Hovering and standing guards: nest defense strategies in a polymorphic stingless bee (Tetragonisca angustula)

Hovering and standing guards: nest defense strategies in a polymorphic stingless bee (Tetragonisca angustula) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Hovering and standing guards: nest defense strategies in a polymorphic stingless bee (Tetragonisca angustula) Luana Guimarães Santos, Bruno Vieira, Jéferson Pedrosa, Fábio do Nascimento This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4915536/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The stingless bee, Tetragonisca angustula, has a sophisticated nest defense strategy carried out by guards that are larger compared to other workers. Guards display two different strategies: flying near the colony entrance (hovering guards) or positioning themselves at the entrance tube (standing guards). To better understand the roles played by each guard behavioral phenotype in nest defense, we investigated whether their behaviors were distinctly displayed when faced with different threats. We used two types of bait (flying and walking) to simulate threats to the colony and compared the behaviors displayed by the guards in relation to the species used as bait and the guard function. We also investigated if the species and the type of bait influenced the number of guards before and after the presentation. We found a significant interaction between the behaviors displayed by the guards and the bait species. Hovering guards were more influenced by flying baits, and standing guards by walking baits. The presence of Lestrimellita limao caused a high proportion of recruitment and aggressive behavioral responses from guards, confirming specialization against this potential enemy. Our results show that the two behavioral phenotypes are capable of recognition and act with complementary behaviors depending on the threat. Biological sciences/Ecology/Behavioural ecology Biological sciences/Ecology/Evolutionary ecology Nest defense resources behavioral plasticity polyethism Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. INTRODUCTION The division of labor is fundamental to the organization of the colony of social insects [ 1 , 2 , 3 ]. Division of labor may be associated with age polyethism (behavior changes throughout life and workers specialize in a subset of tasks according to age), physiological changes (e.g., changes in juvenile hormone titers) and distinct morphotypes among workers [ 4 , 5 , 6 , 7 , 1 , 8 ]. For example, in ants and termites, colony defense is performed mostly by a subcaste of soldiers that are larger and specialized for this task [ 9 , 10 , 1 ]. Thus, workers may present adaptive morphological traits for specific tasks, such as foraging or defense, which improves the colony maintenance and maximizes performance, as these adaptations can improve the individual’s effectiveness [ 11 , 12 ]. In social insects, nest defense is important to both reproductive success and colony survival. Besides the food collected by foragers, the brood and even the nest location are valuable resources and, thus, require protection. Generally, the defense is performed by guards who stay at the colony entrance [ 13 , 4 , 14 , 2 ]. The guards can discriminate nestmates from other individuals via chemical cues, such as cuticular hydrocarbons or volatile ones, which are used to select those familiar individuals to enter the colony while intruders are intercepted at the entrance tube [ 15 , 16 ]. Intruders may be animals of other species, such as wasps and ants, or even other bees, conspecifics or not, as nest pillaging is a common practice in tropical species of stingless bees [ 17 , 14 , 18 , 19 , 20 ]. Previously, there was no evidence suggesting the existence of a morphologically specialized subcaste in eusocial bees, but recent studies managed to determine that at least ten stingless bee species have larger individuals responsible for colony defense. Although the level of polymorphism is not as evident as in ants and termites’ soldiers for example, these guards are significantly larger than other workers [ 21 ]. Phylogenetic reconstruction of the evolutionary history of 28 species suggests that specialized guards have evolved independently five times among these species, linking the appearance of this subcaste with the threat posed by Lestrimelitta Friese (1903) spp. to these species [ 21 ]. Tetragonisca angustula Latreille (1811), commonly known as Jataí, is one of the most common species of eusocial bees native to the Neotropical region, due to being extremely successful in urban environments [ 22 , 23 ]. T. angustula has a guard subcaste with larger heads, is heavier, and has longer legs when compared to foragers [ 12 ]. In addition, guards work relatively more than other workers, also displaying a larger task repertoire. Furthermore, task transition occurs more quickly in these individuals, when in comparison with the minor workers [ 8 , 24 ]. Jataí is one of the few species of stingless bee that presents some resistance to attacks carried by the robber bee Lestrimelitta limao Smith (1863), which is specialized in attacking stingless bee nests to steal pollen and nectar. Therefore, L. limao is considered a major threat to the survivability of Meliponini nests, with attacks often leading to the death of the colony [ 25 , 26 , 27 , 28 ]. L. limao releases a volatile citral compound to disrupt the defensive behavior of the targeted colony, which Jataí recognizes as a threat, triggering its defensive behavior [ 29 ]. The guards display two distinct sets of behaviors at the entrance of the nest, allowing them to be classified into two different behavioral groups: (1) Hovering guards, which hover around the entrance tube, and (2) Standing guards, which remain stationary on the outer wall of the tube. Hovering guards are supposedly responsible for visually inspecting approaching individuals to identify and distinguish between heterospecific and conspecific animals. [ 29 , 30 , 31 , 32 , 33 ]. On the other hand, standing guards are believed to be responsible to distinguish nestmates from non-nestmates [ 32 ]. Since guards from this species display two distinct and possibly complementary set of behaviors, our aim was to study the factors involved in the defensive behavior in a species with a defensive morphologically specialized subcaste in the highly competitive environments in which these bees live. Here, we test whether hovering and standing guards would exhibit distinct behavioral repertoires in relation to different types of intruders (flying and walking baits), to verify a possible specialization and complementation between both guard and intruder types. So, to conduct the experiments, we used each type of bait and analyzed the behavioral repertoire of the hovering and standing guards of T. angustula . We conducted two behavioral tests: Experiment I, which examined the behavioral repertoire of hovering and standing guards in response to the two types of bait, and Experiment II, which measured the number of hovering and standing guards before and after the presentation of different bait types. Our hypothesis is that the number and defensive behavior of guards are directly related to the potential threat encountered. 2. RESULTS 2.1. Number of guards before and after bait presentation We observed that the number of standing guards was always higher than the number of hovering guards before we presented the bait (supplementary table 1 ). We found significant interactions between the bait presentation phase and the type of guards at the entrance of the colony in the following species: L. limao (flying: χ² = 55.78; p < 0.001; walking: χ² = 80.73; p < 0.001; Supplementary Table 1, Fig. 1 a and 1 b), Menemerus sp. (χ² = 7.784; p = 0.005; Supplementary Table 1, Fig. 2 a), A. sexdens (χ² = 10.68; p = 0.001; Supplementary Table 1, Fig. 2 b) and Camponotus sp. (χ² = 14.22; p < 0.001; Table 2, Fig. 2 c). We did not find significant differences in relation to the number of guards before and after bait presentation in S. aff depilis (χ² = 0.37; p = 0.54; Supplementary Table 1) and the piece of paper used as control (walking: χ² = 0.04; p = 0.82; flying: χ² = 0.004; p = 0.94; Supplementary Table 1). In all significant interactions, the number of standing guards was lower after the bait presentation (mean ± standard deviation, before and after presentation, respectively: A. sexdens :18.556 ± 7.294; 12.222 ± 6.717; Camponotus sp.: 18.667 ± 5.625; 9.778 ± 8.128; L. limao (walking): 22.444 ± 5.447; 12.778 ± 6.813; L. limao (flying): 16.944 ± 6.601; 13.444 ± 8.41; Menemerus sp.: 16.705 ± 6.555; 9.882 ± 5.977; Supplementary Table 1, Fig. 1 a − 2c). L. limao was the only species used as bait that increased the number of hovering guards after being presented (mean ± standard deviation, before and after presentation, respectively: L. limao (flying): 5.5 ± 3.185; 13.111 ± 5.738; L. limao (walking): 5.277 ± 2.371; 11.444 ± 4.273; Fig. 1 a and 1 b, Supplementary Table 1). All other species showed no difference in the number of hovering guards before and after presentation. 