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In this study, twenty various insect pheromones based on their structure were applied to determine the attractiveness to V. destructor (Table 1). All of the insect pheromones were above 95% of purity and provided by National Academy of Agricultural Science (NAAS).

For the collection and maintenance of V. destructor, six langstroth bee hives were provided by local beekeepers located in Cheongju from June through August 2022 and 2023, and the honey bee hives were placed in the apiary of the Korean National University of Agriculture and Fisheries during the experiment.

V. destructor were collected from honey bee hives by the sugar powder method (Milbrath, 2016). The method is to use powdered sugar to collect mites from adult bees. First, about 20 g of powdered sugar was poured into a jar containing 300 live bees. Then, a lid made of screen mesh was placed on the container and gently shaked so that the powdered sugar was applied evenly to all the bees. The jar was placed on a hard surface under the shade for 2 minutes to allow the mites dislodged from the bees, and then the jar was shaken upside-down lightly over a white tray for 1 minute. The collected mites were applied to honeybee larvae being capped before brood stage and transferred to a controlled environmental incubator (32°C and 65% relative humidity with no light; 1750 (W)×810 (D)×1760 (H), VS-1203PFC, Korea) in the laboratory. At the following day, the larvae were naturally fallen to the bottom, and they were carefully transferred to 96 well plates to coexist with V. destructor for 2 or 3 days (Fig. 1).

Fig. 1. 
Features of bee larvae (A) and V. destructor, top (B) and bottom (C) views, prepared in this experiment.

To determine the attractant properties of insect pheromones, the ring test method (Rickli et al., 1994; Donzé et al., 1998) was adapted as illustrated in Fig. 2. For the ring test, a petri dish (90 mm×15 mm, SPL Life Sciences, Korea) was equipped with filter paper (Whatman no. 2) with two concentric circles of 35 and 57 mm in diameter. Then, 50 mg of each pheromone, was diluted into 10 μL of ethanol (Sigma, St. Louis, MO, USA), and was applied on the middle circle. Subsequently, 10 V. destructor were placed at the inner circle, allowing them to move freely on the field. Afterward, the petri dish was covered with a lid and was exposed at room temperature (25-28℃) and 70% RH. And then the distribution of the V. destructor on the filter paper was calculated to evaluate the attractive rate. The attractive rate (%) was calculated as [number of mites in Mid Circle/total number of mites applied]×100%. The observations over 4 hours after mite placement were not counted, because the mites might be starved and thirsty which could affect their attracting and repelling behavior (Gashout and Guzmán-Novoa, 2009). Each experiment in this study was repeated in three times with different mites, and ethanol was used as the control treatment in each experiment.

Fig. 2. 
Design of ring test to evaluate attractive effects of the insect pheromones for V. destructor.

To select right insect pheromones of the attractive effect for V. destructor, twenty different insect pheromones were applied to V. destructor at the same concertation as ratios of 50 mg/10 μL for 3 hours at 30℃ and 70% RH under room condition. The attractive effects of twenty different insect pheromones were determined by observing the V. destructor position on the mid circle of the filter paper through visual inspection after 3 hours of the insect pheromone treatments.

A second experiment was conducted to figure out right concentration for the four pheromones, (Z)-8-12:Ac, (E)-8, (E)-10-12:Ac, (E)-11-14Ac and (Z)-11-14Ac, that were highly effective in the first experiment. The concentrations of pheromones were serially prepared with 50 mg, 25 mg, 12.5 mg and 6.25 mg of 10 μL of ethanol, respectively, and then treated for 3 hours at 30℃ and 70% RH under room condition. V. destructor located in the mid circle was counted to determine attractive effects by different concentration of the four insect pheromones.

