboratory of Animal Physiology, School of Biology, Aristotle University of Thessaloniki, 54 124 Thessaloniki, Greece Laboratoire Evolution, Génomes, Spéciation, CNRS UPR 9034, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France Department of Agricultural Sciences, Biotechnology and Food Science, Cyprus University of Technology, p.b. 50329, 3603 Limassol, Cyprus d Vita (Europe) Limited, 21/23 Wote Street, Basingstoke, Hampshire RG21 7NE, UK b c
a r t i c l e
i n f o
Article history: Received 26 April 2011 Accepted 23 August 2011 Available online 27 August 2011 Keywords: Varroa destructor Gravitational reflex Leg flexion Sternum expansion Acaricides Mortality assessment
a b s t r a c t We investigated the effect of several acaricides on Varroa destructor by monitoring the rhythmic expansion of the sternum, followed by a strong flexion of the legs, initiated when the mite was placed in a dorsal side-down position, as an indication of a mite’s vitality. The pulses generated by the force of the rhythmic expansions had an average duration of 3.11 s, force (amplitude) of 73 lN, and frequency of 0.228 Hz. These parameters remained constant for the first 10 h of recording, whilst significant changes occurred after 15 h. The rhythmic sternal expansion is an indication of a Varroa mite’s gravitational reflex, or attempt to return to an upright position; this reflex is observed in all invertebrates and vertebrates. The sternal expansion can be recorded for over 20 h, or for as long as the Varroa mite remains alive, and the expansion stops as soon as the mite is placed in a normal, upright position. Proper function of the chain of proprioceptors, interneurons, motorneurons, neuromuscular junctions, and muscles of Varroa is required for the initiation and maintenance of such a behavioural motor pattern. Any deleterious effect of synthetic chemicals or natural compounds (acaricides) may have a direct effect on one or more of these links, thereby disturbing or even inhibiting the reflex. Topical application of 1.81 103 mg/mite of amitraz completely inhibited the gravitational reflex within 60–70 min for all mites tested. The volatile acaricides formic acid (13.83 mg), thymol crystals (250 mg), and ApiguardÒ (1000 mg) eliminated the reflex within 10–35 min. This bioassay, based on the gravitational reflex, could be a useful tool for accurate assessment of the acaricidal action of numerous compounds under laboratory conditions, saving money and time necessary to conduct field trials. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Over the last four decades, the ectoparasitic mite, Varroa destructor [1], has become the most serious worldwide pest of the honeybee, Apis mellifera L [2,3]. Colony mortality caused by varroosis is close to 100% for untreated apiaries, and colonies might collapse within a few weeks or months if the infestation rate of adult honeybees reaches a level of 30% during the summer [4]. As a result, over the last forty years research has been focused on controlling Varroa infestation, and various products and methods have been tested and applied in laboratory and field trials. Synthetic pesticides acting on Varroa, such as tau-fluvalinate, coumaphos, bromopropylate, amitraz, and flumethrin in numerous products and formulations have been widely applied to beehives around ⇑ Corresponding author. Fax: +30 2310998269. E-mail address: [email protected] (G. Theophilidis). Present address: Department of Agricultural Sciences, Biotechnology and Food Science, Cyprus University of Technology, p.b. 50329, 3603 Limassol, Cyprus. 1
0048-3575/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2011.08.008
the globe [5–7]. Alternatively, many natural products have been used to control varroosis. Organic acids such as formic acid, lactic acid, and oxalic acid are among the most commonly used in applied apiculture [8,9]. In addition, some plant extracts, especially thymol, have demonstrated adequate acaricidal properties [10,11]. A lot of beekeepers currently tend to use thymol-based products in many countries. Research on the development of new acaricides requires a variety of tests, and some of these must be performed under laboratory conditions before field studies can be conducted. The efficacy of each compound tested is assessed in terms of the percent mite mortality following application. The susceptibility and possible development of resistance to current acaricides is also evaluated by estimating the percentage of dead mites after acaricide application in laboratory trials [12–15]. However, accurate assessment of mortality may include a number of challenges. In all cases, the definition of mite mortality is based on visual observation of their mobility [16–18]. However, visual observations carry a risk of inaccurate evaluation or of variability among methodologies. For example, De Guzman et al. [19] considered a mite that showed
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only uncoordinated leg movement dead, while Elzen et al. [20] considered such a mite alive. Moreover, results of studies in which mites are exposed to sublethal concentrations of acaricides appear to be greatly variable, because in some cases mites could be considered alive while in others the same mite would be considered ‘‘paralyzed’’ [16]. These inconsistencies indicate that more accurate methods are required to assess the effects of acaricides used for the control of Varroa. During our preliminary studies on the neuronal control of the legs of Varroa, we noticed that a rhythmic movement of the legs was initiated when a mite was fixed dorsal side-down on a substrate, and that this movement was well coordinated with expansion of the sternal shield. This rhythmic behavioural motor pattern, known as gravitational reflex in vertebrates and invertebrates [21], is completely different from the movement observed in other acarians and insects placed in the same position. Coordination of the central and peripheral nervous system in both invertebrates and vertebrates is required to initiate such a reflex [22–24,21]. The gravitational reflex can last for many hours and for as long as Varroa is alive and fixed in dorsal side-down position, and it was used in our study as an indication that a mite’s nervous system was functioning properly. The first objective of this study was to monitor and quantify the gravitation reflex – the rhythmic movement of legs and sternum of V. destructor – as an indication of the vitality of the mite under specific conditions (fixed in a dorsal side-down position). The second objective was to use this Varroa-preparation to evaluate the deleterious effects of several acaricides: amitraz, formic acid, thymol crystals, and ApiguardÒ – all widely applied for the control of varroosis.