2.2. Behaviors displayed by guards. When exposed to flying baits, we found a significant interaction between the behaviors displayed by each type of guards and the species used as bait (F 2,89 =14.113; p < 0.001). SIMPER analysis indicated that the following behaviors differed between the two types of guards and had the most dissimilarities between them: approach (P) (average contribution ± s.d.; 0.184 ± 0.173; p < 0.001), signaling (S) (0.137 ± 0.19; p < 0.01), evasion (E) (0.155 ± 0.196; p < 0.001) conturbation (C), (0.089 ± 0.157; p = 0.01), attack (A) (0.083 ± 0.124; p < 0.01), recruitment (R) (0.08 ± 0.119; p < 0.01) and grouping (G) (0.046 ± 0.101; p < 0.01). Hovering guards were responsible for displaying more of the P (average abundance (%) of displayed behaviors by hovering x standing guards: 0.6 x 0.088), R (0.32 x 0) A (0.32 x 0) and G (0.18 x 0) behaviors than the hovering ones, while the contrary was shown with the S (0 x 0.377), E (0 x 0.422) and C (0 x 0.266) behaviors. The NMDS plot also displays a strong separation between both types of guards and their displayed behaviors (Fig. 3 ). The guards also displayed a difference of behaviors between them when exposed to the walking baits. Our PERMANOVA results also indicated a significant interaction between the type of guard and the bait species (F 4,124 =5.392; p < 0.001). SIMPER results showed a dissimilarity between the two guard types and their presented behaviors, differing in signaling (S) (average contribution ± s.d.: 0.114 ± 0.146; p < 0.01), conturbation (C) (0.226 ± 0.164; p < 0.01), recruitment (R) (0.086 ± 0.155; p < 0.01), evasion (E) (0.154 ± 0.186; p < 0.001) and grouping (G) (0.036 ± 0.105; p = 0.02). Hovering guards showed more prominently the R (average abundance (%) of displayed behaviors by hovering x standing guards: 0.274 x 0.012), and G (0.117 x 0.012) behaviors, while the standing guards were responsible for displaying more of the C (0 x 0.747), E (0.117 x 0.457) and S (0 x 0.433) behaviors. Similar to what happened with the flying baits, the NMDS indicates a division between which guard displays which behavior (Fig. 4 ). 3. DISCUSSION Our results clearly show that there is task partitioning between the two types of guards in relation to their defensive behavior. When facing different types of threats, the guards can assess the situation and act accordingly. Our results also show that the number and types of guards at the nest entrance is related to what they are facing at the given moment, adjusting their quantity to adapt to the situation. The standing guards are more likely to entrench themselves inside the colony while the hovering guards only increase in numbers when facing real threats. When analyzing their behaviors, it’s also clear that each type of guard specializes in a function when faced with whether the enemy is flying or walking, showing a clear separation of the groups. 3.1. Number of guards before and after bait presentation Our results of this experiment suggest that the number of hovering and standing guards are affected by intruders. Compared to the number of guards before the presentation of the baits, in which the quantity of standing guards was greater than hovering guards, after the presentation we observed that the number of standing guards was lower in all baits presented that had significant differences. It can be argued that the standing guards, upon noticing intruders, place themselves inside the colony. The number of hovering guards was greater only after the presentation of L. limao , possibly meaning that T. angustula displays a specific defense strategy when perceiving the presence of its natural enemy. In the other baits we did not find a significant difference in the number of hovering guards. Therefore, hovering guards would be a more effective and aggressive defense to detect and contain specific intruders. There is a possibility that they can perceive and assess the threat level of suspicious individuals near its entrance. Regarding the species that we found a significant difference before and after the presentation of the baits, there were two species that synthesize the citral compound ( L. limao and A. sexdens ), a spider ( Menemerus sp.) that is a potential predator and an ant ( Camponotus sp.) that is a potential competitor for resources of T. angustula . Despite S. aff. depilis being an aggressive species, we found no significant difference. All other species except S. aff. depilis had a decrease of the quantity of standing guards. When exposed to A. sexdens , a species with volatiles similar to L. limao , like citral, the guards behaved differently than when exposed to L. limao walking individuals. The standing guards followed the same pattern for both, decreasing in numbers at the entrance, as was also observed for the other species with significant results. The hovering guards, however, did not increase in numbers, meaning that the standing ones entered the entrance tube of the nest, contrary to the observed by Karcher and Ratnieks [ 32 ]. Our findings are consistent with those found by Baudier et al. [ 24 ], in which the standing guards never changed to the function of hovering. This behavior of entering the colony help in nest defense, as the narrow entrance may diminish the number of enemies to be repelled at once. Another different behavior from the observed on Karcher and Ratnieks [ 32 ] when exposing the guards to Scaptotrigona bipunctata , a stingless bee from the same genus. In our study, the guards did not react to S. aff depilis , as their numbers did not show a significant difference from before and after the bait presentation, when they found an increase of hovering guards and a decrease of standing ones. With these results we can assume that each guard type is specialized in one function, and that they are complementary to each other when defending the nest. Baits that changed the number of guards at the entrance were different considering the guard type. We can say that there is a threat level assessment by each guard type that takes in consideration whether the enemy is flying or walking and can identify what enemies are more of a threat to the nest. The fact that hovering guards are responsible for the visual discrimination of conspecific and heterospecific individuals [ 29 , 30 , 31 , 32 , 33 ] suggests that they are also capable of recognizing S. aff. depilis as a non-threatening presence. The disturbance of the number of standing guards when the colony was exposed to L. limao can be explained by the strong presence of its kayromone, the citral. Previous studies reported that there is no difference in size in relation to the antennal lobe and the type of guard [ 34 , 35 ]. This explains why despite the bait being presented as flying, standing soldiers also reacted to its presence. We can infer the same about the hovering guards increasing in number when presented with it as a walking bait. 3.2. Behaviors displayed by guards Our findings show a distinct division between the analyzed behaviors, in which hovering and standing guards complement each other. The standing guards acted as signals that some disturbance was happening at the nest and the hovering ones acted as the main force of defense. The signaling and disturbance behaviors were only displayed by standing guards. This division suggests that hovering guards are responsible for detecting heterospecific flying individuals and standing guards act as a last defensive barrier at the entrance of the colony. Our results of this experiment suggest that the behaviors are different between the hovering and standing guards for each type of bait (flying or walking). Therefore, in flying baits we found a greater number of hovering guards showing approach (P), recruitment (R), attack (A) and grouping (G) behavior when compared to standing guards. The standing guards showed signaling (S) and disturbance (C) behaviors. We can infer that the defense mechanism of T. angustula acts in a coordinated way, where flying intruders are recognized and a disturbance occurs in the standing guards, which signal the presence of a potential enemy, promoting agitation in the colony and the beginning of the defense by hovering guards against flying enemies. When exposed to walking baits, we found a greater number of hovering guards showing recruitment (R) and grouping (G) behavior when compared to standing guards. The standing guards showed signaling (S) and disturbance (C) behaviors. We can assess that standing guards signals the presence of the walking enemies and shows disturbance at the entrance nest, which is the cue for the hovering guards to start recruiting other individuals for possible defense against walking enemies. It is also interesting to note that the signaling and conturbation behaviors are only displayed by the standing guards. The main defensive behavior from T. angustula is biting, and usually it involves the death of the defending guard, since they lock their mandibles on the intruder and don’t release it anymore [ 36 ]. The fact that there were no attacks on walking baits can indicate that these threats are not dangerous enough to elicit an agonistic behavior from the guards. T. angustula has an intrinsic evolutionary relationship with L. limao , as reported by Grüter et al. [ 21 ]. The repeated displayed behavior of social parasitism by L. limao was a driving force of the evolution of morphologically specialized guards in T. angustula . As already reported, hovering guards commonly display the behavior of ignoring conspecifics and focusing their attack effort on heterospecific bees, that generally displays a different color [ 37 ]. Recent studies by Baudier et al. [ 34 ] and Valadares et al. [ 35 ] presented results that indicate that there are differences in relation to the type of guard and their brain volume and structure. Hovering guards have a larger medulla, which is the structure responsible for processing the information related to motion detection in a small range [ 38 , 39 ] and color and shape differentiation [ 40 , 41 ]. This is reflected in the behavior observed in this study, since they were the ones who displayed the approach and attack behaviors, mainly when confronted with L. limao. Overall, our results suggest that there is a clear differentiation between hovering and standing guards in relation to their roles on colony defense. Hovering guards are responsible for assessing and acting upon a threat, while standing guards act as a barrier at the entrance tube, narrowing the space and number of possible invaders. We also found that the hovering guards’ number only increased when in the presence of a real threat to the colony, their natural enemy L. limao , which means that they can recognize when to display an agonistic behavior and increase the nest defense in numbers. This is a sophisticated and complementary system that probably evolved along the years to provide a more responsive and cost-efficient defense against raids. 