In the second experiment, two pheromones, (Z)-8-12 : Ac and (E)-8, (E)-10-12:Ac, were highly effective to V. destructor. Therefore, third experiment was conducted the ring test at different time. The two pheromones were applied as the concentration of 50 mg/10 μL with ethanol, and then treated for 1, 2 and 3 hours at 30℃ and 70% RH. V. destructor located in the mid circle at the different time was observed and determined attractive effects by different time of the two insect pheromones.

The attractive effect of insect pheromones for V. destructor was calculated through the mean of three replicated measurements. All experimental data for each treatment were analyzed by ANOVA via SAS (Enterprise Guide 7.1, SAS Institute Inc., Cary, NC, USA), and represented significance differences at P<0.05. Also, Duncan’s multiple range and LSD tests (P<0.05) were conducted to compare any significant difference among various treatments.

The attractive effects of the twenty insect pheromones on V. destructor were observed, and they showed various rates of attractive effects for V. destructor ranged from 10 to 100% (Fig. 3). Among them, the most potent attractive effects were observed with (Z)-8-12:Ac (100%), followed by (E)-8, (E)-10-12:Ac (60%), E11-14 : Ac (57%) and (Z)-11-14 : Ac (57%).

Fig. 3. 
Rate (%) of attractive effects from the insect pheromones for V. destructor. Means within each column followed by the same letters are not significantly different according to Duncan’s multiple range test at P<0.05 (n=10, R=3).

In this study, we hypothesized that different pheromone structures would cause different attractant effect on V. destructor. Indeed, 12-carbon pheromones such as (Z)-8-12:Ac and (E)-8, (E)-10-12:Ac showed higher effects compared to 14-carbon or 16-carbon pheromones. However, some of 12-carbon pheromones showed relatively low attractiveness, this suggested that not all 12-carbon pheromones were equally effective at luring V. destructor. Moreover, there were no discernible differences between the sis and trans forms of the pheromones.

In the first experiment, we selected four pheromones: (Z)-8-12:Ac (E)-8, (E)-10-12:Ac, (E)-11-14Ac and (Z)-11-14Ac, and subsequently assessed their distinct attractive effects at various serially diluted concentration after 3 hours of exposure. Among these pheromones, attraction rates of (Z)-8-12:Ac were demonstrated as 86.7%, 70.0%, 80.0% and 30.0% when applied at concentration of 50 mg, 25 mg, 12.5 mg and 6.25 mg, respectively. Conversely, (E)-8, (E)-10-12:Ac showed attraction rates as 50.0%, 50.0%, 60.0% and 63.3% at the same respective concentration. Additionally, Z11-14Ac displayed attraction rates of 53.3%, 40.0%, 43.3% and 53.3% and E11-14Ac showed attraction rates of 36.7%, 30.0%, 33.3% and 46.7% at the same respective concentration (Fig. 4).

Fig. 4. 
Attractive effects rate (%) by the concentration of the insect pheromones for Varroa destructor. Means within each column followed by the same letters are not significantly different according to Duncan’s multiple range test at P<0.05 (n=10, R=3).

These results suggest that (Z)-8-12Ac exhibited a high attractive effect at concentrations of 50 mg, 25 mg and 12.5 mg; however, its attractiveness decreased at 6.25 mg. On the other hand, (E)-8, (E)-10-12:Ac maintain a relatively high attractive effect even at low concentrations, such as a 6.25 mg. In contrast, both (Z)-8-12:Ac and (E)-11-14Ac showed lower effectiveness compared to the other two pheromones. Therefore, we selected two pheromones, (Z)-8-12:Ac and (E)-8, (E)-10-12:Ac which showed relatively high attractive effect on V. destructor.

Generally, insect pheromones can be influenced by the concentration and many pheromones may not be effective at lower concentrations. However, (E)-8, (E)-10-12:Ac was effective at lower concentrations, and showed higher effect than (Z)-8-12:Ac at 6.25 mg of concentration.