2. Materials and methods 2.1. The Varroa-preparation Experiments were performed on 20 adult female V. destructor. The mites were collected from untreated colonies of A. mellifera macedonica located in the suburbs of city of Thessaloniki, Greece (40°380 30.71500 N, 22°570 8.2300 E). The mites were removed gently by the use of thick needles with retuse edges. None of the compounds used during our pharmacological studies have ever been applied to the sampled colonies for controlling varroosis since their initial establishment, 8 years ago. Honeybees bearing mites were hand-collected and transferred immediately to the laboratory. Mites were fixed dorsal side-down on the top of a plastic cone (pc in Fig. 1A), using non-toxic wax glue (Cenco Scientific Company, USA) and left to settle for over 1 h. The mites were handled using a stereomicroscope. The plastic cone was placed in the centre of a Petri dish (volume of 55 mL) (pd, Fig. 1A). Video recordings of the gravitational reflex were obtained using the stereoscope and a digital video camera (Euromex, DSP, Netherlands) in order to examine the movements of the legs and the sternal shield of a Varroa mite in detail (Movie 1 in the Supplementary Data). To monitor the force generated by either the rhythmic flexion of the legs or the movement of the sternal shield, a sensitive isometric force displacement transducer (FT-03C, Grass Instrument Company, USA) was used. The transducer was mounted on a micromanipulator, while its probe was extended to a micropin (t, in Fig. 1A). The transducer was gently attached either on the surface of the sternal shield or on the tips of the left (or right) legs, usually the 3rd and 4th pairs (Fig. 1B 1st diagram). Due to the very weak signal obtained from the sternal shield, the Varroa-preparation was placed in an anti-vibration table where the recording system was supported by air-strings. This precaution was necessary in order to minimize any background vibrations, which usually
appeared in the recordings as noise. Finally, the whole preparation was placed in a Faraday cage. The analog signal of the transducer was amplified 70,000-fold, using the proper preamplifier and an AC/DC Neurolog NL 106 amplifier (Digitimer, England, UK). Electromyograms (EMGs) from the leg muscles, converging under the sternal shield [25], were obtained using a tungsten metal microelectrode (Harvard Apparatus, Massachusetts USA). The tip of the microelectrode penetrated the thin cuticle of the ventral sternal shield of immobilised Varroa (Fig. 1B diagram 1), and was inserted among the fibres of the leg muscles. Also, the transducer (t, Fig. 1B, 1st diagram) was placed on the legs and was used as an indication of leg flexion. In this way it was possible to simultaneously record the EMGs and the contraction of the flexor muscles. The active input of the microelectrode was connected to an AC differentiated amplifier (Neurolog, NL 104, Digitimer, UK), while the ground electrode of the amplifier, a silver wire with a diameter of 0.010 mm, was placed in a small opening of the sternal shield. The amplified signal from either the transducer or the AC-amplifier was initially filtered (Neurolog NL115), then digitized (1000 Hz) using a data-acquisition card interface (Keithley KPCI-3102, Keithley Instruments, Cleveland, OH, USA) and analyzed using the proper software (Labview 5.1, National Instruments USA). A sample of such recordings is shown in Fig. 1C and D. The entire experimental procedure used to obtain recordings from the sternum via the transducer is presented in Movie 2 (in the Supplementary Data). 