4. METHODS 4.1. Study site and species We performed the experiments at the Universidade de São Paulo, campus Ribeirão Preto, Brazil (21°09'48.2"S, 47°51'37.9"W). We chose T. angustula for this study due to its natural occurrence and abundance at the region, and due to being an important pollinator for native plants. Besides, this species also has a temporal/behavioral specialization between its guards, allowing for an in-depth study of their roles in colony defense. We used eight colonies maintained in wooden boxes of similar size and weight. Data were collected over an 8-month period (March and April, from September to December 2017; January and February 2018). We regularly checked the nest entrance and counted the number of guards to assess colony activity and standardize the colonies used. We only conducted experiments during favorable weather conditions (ambient temperature ≈ 31ºC, 47% humidity and clear sky). 4.2. Bait species selection and presentation To analyze the roles played by both distinct guards’ phenotypes, we exposed them to two different types of baits: walking and flying intruders. We attached a transparent stick (1 meter) to a tripod and positioned it laterally to the colony entrance, so we could freely move it using the tripod head. We anesthetized the individuals through refrigeration (freezer at 5ºC) or with CO 2 (only spiders) and waited for the baits to reanimate before the experiments. The baits were attached to a piece of paper with superglue through the thorax or cephalothorax, leaving the movement of the appendages free. This paper was then placed at the tip of the stick. We simulated a flying intruder by adapting the methodology used by Van Zweden et al. [ 33 ]. We positioned the tripod in front of the colony entrance, and simulated the individual's behavior when flying, never obstructing the nest entrance passage (Fig. 5 a). When testing the flying baits, we moved the stick close to the nest wall, from above the entrance to below it, stopping 3 to 5 centimeters from the entrance, also adapted by Van Zweden et al. [ 33 ] (Fig. 5 b). We replaced the bait whenever it was touched by the guards to prevent the deposition of odors or any other cues that might incite aggressiveness. We chose the following species to simulate walking threats: a) jump spiders ( Menemerus Dufour, 1831), which are high potential predators of T. angustula [ 42 ]; b) carpenter ants ( Camponotus Mayr, 1861), which can be competitors for food resources and responsible for sporadic resources thefts of T. angustula nests [ 43 ]; c) leafcutter ants ( Atta sexdens Linnaeus, 1758), not considered to a threat to T. angustula , but with an interesting characteristic of eliciting odor of citral [ 44 ], a compound found in L. limao , used as a kairomone by T. angustula guards [ 25 ]. We also opted to use d) L. limao individuals as walking baits due to it being T. angustula natural enemy and to the occurrence of citral just like the leafcutter ants, allowing us to infer if the individual morphology has an influence on guards besides their exposition to the compound. We used the following species as flying baits: a) the stingless bee Scaptotrigona aff. depilis (Moure, 1942), potential intruders, competitors and responsible for occasional resource thefts at T. angustula nests [ 17 ]; b) T. angustula natural enemy L. limao , responsible for raids and predation of colonies. All individuals used as bait were collected at the campus facilities, and we kept them alive until their exposure to T. angustula . We used a clean paper with superglue, similar to the one which the baits were glued as control, in both walking and flying assays. 4.3. Behavioral assays Initially, we counted the number of guard bees and verified the standardization of this quantity over time. Subsequently, bait exposure tests were performed in each bee colony to analyze the behavior of the guards (both standing and hovering) walking and flying baits. Before each test, to record the natural activity of the colony, we filmed the guards during 1 minute and counted their number. This initial exposure could also decrease the influence of the observer and the camera on the following responses. During each test, we observed and recorded the behaviors displayed by the guards at the bait presence for 1 minute continuously. Immediately after removing the bait, the number of guards was counted again. The video was recorded for another 2 minutes for further observations of the consequent effects. We repeated the bait exposure 3 times to each colony, with a week interval between them. We analyzed the videos and categorized the displayed behaviors in 7 distinct types (Table 1 ) between both types of guards. Table 1 Behaviors observed during bioassays, abbreviations and operational description. Behavior Abbreviation Description Signaling S Guard bee lifts the abdomen and flaps wings for more than 5 constant seconds. Conturbation D Agitation. Guard bee initiates abnormal and increased movement. Approach P An individual approach the bait but does not touch it. Evasion E Number of guards is at least half as it was before bait presentation. Grouping G Guard bees approach the threat together. Attack A Guard bees lock the jaws on the bait. Or when there are similar attacks to throw the bait on the ground. Recruitment R Number of guard bees double after the presentation of the bait compared to the initial time. The recordings were made at a minimum distance of 1 meter from the colony, to avoid the influence of the observer and the camera in the behavior observations. To improve the contrast in the recordings, a black card paper was placed behind the entrance of the colony, with a 25x25cm size. A Panasonic Ag Ac8 high-definition video camera was used in the recordings. The videos were analyzed with the VLC software, frame-by-frame when necessary. 4.4. Statistical analysis We analyzed the displayed behaviors by the guards (hovering or standing) in relation to the type of bait presented to them using binary variables, 1 for when it happened at least once during each trial and 0 for absence. Each type of bait was tested separately, and the data matrix was elaborated using Jaccard’s distance. We performed Permutational analysis of variance (PERMANOVA) on each bait to investigate if there was difference between each guard type (hovering and standing) behavior regarding the nature of the bait species. Presence or absence of displayed behaviors was utilized as response variable, while type of guard and the bait species were used as explanatory variables. A similarity percentages breakdown (SIMPER) was performed as a post hoc test to analyze if there were differences in each of the displayed behaviors by the guards. We also utilized non-metric multidimensional scaling (NMDS) to assess the distribution of the behaviors based on each type of guard. We also built generalized linear mixed models (GLMM) to investigate if there was a relation between the number of guards defending the colony entrance and the species used as bait. We tested each species by itself, and in the cases of L. limao and the control, we also tested both walking and flying presentations separately. The number of guards was used as response variable; the type of the guard and the recording phase (before or after bait presentation) were employed as fixed explanatory variables. Colony of origin was used as random explanatory variable because we were not interested in variations between colonies. Since we are working with count data, the Poisson family was used, and we tested the models to detect over or under dispersion. Model selection was made via the Likelihood-ratio test (LRT) using a type II Wald Chi-square test, following Zuur et al. [ 45 ]. We performed all the analysis in the software R 4.3.0 [ 46 ] using the packages vegan [ 47 ], ggord [ 48 ], dplyr [ 49 ], multcomp [ 50 ], ggplot2 [ 51 ], lme4 [ 52 , 53 ], ggthemes [ 54 ], car [ 55 , 56 ]. Declarations Competing interests declaration The funders had not influence on the design or interpretation of the study and the authors declare no competing interests. Data availability All data generated or analysed during this study are included in this published article’s supplementary information files. Ethics declaration This study was made in compliance with Brazilian laws and did not require the approval of an ethics committee. Author contributions F. S. N. and L. L. G. S. contributed to the study conception and design. Data collection and video analysis were performed by L. L. G. S. Statistical analyses and Figs preparations were performed by B. 