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Behavioral responses of honey bees, and Apis mellifera , to Vespa mandarinia marking and alarm pheromones

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Behavioral responses of honey bees, Apis cerana and Apis mellifera, to Vespa mandarinia marking and alarm pheromones

Beverly McClenaghan, Marcel Schlaf, Megan Geddes, Joshua Mazza, Grace Pitman, Kaileigh McCallum, Samuel Rawluk, Karen Hand & Gard W. Otis

To cite this article: Beverly McClenaghan, Marcel Schlaf, Megan Geddes, Joshua Mazza, Grace Pitman, Kaileigh McCallum, Samuel Rawluk, Karen Hand & Gard W. Otis (2018): Behavioral responses of honey bees, Apis￿cerana and Apis￿mellifera, to Vespa￿mandarinia marking and alarm pheromones, Journal of Apicultural Research, DOI: 10.1080/00218839.2018.1494917 To link to this article: https://doi.org/10.1080/00218839.2018.1494917

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ORIGINAL RESEARCH ARTICLE Behavioral responses of honey bees, Apis cerana and Apis mellifera,toVespa mandarinia marking and alarm pheromones Beverly McClenaghana , Marcel Schlafb, Megan Geddesa, Joshua Mazzaa, Grace Pitmana, Kaileigh McCalluma, Samuel Rawluka, Karen Handc and Gard W. Otisa aSchool of Environmental Sciences, University of Guelph, Guelph, Canada; bDepartment of Chemistry, University of Guelph, Guelph, Canada; cStrategic Solutions Group, Puslinch, Canada (Received 3 October 2017; accepted 24 May 2018)

The giant , Vespa mandarinia is a predator of honey bees throughout its range in eastern Asia. The ’ behaviors are coordinated through pheromones; a marking pheromone is used to mark nests as a food source, and an alarm pheromone, comprised of three chemical components of their venom, is released when a hornet is attacked by honey bees. The Asian honey bee Apis cerana evolved in sympatry with the giant hornet. In contrast, the European honey bee, Apis mellifera and V. mandarinia evolved in allopatry, although A. mellifera colonies have been intro- duced within the hornet’s range. We investigated the responses of these two honey bee species to the V. mandarinia marking pheromone, two alarm pheromone chemicals individually, and the three alarm pheromone chemicals together. We applied these substances above hive entrances and photographically monitored pre- and post-treatment numbers of bees on the exterior of the hive near the entrance. A. cerana colonies responded to the marking pheromone by quickly retreating into the hive, causing the numbers of bees near the hive entrance to decrease quickly. The bees responded to the other two alarm pheromone components and the 3-compound alarm pheromone treatment by exiting their hive and increasing their activity at the entrance. A. mellifera colonies did not respond to any of the hornet chemical treat- ments. The marked differences in responses of the two honey bee species suggest that Asian honey bees are able to alter their behavior in response to one of their major predators while honey bees that are not exposed to this preda- tor do not. Respuestas del comportamiento de las abejas melıferas, Apis cerana y Apis mellifera, a las feromonas de marcacion y de alarma de Vespa mandarinia El avispon gigante, Vespa mandarinia, es un depredador de las abejas melıferas en toda su area de distribucion en Asia oriental. El comportamiento de los avispones se coordina a traves de las feromonas: una feromona de marcacion se uti- liza para marcar los nidos de las abejas melıferas como fuente de alimento y una feromona de alarma, compuesta de tres componentes quımicos de su veneno, se libera cuando un avispon es atacado por las abejas melıferas. La abeja asia- tica, Apis cerana, evoluciono en simpatrıa con el avispon gigante. En contraste, la abeja europea Apis mellifera y V. man- darinia evolucionaron en alopatrıa, aunque se han introducido colonias de A. mellifera en el area de distribucion del avispon. Investigamos las respuestas de estas dos especies de abejas melıferas a la feromona de V. mandarinia, dos pro- ductos quımicos de feromonas de alarma individualmente y los tres productos quımicos de feromonas de alarma juntos. Aplicamos estas sustancias sobre las entradas de las colmenas y monitoreamos fotograficamente el numero de abejas antes y despues del tratamiento en el exterior de la colmena cerca de la entrada. Las colonias de A. cerana respon- dieron a la feromona de marcado retirandose rapidamente a la colmena, causando que el numero de abejas cerca de la entrada de la colmena disminuyera rapidamente. Las