2.2. Acaricide application 2.2.1. Topical application Experiments were performed on 10 adult female V. destructor, (5 amitraz + solvent and 5 solvent-control). Amitraz (Sigma– Aldrich, Germany), a formamidine insecticide frequently applied against Varroa, was dissolved in dimethyl sulfoxide (DMSO) (Panreac, Spain), and freshly made stocks containing 0.02% amitraz (w/v) were prepared on a daily basis. Both DMSO and amitraz solution were applied topically to Varroa using a micropipette (mp, Fig. 1B 1st diagram). For this purpose, fine-tipped micropipettes were pulled from glass tubing (internal diameter 1.1–1.2 mm, external 1.5–1.6) (Marienfeld, 4360, Germany) using a Flaming/ Brown P-97 pipette puller (Sutter Instruments, Novato, CA, USA). The tips of the micropipettes were broken and backfilled with either pure DMSO, or amitraz 0.02%. Then, the micropipette was connected with the proper tubing to a pressure stimulus controller (Stim. Contr. CS-27, Syntech, Netherlands). Air pulses (100 ms in duration) were applied and fine spherical drops of the experimental solution with a volume 0.078 mm3 were generated at the tip of the micropipette. The entire system including the micropipette and the tubing was mounted on a micromanipulator, which permitted topical application of the drop containing the drug onto the abdomen of Varroa. Approximately 1.81 103 mg of amitraz was applied to each mite. 2.2.2. Volatile products Experiments were performed on 32 adult female V. destructor. Formic acid was applied to 10 Varroa mites while thymol crystals and ApiguardÒ were each applied to 11 mites. To test any possible sublethal effects, eight extra mites were used; four were treated with thymol crystals and four with ApiguardÒ gel. Before the acaricide application, the response and sensitivity of the Varroapreparation were tested on four other separate Varroa mites using CO2, a gas applied extensively as an anaesthetic in insect experiments [26]. CO2 was introduced through a small opening at the rear side of the Petri dish (Fig. 1A). Three volatile products were tested: thymol crystals (Merck Chemicals, USA), ApiguardÒ gel (Vita (Europe) Ltd., UK), and formic acid (Merck Chemicals, USA). The first two, thymol crystals and ApiguardÒ gel, were fixed on
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Fig. 1. Diagrammatic representation of the Varroa-preparation used for recording the rhythmic movements of the legs and the sternum expansion. (A) Varroa (V) was fixed, ventral side–up, on a plastic cone (pc) inside a Petri dish (pd). A micropin attached on the isometric force displacement transducer (t) was placed either on the legs or on the sternal shield (the positioning of the transducer is indicated in B1). A small opening on the top of the Petri dish allowed for contact between the transducer and the mite. The Petri dish was further modified to allow exposure of Varroa to CO2, formic acid, thymol, and ApiguardÒ. (B) Diagrammatic representation of the placement of the transducers (t) on the legs and the sternum of Varroa. A micropipette (mp) was used for the topical application of amitraz. (C) Simultaneous recordings of the pulses generated by the expansion of the sternum (upper trace) and the flexion of legs (lower trace) using two transducers (the position of the transducers is indicated in B1). The massive contraction of leg muscles causes the immediate increase in the volume of the sternum exerting a force on the transducer (B2). As soon as the leg movement stops the abdominal cavity returns to its initial volume (B3). t: time between two sequential pulsed, d: duration of each pulse. (D) Expanded record (in terms of time and amplitude) of leg movement (lower trace) and volume of the sternum (upper trace). The flexion of the legs corresponds in time with the increase in the volume of the sternum, as indicated by the dotted lines. (E) Recordings of the force of the sternum expansion combined with EMGs obtained from the region of the leg muscles. The position of the microelectrode (me) is shown in B1. The expansion of the thorax corresponds with the EMGs recorded from the leg muscles.