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J Chem Ecol 16, 631–641 (1990). Bego, L. R., Zucchi, R. & Mateus, S. Notas sobre a estratégia alimentar (cleptobiose) de Lestrimelitta limao Smith (Hymenoptera, Apidae, Meliponinae). Naturalia 16, 119–127 (1991). Sakagami, S. F., Roubik, D. W. & Zucchi, R. Ethology of the robber stingless bee, Lestrimelitta limao (Hymenoptera: Apidae). Sociobiology 21, 237–277 (1993). Rech, A. R., Schwade, M. A. & Schwade, M. R. M. Abelhas-sem-ferrão amazônicas defendem meliponários contra saques de outras abelhas. Acta Amazon 43, 389–393 (2013). Wittmann, D. Aerial defense of the nest by workers of the stingless bee Trigona (Tetragonisca) angustula (Latreille) (Hymenoptera: Apidae). Behav Ecol Sociobiol 16, 111–114 (1985). Kelber, A. & Zeil, J. A robust procedure for visual stabilisation of hovering flight position in guard bees of Trigona (Tetragonisca) angustula (Apidae, Meliponinae). Journal of Comparative Physiology A 167, 569–577 (1990). Zeil, J. & Wittmann, D. Landmark orientation during the approach to the nest in the stingless bee Trigona (Tetragonisca) angustula (Apidae, Meliponinae). Insectes Soc 40, 381–389 (1993). Kärcher, M. H. & Ratnieks, F. L. W. Standing and hovering guards of the stingless bee Tetragonisea angustula complement each other in entrance guarding and intruder recognition. J Apic Res 48, 209–214 (2009). van Zweden, J. S., Grüter, C., Jones, S. M. & Ratnieks, F. L. W. Hovering guards of the stingless bee Tetragonisca angustula increase colony defensive perimeter as shown by intra- and inter-specific comparisons. Behav Ecol Sociobiol 65, 1277–1282 (2011). Baudier, K. M. et al. Soldier neural architecture is temporarily modality specialized but poorly predicted by repertoire size in the stingless bee Tetragonisca angustula. Journal of Comparative Neurology 530, 672–682 (2022). Valadares, L., Vieira, B. G., Santos do Nascimento, F. & Sandoz, J. Brain size and behavioral specialization in the jataí stingless bee ( Tetragonisca angustula ). Journal of Comparative Neurology 530, 2304–2314 (2022). Shackleton, K. et al. Appetite for self-destruction: Suicidal biting as a nest defense strategy in Trigona stingless bees. Behav Ecol Sociobiol 69, 273–281 (2015). Bowden, R. M., Garry, M. F. & Breed, M. D. Discrimination of Con- and Heterospecific Bees by Trigona (Tetragonisca) angustula Guards. J Kans Entomol Soc 67, 137–139 (1994). Strausfeld, N. J. Beneath the Compound Eye: Neuroanatomical Analysis and Physiological Correlates in the Study of Insect Vision. in Facets of Vision 317–359 (Springer Berlin Heidelberg, Berlin, Heidelberg, 1989). doi:10.1007/978-3-642-74082-4_16. Bausenwein, B. & Fischbach, K. F. Activity labeling patterns in the medulla of Drosophila melanogaster caused by motion stimuli. Cell Tissue Res 270, 25–35 (1992). Bausenwein, B., Dittrich, A. P. M. & Fischbach, K. F. The optic lobe of Drosophila melanogaster - II. Sorting of retinotopic pathways in the medulla. Cell Tissue Res 267, 17–28 (1992). el Jundi, B., Pfeiffer, K. & Homberg, U. A distinct layer of the medulla integrates Sky compass signals in the brain of an insect. PLoS One 6, (2011). Penney, D. & Gabriel, R. Feeding behavior of trunk-living jumping spiders (Salticidae) in a coastal primary forest in the Gambia. J Arachnol 37, 113–115 (2009). Marcolino, M. T., Oliveira-Junior, W. P. & Brandeburgo, M. A. M. Aspectos comportamentais da interação entre formigas Camponotus atriceps Smith (Hymenoptera, Formicidae) e abelhas africanizadas Apis mellifera (L.) (Hymenoptera, Apidae). Naturalia 25, 321–330 (2000). Blum, M. S. Chemical Releasers of Social Behavior. VIII. Citral in the Mandibular Gland Secretion of Lestrimelitta limao (Hymenoptera: Apoidea: Melittidae)1. Ann Entomol Soc Am 59, 962–964 (1966). Zuur, A. F., Ieno, E. N., Walker, N., Saveliev, A. A. & Smith, G. M. Mixed Effects Models and Extensions in Ecology with R. (Springer New York, New York, NY, 2009). doi:10.1007/978-0-387-87458-6. R Core Team (2023). R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. Oksanen, J. et al. vegan: Community Ecology Package. Preprint at https://github.com/vegandevs/vegan (2022). Beck, M. W. ggord: Ordination Plots with ggplot2. Preprint at (2022). Wickham, H., François, R., Henry, L., Müller, K. & Vaughan, D. dplyr: A Grammar of Data Manipulation. Preprint at https://dplyr.tidyverse.org (2023). Hothorn, T., Bretz, F. & Westfall, P. multcomp: Simultaneous Inference in General Parametric Models. Preprint at http://multcomp.R-forge.R-project.org (2023). Wickham, H. Ggplot2: Elegant Graphics for Data Analysis. (Springer-Verlag New York, 2016). Bates, D., Mächler, M., Bolker, B. & Walker, S. Fitting Linear Mixed-Effects Models Using lme4. J Stat Softw 67, 1–48 (2015). Bates, D., Maechler, M., Bolker, B. & Walker, S. lme4: Linear Mixed-Effects Models using Eigen and S4. Preprint at https://github.com/lme4/lme4/ (2023). Arnold, J. B. ggthemes: Extra Themes, Scales and Geoms for ggplot2. Preprint at https://github.com/jrnold/ggthemes (2023). Fox, J. & Weisberg, S. An R Companion to Applied Regression. (Sage, Thousand Oaks CA, 2019). Fox, J., Weisberg, S. & Price, B. car: Companion to Applied Regression. Preprint at https://r-forge.r-project.org/projects/car/ (2023). Additional Declarations No competing interests reported. Supplementary Files data.xlsx suptable1.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4915536","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":357344314,"identity":"21daaa3b-1885-4821-8953-b0fcc9dcaf35","order_by":0,"name":"Luana Guimarães Santos","email":"","orcid":"","institution":"Universidade de São Paulo","correspondingAuthor":false,"prefix":"","firstName":"Luana","middleName":"Guimarães","lastName":"Santos","suffix":""},{"id":357344318,"identity":"37d8041d-aa8c-44af-b397-1cddc888781f","order_by":1,"name":"Bruno Vieira","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4ElEQVRIiWNgGAWjYDACZgaGA0AqgYG9AUgZWJCihQdEGUgQb1kCg0QCiCZCi8Fx5oOHK2oY8uQjn1/d8KNAgoG/vTsBv5bDbAkHzxxjKDa8nVN2swfoMIkzZzfg1SLZzGNwsIGNIXHj7Jy0GzxALQYSuYS08H842PAPqGXmmbSbf4jRws/Mw3CwsY0hcb4E+7HbRNnCz8xmcLCxT6LYgCeH7baMgQQPQb+w8R9+/LHhm02efPvxZzff/LGR42/vxa8FCoDuOcBjAGLxEKMcAuQb2B8Qr3oUjIJRMApGFAAAgmpGwQfH2EoAAAAASUVORK5CYII=","orcid":"","institution":"Universidade de São Paulo","correspondingAuthor":true,"prefix":"","firstName":"Bruno","middleName":"","lastName":"Vieira","suffix":""},{"id":357344319,"identity":"46698a26-588d-4a24-98c1-ffb11a48dee0","order_by":2,"name":"Jéferson Pedrosa","email":"","orcid":"","institution":"Universidade de São Paulo","correspondingAuthor":false,"prefix":"","firstName":"Jéferson","middleName":"","lastName":"Pedrosa","suffix":""},{"id":357344320,"identity":"5bad4965-3d43-4a81-8c80-09d2b246fc96","order_by":3,"name":"Fábio do Nascimento","email":"","orcid":"","institution":"Universidade de São Paulo","correspondingAuthor":false,"prefix":"","firstName":"Fábio","middleName":"do","lastName":"Nascimento","suffix":""}],"badges":[],"createdAt":"2024-08-14 18:29:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4915536/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4915536/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":65069442,"identity":"128d73f3-d328-435c-a366-f45e78594b9c","added_by":"auto","created_at":"2024-09-23 09:35:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":466153,"visible":true,"origin":"","legend":"\u003cp\u003eMean number of guards before and after the presentation of \u003cem\u003eL. limao\u003c/em\u003e as a) flying bait and b) walking bait. Bars represent the standard error of the mean. Dotted lines represent standing guards and solid lines represent hovering guards.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4915536/v1/afae1fd92e05115f9584ed1e.png"},{"id":65069443,"identity":"0d859a0b-40a5-47f3-a300-69c0a635f83c","added_by":"auto","created_at":"2024-09-23 09:35:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":510085,"visible":true,"origin":"","legend":"\u003cp\u003eMean number of guards before and after the presentation of a) \u003cem\u003eMenemerus \u003c/em\u003esp.; b) \u003cem\u003eA. sexdens\u003c/em\u003e and c) \u003cem\u003eCamponotus \u003c/em\u003esp. Bars represent the standard error of the mean. Dotted lines represent standing guards and solid lines represent hovering guards.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4915536/v1/37fa3c6a1162f8701cb26210.png"},{"id":65068780,"identity":"93a83069-ede9-4204-9c52-e54a965342c2","added_by":"auto","created_at":"2024-09-23 09:27:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":333597,"visible":true,"origin":"","legend":"\u003cp\u003enon-metric multidimensional scaling (NMDS) analysis of behaviors displayed by Standing and hovering guards when presented with a flying bait. Each letter represents a specific behavior.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4915536/v1/66dd447454072d6762fb8c59.png"},{"id":65068778,"identity":"f437508c-d4d1-460a-ab5b-213cafe61b62","added_by":"auto","created_at":"2024-09-23 09:27:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":338145,"visible":true,"origin":"","legend":"\u003cp\u003enon-metric multidimensional scaling (NMDS) analysis of behaviors displayed by Standing and hovering guards when presented with a walking bait. Each letter represents a specific behavior.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4915536/v1/b21eb8b556396d285c8adf2d.png"},{"id":65068779,"identity":"58fea738-739b-4278-9a53-7328da602884","added_by":"auto","created_at":"2024-09-23 09:27:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":49110,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentation of the experimental settings. a) flying or b) walking baits positioned in front the nest entrance of \u003cem\u003eT. angustula\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4915536/v1/f89ddc38e16feec85a377dfd.png"},{"id":71734154,"identity":"e6ab1c9d-2d40-4dd9-9d7a-fd8062b35634","added_by":"auto","created_at":"2024-12-18 07:17:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2277621,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4915536/v1/27c9f80f-9608-425d-93a6-17647e7acb23.pdf"},{"id":65068774,"identity":"ed0c4c31-12f3-41d8-a9d4-93ea9cc7075c","added_by":"auto","created_at":"2024-09-23 09:27:07","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":39927,"visible":true,"origin":"","legend":"","description":"","filename":"data.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4915536/v1/7cb9e3dd93b4dd7663b48b9a.xlsx"},{"id":65068775,"identity":"92d6f91e-dd24-4af1-8d98-9709c033b754","added_by":"auto","created_at":"2024-09-23 09:27:07","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":17966,"visible":true,"origin":"","legend":"","description":"","filename":"suptable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4915536/v1/8ff4aa1d0497f2896e339724.