Selected 2 pheromones, (Z)-8-12:Ac and (E)-8, (E)-10-12:Ac, were assessed for their distinct attractive effects at various exposed time at concentration of 50 mg (Fig. 5). As the result, (Z)-8-12:Ac demonstrated attraction rates of 60.8%, 65.8% and 68.3% when applied at exposed time of 1, 2 and 3 hours, respectively. On the other hand, attraction rates of (E)-8, (E)-10-12:Ac were 45.0%, 51.7% and 55.8% at the same exposed time, respectively. This study showed that both (Z)-8-12:Ac and (E)-8, (E)-10-12:Ac remained attractiveness effect on V. destructor, as the exposed time increased.

Fig. 5. 
Attractive effects rate (%) by the exposed time of the insect pheromones, A for (Z)-8-12:Ac and B for (E)-8, (E)-10-12:Ac for V. destructor. Means within each column followed by the same letters are not significantly different according to LSD test at P<0.05 (n=10, R=3).

According to the results of the above experiments, insect pheromones (Z)-8-12:Ac and (E)-8, (E)-10-12:Ac showed attractive effect on V. destructor in laboratory conditions. Notably, (Z)-8-12:Ac demonstrates higher effect than (E)-8, (E)-10-12:Ac. Although (Z)-8-12:Ac was a small effect at low concentration (6.25 mg), the optimal concentration for attractiveness was determined as 12.5 mg, which remained the effectiveness for three hours.

In conclusion, this study suggested two potential insect pheromones as effective attractants of V. destructor, (Z)-7-12:Ac (87%) and (E)-8, (E)-10-12:Ac. These pheromones are relatively small carbon pheromones with 12 carbons and are thought to have a higher attractant effect due to this structural characteristic. Therefore, these pheromones should be further investigated as potential alternative attractant for V. destructor control. In addition, other studies are warranted to determine the synergistic effects among different components, and establish appropriate delivery methods to develop safer and more effective attractant.

In general, insect pheromones are used to attract and kill insects such as moths by utilizing sex pheromones. In particular, (Z)-8-12:Ac is a component of moth sex pheromones used to attract and kill moths (Frerot et al., 1979; Varela et al., 2011). Varela et al. (2011) used (Z)-8-12:Ac to attract and kill the oriental fruit moth and Grapholita molesta (Busck) (Lepidoptera: Tortricidae). Hofmeyer and Calitz (1991) utilized (Z)-8-12:Ac as false codling moth (FCM), Cryptophlebia leucotreta (Meyr.) attractant mixed with (E)-8, (E)-10-12:Ac [trans-8-dodecenyl acetate] as its isomer. Although the attractive effect of the isomer, (E)-8, (E)-10-12:Ac was not investigated in this study, future experiments will be required to analyze the effects of structural differences between these isomers.

(E)-8, (E)-10-12:Ac is also a component of the sex pheromone of moths, and has been studied for its use in attractants (Greenway, 1984; Witzgall et al., 1993). Bengtsson et al. (1994) showed the attraction of the pea moth, Cydia nigricana, to (E)-8, (E)-10-12:Ac and 100 μg of (E)-8, (E)-10-12:Ac (99.8% isomeric purity by GC) was used to attract C. nigricana. This was very low concentration compared to the 6.25-50 mg applied in this study. This was considered as V. destructor does not have the antennae that insects have, so its ability to receive olfactory input is very low, making pheromones less effective in attracting them.

The chemosensory appendages of V. destructor include the forelegs and gnathosoma, which are known to detect odorants using lipid carrier proteins in the organelles (Mani et al., 2022). There have been no studies using pheromones to control V. destructor. The difference arises from V. destructor’s unique olfactory sensing mechanism, which reacts differently from that of insects. First of all, the feature of V. destructor is not able to detect odorants form a long distance would be a big limitation to use insect pheromones to attract them. However, this is the first study to test the effectiveness of using insect pheromones to attract V. destructor, and we believe that it may provide a clue to study the relationship between V. destructor and insect pheromones in the future.