DOI: 10.5061/dryad.np5hqc03n
title: Tool use aids prey-fishing in a specialist predator of stingless bees

description: Tool use is widely reported across a broad range of the animal kingdom,
 yet comprehensive empirical tests of its function and evolutionary drivers
 remain scarce, predominantly focused on a few relatively intelligent
 vertebrate lineages. In this study, we provide a comprehensive examination
 of tool use behavior in the assassin bug Pahabengkakia piliceps, a
 specialist predator of stingless bees that exploits resin droplet from bee
 nest entrance to facilitate hunting. Field behavioral experiments
 demonstrated that resin use is critical for hunting success, as the
 predator uses resin to stimulate the colonial defense of stingless bees,
 luring attacking bees toward its optimal hunting position. Chemical
 analysis revealed that resin processing by the assassin bug enhances the
 emission of volatile compounds, making the resin more attractive and
 stimulating to guard bees. Through these experiments, we empirically
 demonstrate how an invertebrate predator adapts to the colony defense of
 social insects via tool use behavior. We further propose that complex tool
 use can evolve under selective pressures driven by diet specialization.
 Our findings offer a new model for studying the adaptive functions and
 underlying mechanisms of tool use behaviors in animals.
description: # Tool use aids prey-fishing in a specialist predator of stingless bees
 [https://doi.org/10.5061/dryad.np5hqc03n](https://doi.org/10.5061/dryad.np5hqc03n) ## Description of the data and file structure This supplementary dataset, integral to our research paper, contains data crucial for understanding the key findings of our study. It includes all behavior raw data of each assassin bug's interaction with stingless bees, Gas Chromatography-Mass Spectrometer raw data of the total volatiles content, the developmental period of each assassin bug, the number of eggs laid, and the number of eggs hatched during the experiment, and R code for data analysis.  ### Files and variables #### File: Behavior\_Raw\_Data.xlsx **Description:** The raw data of our field experiment about the interaction between each assassin bug from three groups and stingless bees, include the time of assassin bug prey stingless bee or not, and the time stingless bee attack the assassin bug, and the number of times the stingless bees attacked the assassin bugs and the specific body parts targeted during the attacks. All empty cells in the tables indicate that the corresponding events did not occur during the experiments, and therefore no related data were generated. ##### Variables * N-group_Resin on bug's fore & mid legs: “Natural group” assassin bugs applied resin on their fore- and midleg;  * M-group_Resin on bug's hind legs & abdominal tip: “Manipulation-group” assassin bugs were applied resin to hindlegs and pygophore before the experiment; * NR-group_No resin:  “No-resin” group there were no resin on assassin bug's body. * Bug_ID: The assassin bug we used for test. * T0: The time point when the assassin bug first paused before or at the top of the bridge (s). * Ta(1):  The time point when the assassin bug is first attacked by a stingless bee , where an attack is defined as the stingless bee biting or dashing the bug’s body (s). * T1=Ta(1)-T0: Time to first attack (s). * Tp(s):  The time point when predation success (s). * T2=(Tp(s)-T0)/60: Total time spent on predation (s). * Ta(n): The time point of the last attack on the bug by the bee (s). * T3=Ta(n)-T0: The time from the start of the test to the last attack received by the assassin bug (s). * T3=(Ta(n)-T0)/60: The time from the start of the test to the last attack received by the assassin bug (min). * Bee Attacked Number: The total number of times each assassin bug was attacked by stingless bees. * Attack Frequency: The frequency of bee attacks per minute was calculated (F = n / ((Ta(n) - T(0)) / 60)) (times per minute). * Bee Attacked Number on: Stingless bees attacked on bugs' specific body parts, include: Head, Fore legs, Mid legs, Hind legs, Connexivum, Notum, and abdominal tip. * Prey Times: The number of each bug's predation attempts. * Predation Success Rate: Predation success rate of each bug * success: The number of successful predation events by the assassin bug. * fail: The number of failed predation attempts by the assassin bug. * Predation Efficiency: Predation efficiency per minute of each bug (times per minute). * Attack Frequency on Head, Fore legs, Mid legs, Hind legs, Connexivum, Notum, and abdominal tip: The attack frequency of stingless bees per minute on these body parts of the assassin bug (times per minute). #### File: Development Time of Six Groups.xlsx Description: The developmental period from the first instar to adulthood and the survival status of each assassin bug nymph hatched on the same day after being fed different foods. All empty cells in the tables indicate that the assassin bug nymphs died at the corresponding stage during the experiment, and therefore no further data were generated thereafter. ##### Variables * Bug ID T01 to T30: Thirty assassin bugs that fed on stingless bees (stingless bee group). * Bug ID B01 to B30: Thirty assassin bugs that fed on honeybee (honeybee group). * Bug ID F01 to F30: Thirty assassin bugs that fed on housfly (housfly group). * Bug ID C01 to C30: Thirty assassin bugs that fed on cricket (cricket group). * Bug ID Z01 to Z30: Thirty assassin bugs that fed on cockroach (cockroach group). * Bug ID Y01 to Y30: Thirty assassin bugs that fed on yellow-mealworm (yellow-mealworm group). * First Instar: Developmental duration of the first instar stage of the assassin bug (day). * Second Instar: Developmental duration of the second instar stage of the assassin bug (day). * Third Instar: Developmental duration of the third instar stage of the assassin bug (day). * Fourth Instar: Developmental duration of the fourth instar stage of the assassin bug (day). * Final Instar: Developmental duration of the final instar stage of the assassin bug (day). * First Instar to Adult: Developmental duration for the assassin bug from first instar to adulthood (day). * First-Third Instar: Developmental duration for the assassin bug from first to third instar (day). #### File: Dgg Laying Number of Six Groups.xlsx Description: After feeding assassin bugs with different foods, the number of eggs laid by each bug in each group and the hatching success of these eggs. All empty cells in the tables indicate that no eggs were laid or that the eggs failed to hatch during the experiment, and therefore no data were generated. ##### Variables * Bug ID T01 to T10: Ten couples of assassin bugs that fed on stingless bees (stingless bee group). * Bug ID B01 to B10: Ten couples of assassin bugs that fed on honeybee (honeybee group) * Bug ID F01 to F10: Ten couples of assassin bugs that fed on housfly (housfly group). * Bug ID C01 to C10: Ten couples of assassin bugs that fed on cricket (cricket group). * Bug ID Z01 to Z10: Ten couples of assassin bugs that fed on cockroach (cockroach group). * Bug ID Y01 to Y10: Ten couples of assassin bugs that fed on yellow-mealworm (yellow-mealworm group). * Start Date: The start date of the experiment. * Laying Date: The date on which the assassin bug laid eggs. * Egg Number: The number of eggs in each egg mass. * Hatch Date: The date when eggs were hatched. * Hatching Number: The number of eggs hatched in each egg mass. #### File: GC-MS\_Raw\_Data.zip Description: Raw Gas Chromatography-Mass Spectrometer data on the relative content changes of volatiles before and after resin droplet is processed. Data analysis was performed using GC-MS Solution 4.50 (Shimadzu, Japan). The `.qgd` files were generated and can be opened using GC-MS Solution 4.50 (Shimadzu, Japan). The relevant data from these files are already extracted and provided in the Excel files with the same names. ## Code/software Data analysis and visualization were conducted in R 4.2.2 using RStudio (2022.12.0+353). All values are presented as mean ± SD. Welch’s ANOVA followed by Tukey’s HSD test was used to compare attack number, attack frequency, and predation efficiency among groups, as well as the developmental duration under different diets. Chi-square tests were used to analyze feeding preferences and predation success rates. Linear regression was applied to assess the relationship between bee attack frequency on bug body parts and predation efficiency, with weighted residuals to correct for heteroscedasticity.