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Hovering and standing guards: nest defense strategies in a polymorphic stingless bee (Tetragonisca angustula)","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eThe division of labor is fundamental to the organization of the colony of social insects [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Division of labor may be associated with age polyethism (behavior changes throughout life and workers specialize in a subset of tasks according to age), physiological changes (e.g., changes in juvenile hormone titers) and distinct morphotypes among workers [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. For example, in ants and termites, colony defense is performed mostly by a subcaste of soldiers that are larger and specialized for this task [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Thus, workers may present adaptive morphological traits for specific tasks, such as foraging or defense, which improves the colony maintenance and maximizes performance, as these adaptations can improve the individual\u0026rsquo;s effectiveness [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn social insects, nest defense is important to both reproductive success and colony survival. Besides the food collected by foragers, the brood and even the nest location are valuable resources and, thus, require protection. Generally, the defense is performed by guards who stay at the colony entrance [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The guards can discriminate nestmates from other individuals via chemical cues, such as cuticular hydrocarbons or volatile ones, which are used to select those familiar individuals to enter the colony while intruders are intercepted at the entrance tube [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Intruders may be animals of other species, such as wasps and ants, or even other bees, conspecifics or not, as nest pillaging is a common practice in tropical species of stingless bees [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePreviously, there was no evidence suggesting the existence of a morphologically specialized subcaste in eusocial bees, but recent studies managed to determine that at least ten stingless bee species have larger individuals responsible for colony defense. Although the level of polymorphism is not as evident as in ants and termites\u0026rsquo; soldiers for example, these guards are significantly larger than other workers [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Phylogenetic reconstruction of the evolutionary history of 28 species suggests that specialized guards have evolved independently five times among these species, linking the appearance of this subcaste with the threat posed by \u003cem\u003eLestrimelitta\u003c/em\u003e Friese (1903) spp. to these species [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eTetragonisca angustula\u003c/em\u003e Latreille (1811), commonly known as Jata\u0026iacute;, is one of the most common species of eusocial bees native to the Neotropical region, due to being extremely successful in urban environments [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. \u003cem\u003eT. angustula\u003c/em\u003e has a guard subcaste with larger heads, is heavier, and has longer legs when compared to foragers [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In addition, guards work relatively more than other workers, also displaying a larger task repertoire. Furthermore, task transition occurs more quickly in these individuals, when in comparison with the minor workers [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eJata\u0026iacute; is one of the few species of stingless bee that presents some resistance to attacks carried by the robber bee \u003cem\u003eLestrimelitta limao\u003c/em\u003e Smith (1863), which is specialized in attacking stingless bee nests to steal pollen and nectar. Therefore, \u003cem\u003eL. limao\u003c/em\u003e is considered a major threat to the survivability of Meliponini nests, with attacks often leading to the death of the colony [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. \u003cem\u003eL. limao\u003c/em\u003e releases a volatile citral compound to disrupt the defensive behavior of the targeted colony, which Jata\u0026iacute; recognizes as a threat, triggering its defensive behavior [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe guards display two distinct sets of behaviors at the entrance of the nest, allowing them to be classified into two different behavioral groups: (1) Hovering guards, which hover around the entrance tube, and (2) Standing guards, which remain stationary on the outer wall of the tube. Hovering guards are supposedly responsible for visually inspecting approaching individuals to identify and distinguish between heterospecific and conspecific animals. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. On the other hand, standing guards are believed to be responsible to distinguish nestmates from non-nestmates [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSince guards from this species display two distinct and possibly complementary set of behaviors, our aim was to study the factors involved in the defensive behavior in a species with a defensive morphologically specialized subcaste in the highly competitive environments in which these bees live. Here, we test whether hovering and standing guards would exhibit distinct behavioral repertoires in relation to different types of intruders (flying and walking baits), to verify a possible specialization and complementation between both guard and intruder types. So, to conduct the experiments, we used each type of bait and analyzed the behavioral repertoire of the hovering and standing guards of \u003cem\u003eT. angustula\u003c/em\u003e. We conducted two behavioral tests: Experiment I, which examined the behavioral repertoire of hovering and standing guards in response to the two types of bait, and Experiment II, which measured the number of hovering and standing guards before and after the presentation of different bait types. Our hypothesis is that the number and defensive behavior of guards are directly related to the potential threat encountered.\u003c/p\u003e"},{"header":"2. RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Number of guards before and after bait presentation\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eWe observed that the number of standing guards was always higher than the number of hovering guards before we presented the bait (supplementary table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). We found significant interactions between the bait presentation phase and the type of guards at the entrance of the colony in the following species: \u003cem\u003eL. limao\u003c/em\u003e (flying: χ\u0026sup2; = 55.78; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; walking: χ\u0026sup2; = 80.73; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Supplementary Table\u0026nbsp;1, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb), \u003cem\u003eMenemerus\u003c/em\u003e sp. (χ\u0026sup2; = 7.784; p\u0026thinsp;=\u0026thinsp;0.005; Supplementary Table\u0026nbsp;1, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), \u003cem\u003eA. sexdens\u003c/em\u003e (χ\u0026sup2; = 10.68; p\u0026thinsp;=\u0026thinsp;0.001; Supplementary Table\u0026nbsp;1, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb) and \u003cem\u003eCamponotus\u003c/em\u003e sp. (χ\u0026sup2; = 14.22; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Table\u0026nbsp;2, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). We did not find significant differences in relation to the number of guards before and after bait presentation in \u003cem\u003eS.\u003c/em\u003e aff \u003cem\u003edepilis\u003c/em\u003e (χ\u0026sup2; = 0.37; p\u0026thinsp;=\u0026thinsp;0.54; Supplementary Table\u0026nbsp;1) and the piece of paper used as control (walking: χ\u0026sup2; = 0.04; p\u0026thinsp;=\u0026thinsp;0.82; flying: χ\u0026sup2; = 0.004; p\u0026thinsp;=\u0026thinsp;0.94; Supplementary Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eIn all significant interactions, the number of standing guards was lower after the bait presentation (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation, before and after presentation, respectively: \u003cem\u003eA. sexdens\u003c/em\u003e:18.556\u0026thinsp;\u0026plusmn;\u0026thinsp;7.294; 12.222\u0026thinsp;\u0026plusmn;\u0026thinsp;6.717; \u003cem\u003eCamponotus\u003c/em\u003e sp.: 18.667\u0026thinsp;\u0026plusmn;\u0026thinsp;5.625; 9.778\u0026thinsp;\u0026plusmn;\u0026thinsp;8.128; \u003cem\u003eL. limao\u003c/em\u003e (walking): 22.444\u0026thinsp;\u0026plusmn;\u0026thinsp;5.447; 12.778\u0026thinsp;\u0026plusmn;\u0026thinsp;6.813; \u003cem\u003eL. limao\u003c/em\u003e (flying): 16.944\u0026thinsp;\u0026plusmn;\u0026thinsp;6.601; 13.444\u0026thinsp;\u0026plusmn;\u0026thinsp;8.41; \u003cem\u003eMenemerus\u003c/em\u003e sp.: 16.705\u0026thinsp;\u0026plusmn;\u0026thinsp;6.555; 9.882\u0026thinsp;\u0026plusmn;\u0026thinsp;5.977; Supplementary Table\u0026nbsp;1, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea \u0026minus;\u0026thinsp;2c). \u003cem\u003eL. limao\u003c/em\u003e was the only species used as bait that increased the number of hovering guards after being presented (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation, before and after presentation, respectively: \u003cem\u003eL. limao\u003c/em\u003e (flying): 5.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.185; 13.111\u0026thinsp;\u0026plusmn;\u0026thinsp;5.738; \u003cem\u003eL. limao\u003c/em\u003e (walking): 5.277\u0026thinsp;\u0026plusmn;\u0026thinsp;2.371; 11.444\u0026thinsp;\u0026plusmn;\u0026thinsp;4.273; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, Supplementary Table\u0026nbsp;1). All other species showed no difference in the number of hovering guards before and after presentation.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Behaviors displayed by guards.\u003c/h2\u003e \u003cp\u003eWhen exposed to flying baits, we found a significant interaction between the behaviors displayed by each type of guards and the species used as bait (F\u003csub\u003e2,89\u003c/sub\u003e=14.113; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). SIMPER analysis indicated that the following behaviors differed between the two types of guards and had the most dissimilarities between them: approach (P) (average contribution\u0026thinsp;\u0026plusmn;\u0026thinsp;s.d.; 0.184\u0026thinsp;\u0026plusmn;\u0026thinsp;0.173; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), signaling (S) (0.137\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), evasion (E) (0.155\u0026thinsp;\u0026plusmn;\u0026thinsp;0.196; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) conturbation (C), (0.089\u0026thinsp;\u0026plusmn;\u0026thinsp;0.157; p\u0026thinsp;=\u0026thinsp;0.01), attack (A) (0.083\u0026thinsp;\u0026plusmn;\u0026thinsp;0.124; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), recruitment (R) (0.