the cover of the Petri dish just above the Varroa mite (Fig. 1A), since the thymol vapours are heavier than air. To fix thymol crystals and ApiguardÒ gel, we placed a layer of honeybee wax on the top of the Petri dish and the products were easily attached on the wax surface. In apicultural practice, thymol-based anti-Varroa formula-
tions are applied on the top of the frames inside a beehive. In contrast, formic acid was applied at the base of the cone where Varroa was attached and was allowed to evaporate freely (Fig. 1A). To maintain a saturated microenvironment around the mite in all cases, the Petri dish was closed, leaving only a small
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opening of 1–2 mm2 to allow the contact of the probe from the transducer with the restrained Varroa mite (t, in Fig. 1A). The rhythmic expansion of the sternal shield was monitored continuously for 1 h before the application of each compound (control), as described above, and then monitored continuously during and after acaricide application. Each acaricide was applied for 1 h before replacement with air. For the evaluation of possible sublethal effects, the same procedure was followed but the products were removed 20 min post-application. Varroa mites were exposed to 13.83 mg of formic acid (16.2 ll at a concentration of 70% v/v: a dilution commonly applied to colonies by beekeepers). Similarly, the quantity of thymol crystals used for each application was 250 mg, and the quantity of ApiguardÒ gel was 1000 mg (containing 250 mg of thymol), both corresponding to the equivalent dose regularly applied in colonies. 2.3. Data analysis statistics To quantify and validate the gravitational reflex of the Varroapreparation, the following parameters were calculated from the positive pulses of force generated by the rhythmic flexion of the legs (Fig. 1C and D, second trace): (1) the frequency of the pulses was estimated from the period of a full cycle (t, in Fig. 1C; (2) the duration of the pulses was estimated as the time difference between the beginning and the end (onset and offset) of the pulse (d, in Fig. 1C); and (3) the force of the pulse was estimated as the maximum amplitude of the pulse measured from the baseline to the peak (Fig. 1C, vertical calibration mark). The above parameters were measured 1 h after all Varroa-preparations were left to settle. These values served as a control prior to the application of acaricides. Mean values and standard deviations (sd) of frequency, duration, and force of the pulse of tension generated by the expansion of the sternum were then calculated from recordings lasting 100 s at 1 h after fixation (control) and 2 or 5 h time intervals following acaricide application. For vitality experiments, long-term recordings, continuing for more than 20 h, were obtained from five mites while 15 more mites were also recorded for 5 h. For the long-term experiments, frequency, duration and force of the pulses were calculated from recordings lasting 100 s at 1, 5, 10, 15, and 20 h postfixation. The statistical significance of the recordings before and after acaricide application was determined using the Mann–Whitney test for two independent samples. Despite the fact that each individual Varroa mite was considered as an independent experiment, we also compared all parameters between all control mites using non-parametric Kruskal–Wallis one-way analysis of variance. The criterion for all statistical significance was a P value less than 0.05. 3. Results 3.1. The Varroa-preparation Rhythmic movement of the legs began immediately when a Varroa was fixed dorsal side-down (Fig. 1B, 1st diagram). Visual inspection of the Varroa-preparation (Movie 1 in the Supplementary Data) revealed that the leg movement was not random, as it is in insects and arachnids placed in a similar position. There was a simultaneous and coordinated flexion of the legs for a few seconds, followed by a relaxation. The force generated by the flexion of the legs appeared as positive pulses in the recordings shown in Fig. 1C and D (second trace). Detailed analysis of these records revealed the development of small peaks that build on the main pulse (asterisks in Fig. 1D, second trace), which represent extra, short flexions of the leg that are also followed by a short relaxation.
This rhythmic flexion of the legs could be observed only in the last three pairs of legs. The first pair of legs, though also activated during flexion, appeared incapable of either flexion or extension, producing a random movement that, however, was always synchronised in time with the movement of the rest of the legs (Movie 1 in the Supplementary Data). Furthermore, we observed a rhythmic movement of the cuticle of the sternal shield (or sternum) and the abdomen along with the rhythmic flexion of the legs (Movie 1 in the Supplementary Data). Using two tension transducers (Fig. 1B, 1st diagram), it was possible to simultaneously monitor both the pulse of force of the flexion of the legs (Fig. 1C, lower trace) and the pulse of the force generated by the rhythmic expansion of the sternum (Fig. 1C, upper trace). Comparison of the two pulses showed that there was an exact one-to-one correspondence between them. Further detailed analysis of the expanded pulses (Fig. 1D), indicated that even the fine flexions of the legs that built on the main pulse of the flexion (asterisks in the second trace of Fig. 1D) were synchronised, and were well coordinated with the fine expansion of the sternum (dotted lines in Fig. 1D). Furthermore, the EMG recordings from the leg muscles (Fig. 1E, lower trace) indicated that pulse generated by the flexion of the leg muscles also corresponded directly with the pulses of the force generated by the expansion of the sternum (dotted lines, in Fig. 1E). This evidence shows that the rhythmic expansion of the sternum is caused by contraction of the muscles that control flexion of the legs. These muscles converge from the four pair of legs to the region exactly