abejas respondieron a los otros dos componentes de la feromona de alarma y al tratamiento de feromonas de alarma de 3 compuestos saliendo de su colmena y aumentando su actividad en la entrada. Las colonias de A. mellifera no respondieron a ninguno de los tratamientos quımicos del avispon. Las marcadas diferencias en las respuestas de las dos especies de abejas melıferas sugieren que las abejas melıferas asiaticas son capaces de alterar su comportamiento en respuesta a uno de sus principales depredadores, mientras que las abejas melıferas que no estan expuestas a este depredador no lo hacen. Keywords: Alarm pheromone; Apis; chemical synthesis; coevolution; honey bee; kairomone; marking pheromone; Vespa

Introduction Turillazzi, 2010). In addition to feeding on individual for- The giant hornet, Vespa mandarinia is a major honey agers as they return to their nests, the hornets also bee predator that occurs over much of eastern Asia launch mass attacks on entire bee colonies that are (Lien & Carpenter, 2002). These hornets live in large coordinated with pheromones (Ono, Igarashi, Ohno, & colonies with correspondingly large food demands. Sasaki, 1995). After locating a nest of bees, scout hor- Honey bees, with their large colonies, represent a food nets mark it with a pheromone produced by the van bonanza for hornets (Baracchi, Cusseau, Pradella, & der Vecht gland on the last abdominal sternite (Ono

Corresponding author. Email: [email protected]

ß 2018 International Bee Research Association 2 B. McClenaghan et al. et al., 1995). Their nestmates, recruited to the vicinity their marking pheromone, 1-methylbutyl 3-methylbuta- of the nest, orient to the marking pheromone, locate noate (1-M-3-MB; Ono et al., 2003), on the exterior sur- the bee nest, and gradually increase in numbers (Abrol, face of the bees’ nest. It serves as a signal that enables 2013; Ono et al., 1995). If the hornets gain access to other members of the hornet colony to find the food the nest, the bees’ defenses become overwhelmed and (i.e., bees and their brood and honey; Abrol, 2013;Ono the bee colony is killed (A. mellifera; Matsuura & et al., 1995). Ono et al. (1995) demonstrated that A. Sakagami, 1973) or it absconds (A. cerana; Ono et al., cerana responds to the marking pheromone of V. mandar- 1995), in both cases leaving behind a large quantity of inia by increasing the number of bees just inside the hive brood and honey (0.25–0.75 kg for A. cerana; Fuchs & entrance. Additionally, when a V. mandarinia hornet is Tautz, 2011) that the hornets forage upon for several attacked or disturbed it releases an alarm pheromone days afterwards. blend consisting of three chemicals contained within its Apis cerana, the Asian honey bee, has evolved many venom: 2-pentanol (2-P), 3-methyl-1-butanol (3-M-1-B), remarkable defenses to several species of wasp preda- and 1-M-3-MB, the marking pheromone (Ono et al., tors. Bees at the nest entrance “shimmer” in unison in 2003). The hornets may release this alarm pheromone in response to the visual stimulus of a wasp, especially V. response to being heat-balled by honey bees. Additionally, 3-M-1-B is found in honey bee alarm phero- velutina and V. simillima (Fuchs & Tautz, 2011; Oldroyd & mones (Wang et al., 2016b;Wenetal.,2017). Wongsiri, 2006). Shimmering involves a coordinated Hornet pheromones may become enemy-avoidance rapid shaking of the bees’ abdomens that is repeated at kairomones (Ono et al. 2003; Ruther, Meiners, & Steidle, approximately one-second intervals (Tan, Wang, Chen, 2002) if detected and acted upon by the bees. In many Hu, & Oldroyd, 2013). This “Iseeyou” display informs predator-prey systems, prey species have evolved incoming forager bees of the presence of the wasp, and responses to pheromones of their predators. For may inform the wasp that it has been detected (Tan example, Daphnia, after detecting chemicals emitted by et al., 2012). In response to a wasp, some of the bees predatory midge larvae, move to greater depths and are emit vibrational “stop signals” that are tuned to the stimulated to grow antipredator “neckteeth” (Lampert, severity of the threat (i.e., the species of wasp; Tan Tollrian, & Stibor, 1994; Tollrian & Dodson, 1999). et al., 2016). Bees can detect the odor of live wasps Stingless bees, Trigona (Tetragonisca) angustula, detect cit- while foraging, and it reduces their ability to learn floral ral secreted by attacking Lestrimelitta limao bees and odors (Wang et al., 2016a). Furthermore, when V. man- respond by increasing the number of aerial defenders near darinia hornets arrive at a colony of A. cerana,worker their nest entrance (Wittmann, Radtke, Zeil, Lubke,€ & bees retreat into the nest and aggregate near the Francke, 1990). Within the honey bees, has entrance (Ono, Terabe, Hori, & Sasaki, 2003). If a wasp been shown to detect and avoid pheromone trails left by of any species gets too close to the bees, they rush out weaver ants (Oecophylla smargdina), a major predator of and surround it in a tight ball, then generate heat with bees throughout tropical Asia (Li, Wang, Tan, Qu, & Nieh, their flight muscles (Ken et al., 2005; Ono et al., 1995). 