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.119; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and grouping (G) (0.046\u0026thinsp;\u0026plusmn;\u0026thinsp;0.101; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Hovering guards were responsible for displaying more of the P (average abundance (%) of displayed behaviors by hovering x standing guards: 0.6 x 0.088), R (0.32 x 0) A (0.32 x 0) and G (0.18 x 0) behaviors than the hovering ones, while the contrary was shown with the S (0 x 0.377), E (0 x 0.422) and C (0 x 0.266) behaviors. The NMDS plot also displays a strong separation between both types of guards and their displayed behaviors (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe guards also displayed a difference of behaviors between them when exposed to the walking baits. Our PERMANOVA results also indicated a significant interaction between the type of guard and the bait species (F\u003csub\u003e4,124\u003c/sub\u003e=5.392; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). SIMPER results showed a dissimilarity between the two guard types and their presented behaviors, differing in signaling (S) (average contribution\u0026thinsp;\u0026plusmn;\u0026thinsp;s.d.: 0.114\u0026thinsp;\u0026plusmn;\u0026thinsp;0.146; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), conturbation (C) (0.226\u0026thinsp;\u0026plusmn;\u0026thinsp;0.164; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), recruitment (R) (0.086\u0026thinsp;\u0026plusmn;\u0026thinsp;0.155; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), evasion (E) (0.154\u0026thinsp;\u0026plusmn;\u0026thinsp;0.186; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and grouping (G) (0.036\u0026thinsp;\u0026plusmn;\u0026thinsp;0.105; p\u0026thinsp;=\u0026thinsp;0.02). Hovering guards showed more prominently the R (average abundance (%) of displayed behaviors by hovering x standing guards: 0.274 x 0.012), and G (0.117 x 0.012) behaviors, while the standing guards were responsible for displaying more of the C (0 x 0.747), E (0.117 x 0.457) and S (0 x 0.433) behaviors. Similar to what happened with the flying baits, the NMDS indicates a division between which guard displays which behavior (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3. DISCUSSION","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eOur results clearly show that there is task partitioning between the two types of guards in relation to their defensive behavior. When facing different types of threats, the guards can assess the situation and act accordingly. Our results also show that the number and types of guards at the nest entrance is related to what they are facing at the given moment, adjusting their quantity to adapt to the situation. The standing guards are more likely to entrench themselves inside the colony while the hovering guards only increase in numbers when facing real threats. When analyzing their behaviors, it\u0026rsquo;s also clear that each type of guard specializes in a function when faced with whether the enemy is flying or walking, showing a clear separation of the groups.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e3.1. \u003cem\u003eNumber of guards before and after bait presentation\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eOur results of this experiment suggest that the number of hovering and standing guards are affected by intruders. Compared to the number of guards before the presentation of the baits, in which the quantity of standing guards was greater than hovering guards, after the presentation we observed that the number of standing guards was lower in all baits presented that had significant differences. It can be argued that the standing guards, upon noticing intruders, place themselves inside the colony.\u003c/p\u003e \u003cp\u003eThe number of hovering guards was greater only after the presentation of \u003cem\u003eL. limao\u003c/em\u003e, possibly meaning that \u003cem\u003eT. angustula\u003c/em\u003e displays a specific defense strategy when perceiving the presence of its natural enemy. In the other baits we did not find a significant difference in the number of hovering guards. Therefore, hovering guards would be a more effective and aggressive defense to detect and contain specific intruders. There is a possibility that they can perceive and assess the threat level of suspicious individuals near its entrance.\u003c/p\u003e \u003cp\u003eRegarding the species that we found a significant difference before and after the presentation of the baits, there were two species that synthesize the citral compound (\u003cem\u003eL. limao\u003c/em\u003e and \u003cem\u003eA. sexdens\u003c/em\u003e), a spider (\u003cem\u003eMenemerus\u003c/em\u003e sp.) that is a potential predator and an ant (\u003cem\u003eCamponotus\u003c/em\u003e sp.) that is a potential competitor for resources of \u003cem\u003eT. angustula\u003c/em\u003e. Despite \u003cem\u003eS.\u003c/em\u003e aff. \u003cem\u003edepilis\u003c/em\u003e being an aggressive species, we found no significant difference. All other species except \u003cem\u003eS.\u003c/em\u003e aff. \u003cem\u003edepilis\u003c/em\u003e had a decrease of the quantity of standing guards.\u003c/p\u003e \u003cp\u003eWhen exposed to \u003cem\u003eA. sexdens\u003c/em\u003e, a species with volatiles similar to \u003cem\u003eL. limao\u003c/em\u003e, like citral, the guards behaved differently than when exposed to \u003cem\u003eL. limao\u003c/em\u003e walking individuals. The standing guards followed the same pattern for both, decreasing in numbers at the entrance, as was also observed for the other species with significant results. The hovering guards, however, did not increase in numbers, meaning that the standing ones entered the entrance tube of the nest, contrary to the observed by Karcher and Ratnieks [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur findings are consistent with those found by Baudier et al. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], in which the standing guards never changed to the function of hovering. This behavior of entering the colony help in nest defense, as the narrow entrance may diminish the number of enemies to be repelled at once. Another different behavior from the observed on Karcher and Ratnieks [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] when exposing the guards to \u003cem\u003eScaptotrigona bipunctata\u003c/em\u003e, a stingless bee from the same genus. In our study, the guards did not react to \u003cem\u003eS.\u003c/em\u003e aff \u003cem\u003edepilis\u003c/em\u003e, as their numbers did not show a significant difference from before and after the bait presentation, when they found an increase of hovering guards and a decrease of standing ones.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eWith these results we can assume that each guard type is specialized in one function, and that they are complementary to each other when defending the nest. Baits that changed the number of guards at the entrance were different considering the guard type. We can say that there is a threat level assessment by each guard type that takes in consideration whether the enemy is flying or walking and can identify what enemies are more of a threat to the nest.\u003c/p\u003e\u003cp\u003eThe fact that hovering guards are responsible for the visual discrimination of conspecific and heterospecific individuals [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] suggests that they are also capable of recognizing \u003cem\u003eS.\u003c/em\u003e aff. \u003cem\u003edepilis\u003c/em\u003e as a non-threatening presence. The disturbance of the number of standing guards when the colony was exposed to \u003cem\u003eL. limao\u003c/em\u003e can be explained by the strong presence of its kayromone, the citral. Previous studies reported that there is no difference in size in relation to the antennal lobe and the type of guard [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. This explains why despite the bait being presented as flying, standing soldiers also reacted to its presence. We can infer the same about the hovering guards increasing in number when presented with it as a walking bait.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.2. \u003cem\u003eBehaviors displayed by guards\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eOur findings show a distinct division between the analyzed behaviors, in which hovering and standing guards complement each other. The standing guards acted as signals that some disturbance was happening at the nest and the hovering ones acted as the main force of defense. The signaling and disturbance behaviors were only displayed by standing guards. This division suggests that hovering guards are responsible for detecting heterospecific flying individuals and standing guards act as a last defensive barrier at the entrance of the colony.\u003c/p\u003e \u003cp\u003eOur results of this experiment suggest that the behaviors are different between the hovering and standing guards for each type of bait (flying or walking). Therefore, in flying baits we found a greater number of hovering guards showing approach (P), recruitment (R), attack (A) and grouping (G) behavior when compared to standing guards. The standing guards showed signaling (S) and disturbance (C) behaviors. We can infer that the defense mechanism of \u003cem\u003eT. angustula\u003c/em\u003e acts in a coordinated way, where flying intruders are recognized and a disturbance occurs in the standing guards, which signal the presence of a potential enemy, promoting agitation in the colony and the beginning of the defense by hovering guards against flying enemies.