under the mite’s sternum. All of the flexor muscles are confined within the small volume of the sternum cavity: their simultaneous contraction pushes the sternal shield and increases the sternal volume, as shown in the 2nd diagram of Fig. 1B. When the flexor muscles are relaxed, the sternum returns to its initial volume (Fig. 1B, 3rd diagram). Comparison of the recordings shown in Fig. 1C reveals that the pulses of the force recorded from the transducer attached on the sternum (upper trace) were more stable than those obtained from the transducer placed on the legs (lower trace). This is because the sternal shield has a stable surface to which the probe of the transducer can be firmly attached, but the tips of the legs can flex with a phase difference, distorting the final force output of the transducer and therefore producing less stable and less accurate recordings. Therefore, in order to monitor the rhythmic contraction of a mite’s leg muscles – a behavioural motor pattern generated by the central nervous system (CNS) – we chose to record the stable pulses generated by the expansion of the sternal shield, rather than the unstable force of the flexion of the legs, even though the force of the latter is about 10 times larger than that of the former. The rhythmic expansion of the sternal shield is a behavioural motor pattern that persists for hours – as long as the Varroa mite remains in the dorsal side-down position. Since the recording technique was non-invasive and caused no apparent injury, it was possible to obtain measurements over a prolonged period of time from the same Varroa mite (see the sample recordings of the force of the sternum expansion in Fig. 2A obtained at time t1 = 1 h, t2 = 5 h, t3 = 10 h, t4 = 15 h and t5 = 20 h). The parameters of the pulses were stable for each individual Varroa during the first 10 h (frequency, P = 0.3672; duration, P = 0.9653; force, P = 0.1283). Surprisingly, the rhythmic expansions of the sternum of an immobilised Varroa mite fixed dorsal side-down were maintained for 20–24 h (recordings overnight). Comparison of parameters after more than 10 h showed a slight but non-significant decrease in frequency (P = 0.1659) and force (P = 0.2545); this decrease was still not significant after 20 h of recording. However, the pulses were significantly shorter (P < 0.0001) after the 15th hour of continuous recording (Fig. 2A, t4).
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Fig. 2. (A) Monitoring the force generated by the rhythmic expansion of the sternum of an immobilized Varroa. Recordings were obtained at time t1 = 1 h, t2 = 5 h, t3 = 10 h, t4 = 15 h, and t5 = 20 h. (B) Monitoring the rhythmic expansion of the sternum of Varroa before, during, and after application of CO2. The first arrow indicates the application of CO2 and the second the replacement with air.
For the 20 mites examined at time t = 1 h, the mean force was 73 lN (sd = 1.041), the mean frequency was 0.228 Hz (sd = 1.424), and the average duration was 3.112 s (sd = 0.972). There were no significant differences between individual mites recorded at time t = 1 h and those recorded at time t = 10 h (force, 0.921 < P < 0.111; frequency, 0.933 < P < 0.082; duration, 0.889 < P < 0.075). When all mites (n = 20) were considered as a group, comparison of parameters of the pulses at time t = 1 h showed that there were significant differences between mites in pulse frequency (P < 0.0001, Nonparametric ANOVA – Kruskal–Wallis Test, KW = 48.003), duration (P < 0.0001, Nonparametric ANOVA – Kruskal– Wallis Test, KW = 57.024) and force (P < 0.0001, Nonparametric ANOVA – Kruskal–Wallis Test, KW = 59.089). 3.2. Acaricide application The pulses of the rhythmic expansions of the sternum can be recorded for many hours and for as long the mite is left in dorsal side-down position. However, recordings obtained after 15–20 h, though similar to the initial recordings at time t = 1 h and t = 5 h,
presented significant differences in some of the main parameters. Therefore, recordings after 15 h can be used to verify the vitality of a Varroa mite, but cannot be used to quantify the effects of acaricides. Recordings obtained within the first 3 h following application were used to assess acaricide toxicity. Before application of any acaricide, rhythmic sternum expansions were recorded for 1 h as a control (Fig. 2B), after the Varroa mite had been left for 1 h to settle. 3.2.1. Topical application (amitraz) Amitraz, 1.81 103 mg/mite, was applied to the surface of the abdomen or sternum (Fig. 3B, first arrow). The force of the pulses of sternal expansion gradually decreased. The frequency of these pulses, however, drastically increased, resulting in a decrease in their duration (see Fig. 3B, the comparison of the expanded records at t = 3 min [control] and at time t = 60 min after exposure to amitraz). After 120 min of continuous exposure to amitraz, the pulses of the sternal expansion of all mites (n = 5) ceased (expanded record at time t = 120 min in Fig. 3B). There was no recovery after 10 h of recordings.
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Fig. 3. (A) Monitoring the force generated by the rhythmic expansion of the sternum of an immobilized Varroa starting 20 min before and continuing after topical application of DMSO using the micropipette. DMSO had no effect on the rhythmic expansion of the sternum even after 180 min. The parallel lines on the record indicate a time gap of 120 min. (B) Monitoring the force generated by the rhythmic expansion of the sternum of an immobilized Varroa starting 20 min before and continuing after topical application of 0.02% (w/v) amitraz on the cuticle of Varroa using a micropipette. The topical application of amitraz completely eliminated the rhythmic expansion of the sternum 60–70 min post-application. The region of the record marked with boxes indicates the time when the expanded records were extracted from the main record. The parallel lines on the record indicate a time gap of 120 min.