2014). Moreover, A. cerana “eavesdrop” on other honey Due to a higher temperature tolerance in A. cerana com- bee species by detecting their alarm pheromones and pared to that of wasps, the honey bees are able to raise avoiding associated food resources (Wang et al., 2016b). ’ their body temperature above the wasp s lethal limit (Ono, We qualitatively investigated the responses of A. mel- Okada, & Sasaki, 1987). Heat and carbon dioxide generated lifera from Canada and A. cerana from Vietnam to the by the bees kill the wasp in the bee ball (Sugahara & marking pheromone and alarm pheromone chemicals of Sakamoto, 2009). However, if the wasps manage to over- the Asiatic hornet V. mandarinia. We hypothesized that whelm the bees’ defenses, the bees abscond. A. cerana in Vietnam, living sympatrically with V. mandari- Apis mellifera has been transported worldwide and, in nia, would respond defensively to both alarm and mark- the past 50 years, has become widespread in many parts ing pheromones of the hornets. In contrast, we of Asia where they are now exposed to novel wasp hypothesized that A. mellifera in Canada, having never predators (Abrol, 2006). In contrast to the suite of been exposed to V. mandarinia, would exhibit either defensive responses of A. cerana, European A. mellifera weak or no responses to the hornet pheromones. We colonies, having evolved in the absence of strong wasp tested the responses of these two species of bees by predators, exhibit weak nest defense and their colonies quantifying the numbers of worker bees on the front of generally are overwhelmed by attacking hornets their hive both before and after applying the alarm and (Matsuura & Sakagami, 1973; Ono et al., 1995). This marking pheromones of the hornet. weak defense is evident both in Asia (Matsuura & Sakagami, 1973) and in western Europe where the intro- Materials and methods duced Asian wasp V. velutina has devastated colonies of European honey bees since its initial detection in south- Experimental organisms ern France in 2004 (Monceau, Bonnard, & Thiery, 2014). We studied 25 A. cerana colonies on 3–4 May 2013, at As mentioned, V. mandarinia hornets coordinate their an apiary in Cuc Phuong, Ninh Binh Province, northern predatory behaviors with pheromones. They deposit Vietnam (20.238N, 105.722E). These bees were locally Honey bee responses to pheromones 3 sourced. We observed no giant hornets during our entrance; on A. mellifera hives, we applied the chemical study. Each hive was presented only one treatment per in three locations above the wide hive entrance that day and no hive was given the same treatment twice. extended nearly the width of the hive (i.e., the larger A. We studied 30 A. mellifera colonies on 20 June 2013, at mellifera colonies received 3 times as much of each the University of Guelph Honey Bee Research Centre chemical; see Online Supplementary Material Figure S1). in Guelph, Ontario, Canada (43.536N, 80.214W). The amount of each chemical typically released by V. Some hives were tested twice, however 5 h transpired mandarinia when attacking a bee colony is unknown. between treatments and no hives were treated twice Because the exact volume we applied with the chop- with the same chemical. The A. mellifera colonies were stick was not quantified, our study of the bees’ of northern European origin (predominantly of the sub- responses was qualitative. After the application of the species A. m. carnica and A. m. ligustica; P G Kelly, pers. chemical(s), we took a photo of the hive front every comm.) and, being studied in Canada, they had never 10 s for the first two minutes and then every minute up previously come into contact with any Asian hornet to five minutes. Notes on the behaviors of the bees species, including V. mandarinia. Because there were no were recorded throughout the five-minute trial interval. European honey bees near Cuc Phuong, Vietnam, we could not conduct trials on A. mellifera and A. cerana Data analysis colonies in the same location (i.e., under the same environmental conditions). We counted the number of bees on the hive front within 5 cm of the hive entrance in the photos taken before and after the treatments had been applied. The Experimental protocols two species differed greatly in numbers of bees near We experimented with synthetic V. mandarinia marking the entrance (i.e., A. mellifera colonies had 10–30 and alarm chemicals. 