\u003c/p\u003e \u003cp\u003eWhen exposed to walking baits, we found a greater number of hovering guards showing recruitment (R) and grouping (G) behavior when compared to standing guards. The standing guards showed signaling (S) and disturbance (C) behaviors. We can assess that standing guards signals the presence of the walking enemies and shows disturbance at the entrance nest, which is the cue for the hovering guards to start recruiting other individuals for possible defense against walking enemies.\u003c/p\u003e \u003cp\u003eIt is also interesting to note that the signaling and conturbation behaviors are only displayed by the standing guards. The main defensive behavior from \u003cem\u003eT. angustula\u003c/em\u003e is biting, and usually it involves the death of the defending guard, since they lock their mandibles on the intruder and don\u0026rsquo;t release it anymore [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The fact that there were no attacks on walking baits can indicate that these threats are not dangerous enough to elicit an agonistic behavior from the guards.\u003c/p\u003e \u003cp\u003e \u003cem\u003eT. angustula\u003c/em\u003e has an intrinsic evolutionary relationship with \u003cem\u003eL. limao\u003c/em\u003e, as reported by Gr\u0026uuml;ter et al. [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The repeated displayed behavior of social parasitism by \u003cem\u003eL. limao\u003c/em\u003e was a driving force of the evolution of morphologically specialized guards in \u003cem\u003eT. angustula\u003c/em\u003e. As already reported, hovering guards commonly display the behavior of ignoring conspecifics and focusing their attack effort on heterospecific bees, that generally displays a different color [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecent studies by Baudier et al. [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] and Valadares et al. [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] presented results that indicate that there are differences in relation to the type of guard and their brain volume and structure. Hovering guards have a larger medulla, which is the structure responsible for processing the information related to motion detection in a small range [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] and color and shape differentiation [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. This is reflected in the behavior observed in this study, since they were the ones who displayed the approach and attack behaviors, mainly when confronted with \u003cem\u003eL. limao.\u003c/em\u003e\u003c/p\u003e \u003cp\u003eOverall, our results suggest that there is a clear differentiation between hovering and standing guards in relation to their roles on colony defense. Hovering guards are responsible for assessing and acting upon a threat, while standing guards act as a barrier at the entrance tube, narrowing the space and number of possible invaders. We also found that the hovering guards\u0026rsquo; number only increased when in the presence of a real threat to the colony, their natural enemy \u003cem\u003eL. limao\u003c/em\u003e, which means that they can recognize when to display an agonistic behavior and increase the nest defense in numbers. This is a sophisticated and complementary system that probably evolved along the years to provide a more responsive and cost-efficient defense against raids.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. METHODS","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e4.1. Study site and species\u003c/h2\u003e\n \u003cp\u003eWe performed the experiments at the Universidade de S\u0026atilde;o Paulo, \u003cem\u003ecampus\u003c/em\u003e Ribeir\u0026atilde;o Preto, Brazil (21\u0026deg;09\u0026apos;48.2\u0026quot;S, 47\u0026deg;51\u0026apos;37.9\u0026quot;W). We chose \u003cem\u003eT. angustula\u003c/em\u003e for this study due to its natural occurrence and abundance at the region, and due to being an important pollinator for native plants. Besides, this species also has a temporal/behavioral specialization between its guards, allowing for an in-depth study of their roles in colony defense.\u003c/p\u003e\n \u003cp\u003eWe used eight colonies maintained in wooden boxes of similar size and weight. Data were collected over an 8-month period (March and April, from September to December 2017; January and February 2018). We regularly checked the nest entrance and counted the number of guards to assess colony activity and standardize the colonies used. We only conducted experiments during favorable weather conditions (ambient temperature\u0026thinsp;\u0026asymp;\u0026thinsp;31\u0026ordm;C, 47% humidity and clear sky).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e4.2. Bait species selection and presentation\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eTo analyze the roles played by both distinct guards\u0026rsquo; phenotypes, we exposed them to two different types of baits: walking and flying intruders. We attached a transparent stick (1 meter) to a tripod and positioned it laterally to the colony entrance, so we could freely move it using the tripod head. We anesthetized the individuals through refrigeration (freezer at 5\u0026ordm;C) or with CO\u003csub\u003e2\u003c/sub\u003e (only spiders) and waited for the baits to reanimate before the experiments. The baits were attached to a piece of paper with superglue through the thorax or cephalothorax, leaving the movement of the appendages free. This paper was then placed at the tip of the stick.\u003c/p\u003e\n \u003cp\u003eWe simulated a flying intruder by adapting the methodology used by Van Zweden et al. [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e]. We positioned the tripod in front of the colony entrance, and simulated the individual\u0026apos;s behavior when flying, never obstructing the nest entrance passage (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea). When testing the flying baits, we moved the stick close to the nest wall, from above the entrance to below it, stopping 3 to 5 centimeters from the entrance, also adapted by Van Zweden et al. [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e] (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eb). We replaced the bait whenever it was touched by the guards to prevent the deposition of odors or any other cues that might incite aggressiveness.\u003c/p\u003e\n \u003cp\u003eWe chose the following species to simulate walking threats: \u003cstrong\u003ea)\u003c/strong\u003e jump spiders (\u003cem\u003eMenemerus\u003c/em\u003e Dufour, 1831), which are high potential predators of \u003cem\u003eT. angustula\u003c/em\u003e [\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e]; \u003cstrong\u003eb)\u003c/strong\u003e carpenter ants (\u003cem\u003eCamponotus\u003c/em\u003e Mayr, 1861), which can be competitors for food resources and responsible for sporadic resources thefts of \u003cem\u003eT. angustula\u003c/em\u003e nests [\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e]; \u003cstrong\u003ec)\u003c/strong\u003e leafcutter ants (\u003cem\u003eAtta sexdens\u003c/em\u003e Linnaeus, 1758), not considered to a threat to \u003cem\u003eT. angustula\u003c/em\u003e, but with an interesting characteristic of eliciting odor of citral [\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e], a compound found in \u003cem\u003eL. limao\u003c/em\u003e, used as a kairomone by \u003cem\u003eT. angustula\u003c/em\u003e guards [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e]. We also opted to use \u003cstrong\u003ed)\u003c/strong\u003e \u003cem\u003eL. limao\u003c/em\u003e individuals as walking baits due to it being \u003cem\u003eT. angustula\u003c/em\u003e natural enemy and to the occurrence of citral just like the leafcutter ants, allowing us to infer if the individual morphology has an influence on guards besides their exposition to the compound.\u003c/p\u003e\n \u003c/div\u003e\n \u003cp\u003eWe used the following species as flying baits: \u003cstrong\u003ea)\u003c/strong\u003e the stingless bee \u003cem\u003eScaptotrigona\u003c/em\u003e aff. \u003cem\u003edepilis\u003c/em\u003e (Moure, 1942), potential intruders, competitors and responsible for occasional resource thefts at \u003cem\u003eT. angustula\u003c/em\u003e nests [\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e]; \u003cstrong\u003eb)\u003c/strong\u003e \u003cem\u003eT. angustula\u003c/em\u003e natural enemy \u003cem\u003eL. limao\u003c/em\u003e, responsible for raids and predation of colonies. All individuals used as bait were collected at the \u003cem\u003ecampus\u003c/em\u003e facilities, and we kept them alive until their exposure to \u003cem\u003eT. angustula\u003c/em\u003e. We used a clean paper with superglue, similar to the one which the baits were glued as control, in both walking and flying assays.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e4.3. Behavioral assays\u003c/h2\u003e\n \u003cp\u003eInitially, we counted the number of guard bees and verified the standardization of this quantity over time. Subsequently, bait exposure tests were performed in each bee colony to analyze the behavior of the guards (both standing and hovering) walking and flying baits. Before each test, to record the natural activity of the colony, we filmed the guards during 1 minute and counted their number. This initial exposure could also decrease the influence of the observer and the camera on the following responses. During each test, we observed and recorded the behaviors displayed by the guards at the bait presence for 1 minute continuously. Immediately after removing the bait, the number of guards was counted again. The video was recorded for another 2 minutes for further observations of the consequent effects. We repeated the bait exposure 3 times to each colony, with a week interval between them. We analyzed the videos and categorized the displayed behaviors in 7 distinct types (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) between both types of guards.\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eBehaviors observed during bioassays, abbreviations and operational description.