The solvent (DMSO), when tested alone, had a minor effect on the rhythmic expansion of the sternal shield over the first 60 min of continuous exposure (t = 60 min, Fig. 3A). However, there were no significant differences in any of the parameters measured before and after 180 min (Fig. 3A, insert record at t = 180 min) of topical application of DMSO: force (0.5763 < P < 0.9216); frequency (0.7391 < P < 0.9104); duration (0.6239 < P < 0.9216) of the movements for all mites exposed to DMSO (n = 5). 3.2.2. Application of volatile products (formic acid, crystal thymol, and ApiguardÒ) The response of the Varroa-preparation to volatile products was tested using CO2. When Varroa mites were exposed to CO2 for 40 s, the rhythmic pulses of the sternal shield stopped completely (Fig. 2B, first arrow), and negative pressure developed in the sternal shield – an indication of complete relaxation of the leg muscles. When CO2 was removed (Fig. 2 B, second arrow), there was gradual recovery of the rhythmic expansions of the sternum within 3–5 min for all mites tested (n = 4). When Varroa mites were exposed to formic acid (Fig. 4A, first arrow), the pulses of the rhythmic sternal expansion, or the rhythmic contraction of the flexor muscles, gradually decreased. At around 6–8 min there was a massive tetanic contraction of the leg muscles causing drastic expansions of the sternal shield over a few seconds (Fig. 4A). As soon as the expansion reached its maximum, activity of the flexor muscles ceased completely and the muscles relaxed completely, causing a vacuum in the body cavity (indicated by the asterisks in Fig. 4A). The rhythmic pulses ceased completely after 8.74–11.37 min (n = 10, mean = 9.63 min, sd = 1.07) of exposure. When formic acid was removed from the chamber and replaced with air (Fig. 4A, second arrow) there was
no recovery of sternal movement recorded, even after 10 h, indicating that all mites were dead (n = 10). Application of thymol crystals drastically increased the frequency of the pulses of sternal expansion within 3–5 min (Fig. 4B, first arrow). The rhythmic pulses were eventually eliminated completely after 18.37–51.08 min (n = 11, mean = 33.43 min, sd = 10.87) of exposure (see the expanded record: Fig. 4B, t2). When the thymol crystal vapours were replaced with air (Fig. 4B, second arrow), there was no recovery of the pulses (see the expanded record: Fig. 4B, t3). Inspection of all mites 10 h post-application confirmed that all mites were dead (n = 11). During the extra tests for the evaluation of possible sublethal effects, three out of the eight mites (37.5%) showed no recovery after the removal of thymol crystals (20 min post application), while the rest demonstrated sternal movements that lasted for four up to more than 20 h, signalling their recovering. However, in all cases the parameters of the contraction (frequency, force and duration) differed significantly from the recordings before application (P < 0.001 for frequency, force and duration), indicating some possible sublethal effects of the application. Before application (control) the average force was 70 lN (sd = 1.186), the average frequency was 0.203 Hz (sd = 1.610) and the average duration was 3.301 s (sd = 1.522). Two hours after the removal of thymol crystals the average force was 48 lN (sd = 4.339), the average frequency was 0.019 Hz (sd = 4.558) and the average duration was 1.674 s (sd = 6.237). When the ApiguardÒ gel was applied (Fig. 4C, first arrow), there was a temporal excitation of Varroa with an increase in both force and frequency of the pulses of rhythmic sternal expansions. This excitation lasted for about 10 min and was followed by a drastic decrease in both force and frequency of the pulses (see the expanded record in Fig. 4C, t2). The pulses of sternal expansions
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Fig. 4. (A) Monitoring the force generated by the rhythmic expansions of the sternum from an immobilized Varroa starting 20 min before evaporation of formic acid, 20 min during, and continuing immediately after replacement with air. (B and C) Application of thymol crystals (B), and ApiguardÒ (C) starting 20 min before evaporation of the acaricides, 1 h during, and continuing immediately after replacement of each acaricide with air. In all records, the first arrow indicates application of each acaricide while the second arrow indicates replacement with air. The expanded record for (A) was obtained at time t1 = 15 min, t2 = 15 min post-application of formic acid, and t3 = 10 min in air. The expanded record for (B) was obtained at time t1 = 19 min, t2 = 2 min post-application of thymol crystals, and t3 = 2 min in air. The expanded record for (C) was obtained at time t1 = 18 min, t2 = 8 min post-application of ApiguardÒ, and t3 = 2 min in air. Horizontal scale bar at (A) corresponds to 2 min for upper trace and 20 s for lower trace. Horizontal scale bar at (B) and (C) corresponds to 1 min for upper trace and 10 s for lower trace. The parallel lines on the record indicate a time gap of 30 min.