3-M-1-B (> 98%, iso-amylalcohol, more bees near their wider hive entrances). CAS#123-51-3) and R/S-2-P (> 98%, CAS # 6032-29-7) Consequently, we calculated the bees’ responses rela- were commercially sourced from Sigma-Aldrich tive to pre-treatment activity in order to facilitate com- (Oakville, Ontario, Canada) and used as received. The parisons between the two species. The six pre- racemic ester R/S-1-M-3-MB (CAS # 117421-34-8) was treatment values for number of bees at the hive synthesized in a novel procedure by Dean-Stark con- entrance were averaged and assigned a relative value of densation of 2-P and isovaleric acid (>98%, 3-methylbu- 1.0 at –30 s; post-treatment values were plotted relative tanoic acid, CAS# 503-74-2; also commercially sourced; to this pre-treatment value. We applied the chemical see Online Supplementary Material for details of the and sham treatments at time zero. Due to limitations synthesis). We tested bee colony responses to each of imposed by the number of trials performed and result- the three pheromone chemicals individually and, to ing low degrees of freedom, we condensed the data on approximate the full alarm pheromone, in combination. post-treatment bee numbers by averaging the data from We also performed sham trials with water to ensure two consecutive photo scans for chemical trials and that any responses we observed were due to phero- from four consecutive scans for the sham trials. mone treatments and not our experimental procedure. Consequently, we analyzed the data representing nine We performed 10 replicates of each of the four time points for the chemical treatments and five time chemical treatments and five replicates of the sham points for the sham treatment. Based on the residual treatment on each Apis species. We performed fewer analysis, we log transformed the response variables. To sham trials because we were limited by time and the test for the effects of time, species, treatments, and number of hives we had access to in Vietnam and we interactions, we used a repeated measures ANOVA wanted to avoid treating any hive more than once in a with an uncorrected error structure; p-values are pre- day. We monitored bee behavior at the hives through- sented to indicate statistical significance at a ¼ 0.05. In out each hive trial by continuous visual observations order to assess the differences between the time and photographic scan sampling. For one minute prior points, the sham trial data were analyzed in a separate to applying a chemical treatment we took photos every model from the treatments because after condensing 10 s to determine the basal activity level of the bees the data, the time intervals differed between treatment around the hive entrance. Due to our remote location and sham trials. and the lack of access to a micropipette, we applied a small spot of a chemical directly above the main hive entrance using the tip of a wooden chopstick that had Results been dipped in one of the liquid chemicals (3–5 ml; The analysis of the sham trials demonstrated that our determined by weighing chopsticks that had been treatment method had no effect on the number of bees dipped in water, then again after applying a liquid spot). at the entrances over time (F(4,8) ¼ 0.44, p ¼ 0.77; For the alarm pheromone blend, we applied a spot of Supplementary Figure S2). In contrast, our analysis of each of the three chemicals (ratio of 1:1:1). On A. the pheromone treatments indicated that the number cerana hives, we applied the chemical above the narrow of bees at the hive entrance was affected by the species 4 B. McClenaghan et al.

Figure 1. Response of honey bees to (a) the marking pheromone (1-M-3-MB), (b) alarm pheromone component (3-M-1-B), (c) alarm pheromone component (2-P), (d) alarm pheromone blend of all three chemicals. Asterisks ( ) indicate significant differences in the number of bees on hive fronts relative to the pre-treatment average (assigned a value of 1.0) within each species, based on a repeated measures ANOVA at a ¼ 0.05. Refer to Table 1 (Panel A) for details of statistical significance.