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBehavior\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAbbreviation\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDescription\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSignaling\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGuard bee lifts the abdomen and flaps wings for more than 5 constant seconds.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eConturbation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAgitation. Guard bee initiates abnormal and increased movement.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eApproach\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAn individual approach the bait but does not touch it.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEvasion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNumber of guards is at least half as it was before bait presentation.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGrouping\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGuard bees approach the threat together.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAttack\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGuard bees lock the jaws on the bait. Or when there are similar attacks to throw the bait on the ground.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRecruitment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNumber of guard bees double after the presentation of the bait compared to the initial time.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003eThe recordings were made at a minimum distance of 1 meter from the colony, to avoid the influence of the observer and the camera in the behavior observations. To improve the contrast in the recordings, a black card paper was placed behind the entrance of the colony, with a 25x25cm size. A Panasonic Ag Ac8 high-definition video camera was used in the recordings. The videos were analyzed with the VLC software, frame-by-frame when necessary.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e4.4. Statistical analysis\u003c/h2\u003e\n \u003cp\u003eWe analyzed the displayed behaviors by the guards (hovering or standing) in relation to the type of bait presented to them using binary variables, 1 for when it happened at least once during each trial and 0 for absence. Each type of bait was tested separately, and the data matrix was elaborated using Jaccard\u0026rsquo;s distance. We performed Permutational analysis of variance (PERMANOVA) on each bait to investigate if there was difference between each guard type (hovering and standing) behavior regarding the nature of the bait species. Presence or absence of displayed behaviors was utilized as response variable, while type of guard and the bait species were used as explanatory variables. A similarity percentages breakdown (SIMPER) was performed as a post hoc test to analyze if there were differences in each of the displayed behaviors by the guards. We also utilized non-metric multidimensional scaling (NMDS) to assess the distribution of the behaviors based on each type of guard.\u003c/p\u003e\n \u003cp\u003eWe also built generalized linear mixed models (GLMM) to investigate if there was a relation between the number of guards defending the colony entrance and the species used as bait. We tested each species by itself, and in the cases of \u003cem\u003eL. limao\u003c/em\u003e and the control, we also tested both walking and flying presentations separately. The number of guards was used as response variable; the type of the guard and the recording phase (before or after bait presentation) were employed as fixed explanatory variables. Colony of origin was used as random explanatory variable because we were not interested in variations between colonies. Since we are working with count data, the Poisson family was used, and we tested the models to detect over or under dispersion. Model selection was made via the Likelihood-ratio test (LRT) using a type II Wald Chi-square test, following Zuur et al. [\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e]. We performed all the analysis in the software R 4.3.0 [\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e] using the packages \u003cem\u003evegan\u003c/em\u003e [\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e], \u003cem\u003eggord\u003c/em\u003e [\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e], \u003cem\u003edplyr\u003c/em\u003e [\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e], \u003cem\u003emultcomp\u003c/em\u003e [\u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e], \u003cem\u003eggplot2\u003c/em\u003e [\u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e], \u003cem\u003elme4\u003c/em\u003e[\u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e], \u003cem\u003eggthemes\u003c/em\u003e [\u003cspan class=\"CitationRef\"\u003e54\u003c/span\u003e], \u003cem\u003ecar\u003c/em\u003e [\u003cspan class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e56\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cem\u003eCompeting interests declaration\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe funders had not influence on the design or interpretation of the study and the authors declare no competing interests.\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eData availability\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article\u0026rsquo;s supplementary information files.\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEthics declaration\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis study was made in compliance with Brazilian laws and did not require the approval of an ethics committee.\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAuthor contributions\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eF. S. N. and L. L. G. S. contributed to the study conception and design. Data collection and video analysis were performed by L. L. G. S. Statistical analyses and Figs preparations were performed by B. G. V. The manuscript was written by B. G. V. and J. P. S. and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAcknowledgments\u003c/p\u003e\n\u003cp\u003eFinancial support was provided by the S\u0026atilde;o Paulo Research Foundation (FAPESP #2022/01427-7; #2019/07885-4; #2021/05598-8; 2023/03122-1), the National Council for Scientific and Technological Development (CNPq #130143/2016-2) and the Coordination of Superior Level Staff Improvement (Capes #001).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRatnieks, F. L. W. \u0026amp; Anderson, C. Task partitioning in insect societies. 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Insectes Soc 40, 381\u0026ndash;389 (1993).\u003c/li\u003e\n\u003cli\u003eK\u0026auml;rcher, M. H. \u0026amp; Ratnieks, F. L. W. Standing and hovering guards of the stingless bee Tetragonisea angustula complement each other in entrance guarding and intruder recognition. J Apic Res 48, 209\u0026ndash;214 (2009).\u003c/li\u003e\n\u003cli\u003evan Zweden, J. S., Gr\u0026uuml;ter, C., Jones, S. M. \u0026amp; Ratnieks, F. L. W. Hovering guards of the stingless bee Tetragonisca angustula increase colony defensive perimeter as shown by intra- and inter-specific comparisons. Behav Ecol Sociobiol 65, 1277\u0026ndash;1282 (2011).\u003c/li\u003e\n\u003cli\u003eBaudier, K. M. et al. Soldier neural architecture is temporarily modality specialized but poorly predicted by repertoire size in the stingless bee Tetragonisca angustula. Journal of Comparative Neurology 530, 672\u0026ndash;682 (2022).\u003c/li\u003e\n\u003cli\u003eValadares, L., Vieira, B. 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Preprint at https://r-forge.r-project.org/projects/car/ (2023).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Nest defense, resources, behavioral plasticity, polyethism","lastPublishedDoi":"10.21203/rs.3.rs-4915536/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4915536/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"The stingless bee, Tetragonisca angustula, has a sophisticated nest defense strategy carried out by guards that are larger compared to other workers. Guards display two different strategies: flying near the colony entrance (hovering guards) or positioning themselves at the entrance tube (standing guards). To better understand the roles played by each guard behavioral phenotype in nest defense, we investigated whether their behaviors were distinctly displayed when faced with different threats. We used two types of bait (flying and walking) to simulate threats to the colony and compared the behaviors displayed by the guards in relation to the species used as bait and the guard function. We also investigated if the species and the type of bait influenced the number of guards before and after the presentation. We found a significant interaction between the behaviors displayed by the guards and the bait species. Hovering guards were more influenced by flying baits, and standing guards by walking baits. The presence of Lestrimellita limao caused a high proportion of recruitment and aggressive behavioral responses from guards, confirming specialization against this potential enemy. Our results show that the two behavioral phenotypes are capable of recognition and act with complementary behaviors depending on the threat.","manuscriptTitle":"Hovering and standing guards: nest defense strategies in a polymorphic stingless bee (Tetragonisca angustula)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-23 09:27:02","doi":"10.21203/rs.3.rs-4915536/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"554a82cd-3c0d-4919-9829-d77b7ad0c4dd","owner":[],"postedDate":"September 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":38013943,"name":"Biological sciences/Ecology/Behavioural ecology"},{"id":38013944,"name":"Biological sciences/Ecology/Evolutionary ecology"}],"tags":[],"updatedAt":"2024-12-18T07:09:07+00:00","versionOfRecord":[],"versionCreatedAt":"2024-09-23 09:27:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4915536","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4915536","identity":"rs-4915536","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}