were eliminated completely within 12.33–15.45 min postapplication (n = 11, mean = 14.01 min, sd = 1.11) and there was no recovery when this acaricide was replaced by air (Fig. 4C, second arrow). Inspection of all mites 10 h post-application confirmed that all mites were dead (n = 11). During the tests for the evaluation of possible sublethal effects five out of the eight mites (54.55%) showed no recovery after the removal of ApiguardÒ, while the rest demonstrated sternal movements that lasted for two up to more than 18 h. Similarly to crystal thymol, the parameters of the contraction differed from the recordings before the application of ApiguardÒ (P < 0.001 for frequency, force and duration). Before application (control) the average force was 75 lN (sd = 0.933), the average frequency was 0.261 Hz (sd = 1.171) and the average duration was 3.465 s (sd = 1.772). Two hours after the removal of ApiguardÒ the average force was 39 lN (sd = 3.018), the average frequency was 0.025 Hz (sd = 3.211) and the average duration was 1.504 s (sd = 4.673).
4. Discussion Our assessment of the toxicity of certain acaricides to Varroa, and our attempt to locate any rhythmic neural response as an indication of vitality, involved recording rhythmic movement of the four pairs of legs. This movement was only initiated when the mite was placed in a dorsal side-down position. It is worth noting that while the last three pairs of legs created a well- coordinated rhythmic flexion, the movement of the first pair was synchronised but never identical to the others, and instead moved randomly during the period of flexion. This independent action of the first pair of legs was previously described by Rickli et al. [27]. Rhythmic flexion of the legs appears to be an attempt by the Varroa mite to recover its normal, dorsal side-up, position – a behavioural motor pattern observed in almost all insects and arachnids. However, for Varroa this particular motor activity has a rhythmic and continuous pattern that is maintained for as long
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as Varroa was alive in this position – in some cases for over 20 h. The synchronized contraction of the flexor muscles, converging under the sternal shield, generated an expansion of the flexile sternum that also was monitored as a pulse. It is worth noting that during the flexion of the legs both expansion of the sternum and electrical activity of the flexor muscles were recorded, indicating strong motor activity; no equivalent activity was ever recorded during the extension of the legs. We assume that this extension is caused by a hydraulic pressure build-up inside the body, as has been observed in other arachnids [28]. This rhythmic pulse may play in important role in physiology of Varroa, a very specialized parasite with very little profound results on various aspects of physiology. For example, the movements could aid respiration, since the main tracheal system of Varroa is located directly under the sternum [29]. In addition, many arachnids lack extensor muscles at the femoropatellar (knee) joint of their legs and they extend this joint with hydraulic pressure during locomotion; a certain level a continuous pumping is required for the maintenance of the prosomal pressure. In Varroa, it is possible that this regulation in pressure could be achieved through rhythmic movement of the sternum and the abdomen. However, the rhythmic pulse ceases completely as soon as Varroa is placed in a normal, dorsal side-up position; the legs instantly engage in another behavioural motor activity, i.e. walking. This is an indication that the specific motor pattern is not related to either respiration or prosomal pressure, since neither system can be turned off under any circumstances. The only behavioural motor pattern that can be initiated and maintained continuously by placing an animal in a dorsal side-down position is the gravitational reflex in both invertebrates and vertebrates [21–24]. This is probably the first time that such a powerful gravitation reflex has been reported in such a small organism. The parameters characterizing the recordings of this gravitational reflex (frequency, force and duration) remained stable for periods reaching 20 h of continuous recordings in some cases. The high variability amongst the total of tested mites could be a result of the sampling method. Since we collected mites from adult honeybees, we could either pick a female which has been mated and laid within a brood cell or we could pick a virgin female Varroa which has exited the brood cell, before its re-entrance to a new cell for reproduction. During additional experiments we tested the gravitational reflex using Varroa mites obtained from sealed brood cells just before the emerging of honeybees (Data not shown). The gravitational reflex of the mated female differed significantly from its first offspring though there were no differences between all mated mites and all offspring tested. This area is a field of future research and application of this methodology. Additionally, the impact of other factors like the nutritional conditions of mites could not be excluded as a source of extra variation. Nevertheless, what is most important during the application of this method is the recording of each individual and the changes presented before and after the application of any tested compound. Whether the Varroa-preparation was suitable for assessing the toxic effects of acaricides was initially tested using CO2 – a gas that probably remains the most popular anaesthetic in entomological research. There was strong and instant inhibition on the gravitational reflex by CO2. The effect of CO2 on the nervous system of invertebrates has been elucidated elsewhere [26–30]. The instant response of the Varroa-preparation to CO2 indicates that the same preparation could be vulnerable to other toxic compounds, like acaricides. For example, topical application of 1.81 103 mg amitraz/mite caused a drastic decrease in the force of the pulses generated by the rhythmic expansions of the sternum, but a drastic simultaneous increase in the frequency of the pulses. Then, within 180 min, all Varroa activity was eliminated completely. The main target of this acaricide in Varroa is unknown,