(F(1,72) ¼ 170.21, p < 0.0001), the time elapsed since response to 3-M-1-B and four-fold in response to 2-P. treatment (F(8,72) ¼ 5.38, p < 0.0001) and the interaction A. mellifera colonies, in contrast, failed to respond to between treatment and time (F(24,72) ¼ 3.39, p < 0.0001) these same two alarm pheromone components indicating that the pattern of change in number of bees (p > 0.05 for both chemicals; Figure 1b,c). When all at the entrance over time differed between treatments. three of the hornet alarm pheromone chemicals were See Supplementary Figures S3 and S4 for the untrans- applied simultaneously (Figure 1d), A. cerana workers formed data for all treatments in A. mellifera and A. again responded by exiting the hive and there was a sig- cerana colonies, respectively. nificant 2.5-fold increase in the number of bees at the In response to the V. mandarinia marking pheromone hive entrance within the first minute. The initial (1-M-3-MB), A. cerana retreated into their hive; there response was less intense but the return to baseline was a 50% decrease in activity at the hive entrance activity was slower than with the individual chemical within 25 s of its application, and at the end of the five- treatments. A. mellifera showed a slight numerical but minute trial period bee numbers at the entrance had statistically nonsignificant increase in response to the 3- not begun to recover (Figure 1a). In contrast, A. melli- chemical treatment. fera colonies exhibited a slight numerical but non-signifi- Table 1 displays statistical differences between all cant decrease in the number of bees at the entrance in possible pairs of time points for all four treatments applied to A. cerana hives. Statistical differences are not response to the hornet marking pheromone (Figure 1a). shown for A. mellifera since there were no significant In response to 3-M-1-B and 2-P, both components of differences between any time points for any treatments. the V. mandarinia alarm pheromone, when applied indi- vidually, A. cerana exited the hive and the bees became much more active (Figure 1b,c, respectively), walking Discussion and turning on the hive front near the entrance. There Apis cerana responded to V. mandarinia marking and was a rapid and significant increase in the number of A. alarm pheromones by rapidly altering their level of cerana bees at hive entrances in response to both activity at the hive entrance. The hornet marking phero- chemicals. Within 25 s of applying a chemical, the num- mone, 1-M-3-MB, caused worker bees to retreat into ber of bees on the hive front had increased eight-fold in their hives, which led to a reduction in the numbers of Honey bee responses to Asian hornet pheromones 5

Table 1. p-Values between all possible pairs of time points for A. cerana trials, based on a repeated measures ANOVA for: (Panel A) marking pheromone (1-M-3-MB); (Panel B) alarm pheromone component 3-M-1-B; (Panel C) alarm pheromone component 2-P; (Panel D) alarm pheromone mixture of 3-M-1-B, 2-P, and 1-M- 3-MB, as reported by Ono et al. (2003). p-Values indicating significant differences (p < 0.05) are shown in bold font. Panel A Times post-treatment (s) 5 25 45 65 85 105 150 270 30 0.3667 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 0.0001 0.0176 5 – 0.4475 0.2626 0.3563 0.5179 0.5845 0.5161 0.5298 25 –– 0.0543 0.3605 0.8100 0.5666 0.8756 0.9372 45 –– – 0.2958 0.0738 0.0541 0.1932 0.6814 65 –– – – 0.1276 0.0957 0.3621 0.8533 85 –––––0.5561 0.94 0.8666 105 ––––––0.5059 0.7463 150 –––––––0.8784 Panel B Times post-treatment (s) 5 25 45 65 85 105 150 270 30 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 0.0561 5 – <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 25 ––0.0537 0.8691 0.6912 0.5802 0.3344 0.2870 45 –––0.0579 0.2518 0.383 0.0446 0.1290 65 ––––0.6859 0.5873 0.224 0.2334 85 –––––0.7718 0.0633 0.2067 105 ––––––0.0077 0.1420 150 –––––––0.4422 Panel C Times post-treatment (s) 5 25 45 65 85 105 150 270 30 0.522 0.0044 <0.0001 <0.0001 <0.0001 <0.0001 0.001 0.4655 5 – 0.5962 0.291 0.2333 0.2292 0.3221 0.5233 0.3943 25 –– 0.0072 0.0015 0.0044 0.0386 0.5722 0.0401 45 –– – 0.4203 0.4052 0.9747 0.2534 0.008 65 –– – – 0.7251 0.4972 0.0474 0.0019 85 –– – – – 0.1965 0.0057 0.0018 105 ––––––0.0162 0.0015 150 –––––––0.0131 Panel D Times post-treatment (s) 5 25 45 65 85 105 150 270 30 0.8612 0.1904 0.0766 0.0021 0.0008 0.0054 0.1308 0.3176 5 – 0.4982 0.3828 0.1914 0.1766 0.2885 0.4943 0.5101 25 ––0.2774 0.0022 0.0037 0.0678 0.8094 0.8150 45 –––0.0159 0.0286 0.3251 0.6612 0.9641 65 –––– 0.5487 0.3826 0.0254 0.4978 85 –––– – 0.0699 0.0012 0.4222 105 –––– – – 0.0100 0.6394 150 –––––––0.8759