but the mode of action of formamidine pesticides like amitraz in insects is believed to be via the toxic effects on a G protein-coupled receptor for a neuromodulator, octopamine [31]. It seems that each acaricide has its own pattern of action on the gravitational reflex (or on the vitality of mites); Varroa responded in a completely different manner when exposed to each acaricide. Vapours of formic acid led to massive contraction of the flexor muscles, followed by an intense relaxation that culminated as a permanent negative phase. The effect was so strong that less than 10 min of continuous exposure was enough to kill Varroa mites. The effects of thymol crystals and ApiguardÒ (a thymol-based product) vapours were milder, but both completely inhibited the gravitational reflex within 15–35 min. In insects and mammals, thymol acts on the nervous system by binding to c-aminobutyric acid (GABA) receptors and potentiating their response [32]. The action of thymol crystals and ApiguardÒ was relatively slow. Initially, an increase in pulse force and frequency could be observed as an indication of the withdrawal of any inhibitory input from the neural network. Then, almost all activity ceased, an effect that could either be related to inhibitory synapses in the CNS or in the muscles. The application of ApiguardÒ gel on Varroa mites resulted in faster mortality than the application of thymol crystals. This difference may be related to more controlled release of thymol from the ApiguardÒ matrix-gel. From the three volatile compounds examined using the novel method, it seems that formic acid is the most potent, inducing faster the death of Varroa at the lowest quantity (13.8 mg). Indeed, the time of death induced by Apiguard and thymol crystals (250–1000 mg) was 1.45–3 times higher compared to formic acid. These clear differences in time of death in relation to quantity applied are strong indications that the method presented here can successfully detect the potency of different compounds examined. However, for more accurate assessment of the toxicity of the compounds examined here or during future experiments, detailed dose–response curves (time of death vs. concentration) should be prepared. Results obtained from this bioassay showed that the inconsistency in mite death measured using visual observations no longer exists when using recordings of the gravitational reflex. Recordings from the toxicological tests showed that by using the Varroapreparation we can define the exact time of death caused by the compounds tested. A mite can be considered dead once no sternal movement is recorded. This bioassay could be used as a rapid, low cost, and accurate screening test for new substances that possess possible acaricidal properties. The application of these laboratory methods can provide some indication of the acaricide action of a compound, and this could save time, money and effort required for complicated field studies. An unlimited range of acaricide’s quantity, administered per mite, could be tested either topically, through feeding, or through fumigation or evaporation. Furthermore, honeybees could easily be tested for tolerance to the same quantities of acaricides by applying a similar, non-invasive in vivo method using respiratory rhythm as an indication of the honeybee’s vitality [33]. As demonstrated by some preliminary experimental results, one of the advantages of the Varroa-preparation is that it not only allows for precise measurement of the time required to ensure Varroa death, but also for detection of sublethal effects of low concentration of acaricides, provided that future studies incorporate more accurate recordings that use extremely sensitive force transducers and more complex mathematical analysis of the sternum pulses. The study of sublethal effects on mites could be significant because many compounds, especially natural acaricides, may also cause irreversible but non-lethal damage to Varroa [10,34]. Furthermore, this type of detailed analysis of the pulses of the sternum expansion due to the contraction of the flexor
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muscles controlled by the CNS could be used to assess possible acaricide resistance by comparing the response of resistant and non-resistant mites. Development of resistance is a major problem that has already been detected for many anti-Varroa products, such as tau-fluvalinate, flumethrin, and amitraz [13,16,35,36]. Acknowledgments We thank Tasos Gavriilidis for providing honeybees and Varroa mites from his apiary during experiments. We are grateful to Gerard Arnold for reviewing the manuscript. We are indebted to Lev Paraskevopoulos from L-Studio (www.l-studio.gr) who created the 3D animations. We dedicate this research to the memory of Jérôme Trouiller, who continuously supported our research on Varroa.
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