bees on hive fronts that persisted for the full five- bees exiting their hives and accumulating in increased minute study period. Observations by Ono et al. (1995) numbers on the hive fronts. Numbers of bees rapidly parallel ours; they observed worker bees to retreat increased when 3-M-1-B and 2-P were applied individu- into their hive after it was marked by a foraging hornet. ally, whereas the three-chemical alarm pheromone In contrast, 3-M-1-B and 2-P, two alarm pheromone treatment caused a weaker but more sustained increase components, as well as the combination of all three- in the numbers of bees on the hive front. This weaker alarm pheromone components resulted in A. cerana response may be due to the ratio with which we 6 B. McClenaghan et al. applied the alarm pheromone components (equal parts predator-prey systems ( Li et al., 2014; Matthews & of all three), while in venom extracted from the venom Matthews, 2010; Wang et al., 2016a; Wittmann et al., gland of V. mandarinia 2-P was shown to be a major 1990). In our honey bee-hornet system, hornet phero- component compared to 3-M-1-B and 1-M-3-MB (Ono mones provide honest cues to honey bees of hornet et al., 2003). alarm and predatory behaviors. 3-M-1-B is also a com- Although A. mellifera showed slight behavioral ponent of the honey bee alarm pheromone. A. cerana responses in the same directions as A. cerana, none of foragers avoided the alarm pheromone mixtures from their responses were significant. The honey bees we congenerics, however, they did not avoid 3-M-1-B on experimented within Canada have never been exposed their own (Wang et al., 2016b). This suggests that A. to wasp predators and, therefore, could not have cerana may exhibit a different response to the same learned to respond to hornet pheromones. It is possible chemical when foraging and when at the hive. that introduced A. mellifera colonies that are sympatric The lack of response of European A. mellifera to the with V. mandarinia show some level of learned response pheromones of Asian V. mandarinia could reflect the to hornet pheromones. Alternatively, it is possible that absence of selection pressures of wasps on the bees the response we observed here is an innate response due to their non-overlapping ranges during their evolu- and A. mellifera, having evolved in allopatry with V. man- tionary history (Ken et al., 2005) or an absence of darinia, has not evolved a similar response. learned behavior due to the lack of exposure to hornet These qualitative results support our hypothesis that predators. Where wasp predators are more abundant A. cerana would respond to both the marking and alarm in the native range of A. mellifera, the bees do exhibit pheromones of V. mandarinia. Their responses seem some responses to the local wasp species (Baracchi adaptive. By retreating when a hornet has marked the et al., 2010; Kandemir et al., 2012). These behaviors colony as a food source, the number of bees directly include massing at the entrance, flying at the wasps, and exposed to recruited hornets on the outside of the the formation of bee balls coupled with heat generation. hive is reduced. In Japan, after exposure to the marking There is no need for anti-wasp behaviors in North pheromone, A. cerana workers retreat inside their hive America, Australia, and, until recently, much of Europe, and congregate near the hive entrance (Ono et al., where there have been no significant wasp predators of 2003), prepared to heat-ball hornets should they have honey bees (following paragraphs discuss V. velutina in an opportunity to do so. A. cerana in Vietnam may have Europe). A. mellifera colonies originating from those acted in this same way, however, the hive design did regions have been introduced and are widely managed not allow us to observe within-nest behaviors. In con- in Asia for their prolific honey production. They lack trast, when a hornet is being heat-balled and tries to the shimmering (Fuchs & Tautz, 2011; Oldroyd & sting the attacking bees, small releases of venom that Wongsiri, 2006), strong heat-balling (Abrol, 2013;Ono contain the alarm pheromone would stimulate worker et al., 1995) and kairomonal responses to hornet phero- bees to exit their nest to aid-in defense of the colony mones (Ono et al., 1995; this study) of A. cerana. against the hornet. Introduced A. mellifera bees are consequently much These adaptive responses may reflect coevolution more susceptible to giant hornet attacks in Asia. In between A. cerana and their predator V. mandarinia or Vietnam, beekeepers must live in A. mellifera apiaries, to they may reflect a learned behavior. We did not test if protect the colonies by killing predatory wasps (GWO, this response was innate, however, it is unlikely that the pers. obs.). honey bees we experimented on in Vietnam were Recently, A. mellifera has become exposed to attacks exposed to wasp predators previously. Wasps are by the Asian hornet V. velutina within its native range in active predators of honey bees in late-summer and fall Europe (Villemant, Haxaire, & Streito, 2006). V. velutina and we performed our experiments in the early May has expanded its range rapidly from an inferred location (Matsuura & Sakagami, 1973). Honey bees workers typ- in southwestern France (Robinet, Suppo, & Darrouzet, ically do not live longer than a few months suggesting 2017). It preys extensively on honey bee colonies that that it is unlikely these honey bees had previously inter- lack protective defensive behaviors (Arca et al., 2014; acted with wasp predators (Neukirch, 1982; Monceau et al., 2014). This situation in Europe is analo- Woyciechowski & Moron, 2009). The hornets have gous to that of introduced A. mellifera colonies in Asia evolved pheromones that they use to recruit their nest- that are exposed to a number of predatory Vespa spe- mates to food (marking pheromone) or to defend cies. Some local populations of honey bees in Europe themselves from attackers (alarm pheromone). In have evolved heat-balling behavior in response to wasp response, Asian honey bees have developed the ability predators, however, most populations have yet to to detect and respond to these hornet-produced chemi- develop effective defenses against these novel predators cals by avoiding the marking pheromone through (Arca et al., 2014; Papachristoforou et al., 2007). retreat and by becoming more excited by the alarm In this study, we demonstrated strong responses by pheromone. The use of kairomones by prey species to Asian A. cerana to the chemicals that constitute the detect their predators has been observed in many marking and alarm pheromones of the Asian hornet, V. Honey bee responses to Asian hornet pheromones 7 mandarinia. In sharp contrast, we observed no significant Fuchs, S., & Tautz, J. (2011). Colony defense and natural ene- changes in behavior of European A. mellifera to these mies. In R. Hepburn & S. E. Radloff (Eds.), Honey bees of – same chemicals. Although the doses of hornet phero- Asia (pp. 369 395). New York: Springer. Kandemir, I., Cakmak, I., Abramson, C. I., Cakmak, S. S., mone chemicals we applied to hives were approximate Serrano, E., Song, D. N., … Wells, H. (2012). A colony and the amounts of the hornet pheromone compounds defense difference between two honey bee subspecies (Apis emitted naturally have never been quantified, our results mellifera cypria and Apis mellifera cacasica). Journal of do demonstrate strong qualitative interspecific differen- Apicultural Research, 51, 169–173. doi:10.3896/IBRA.1.51.2.0. ces. We interpret the A. cerana behaviors as kairomonal Ken, T., Hepburn, H. R., Radloff, S. E., Yusheng, Y., Yiqiu, L., Danyin, Z., & Neumann, P. (2005). Heat-balling wasps by responses to their natural predator V. mandarinia (c.f., honeybees. Naturwissenschaften, 92, 492–495. doi:10.1007/ Kandemir et al., 2012). Future studies utilizing treat- s00114-005-0026-5. ments that match the true amounts of the chemicals Lampert, W., Tollrian, R., & Stibor, H. (1994). Chemical induc- released by the hornets and utilizing venom directly tion of defense mechanisms in freshwater . – extracted from hornets are required to confirm and Naturwissenschaften, 81, 375 382. Li, J. J., Wang, Z. W., Tan, K., Qu, Y. E., & Nieh, J. C. (2014). extend our results. Giant Asian honey bees use olfactory eavesdropping to detect and avoid ant predators. Behavior, 97,69–76. 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