Hive entrance smearing behavior of Apis cerana japonica in response only to Vespa mandarinia attacks: First report on the use of diverse organisms by worker bees in emergency situations

Abstract

　Vespa mandarinia is a formidable predator that attacks the hives of Apis mellifera and Apis cerana japonica in the autumn, sometimes resulting in their complete destruction. A. c. japonica is known to defend their hives from V. mandarinia by forming a "hot defensive bee ball" to kill the scout hornets and subsequently repelling the devastating mass attacks. Since 2011, the authors’ ongoing investigations have revealed that immediately after the attack of V. mandarinia scouts at the hive, worker bees of A. c. japonica collect and smear leaves, buds, and other parts of several plant species around the hive entrance and perform a unique emergency dance (Fujiwara et al. 2016, 2017, Fujiwara 2020). Furthermore, we demonstrated that these behaviors are exclusively observed in response to V. mandarinia and not exhibited towards other hornet species (Fujiwara et al. 2016, 2017).

　In this study, from 2011 to 2015, the smearing behavior of worker bees was recorded and verified using video cameras. In the autumn of 2015, smearing materials were observed using a microscope, multiple species of insects and a large number of materials from unknown organisms were found. Additionally, in the autumn of 2016, following a simulated attack by V. mandarinia, it was observed through field surveys and marking of individual worker bees that worker bees gnawed mushrooms growing near their hives and subsequently returned to the hive to smear them around the hive entrance.

　In the autumn of 2017, it was confirmed that worker bees carried larva of moth and Diptera and pupa of Diptera in their mandibles back to the hive and smeared them around the hive entrance. These insect individuals were all in a fresh state, and individuals with bodily fluids emanating from wounds were also observed. These evidences strongly supported the conclusion that the insects were alive immediately prior to capture. The behavior of A. c. japonica capturing live insects in the field to defend their hives against V. mandarinia and bringing them back to smear around the hive entrance was, to the best of the authors' knowledge, reported for the first time in this study.

　To comprehensively understand the organisms utilized worker bees for nest defense, smearing materials were collected from six colonies maintained at three survey sites with distinct surrounding environments in the autumn of 2021, and DNA analysis was conducted. In this study, to minimize the risk of contamination from other organisms or their fragments and to prevent DNA degradation when collecting materials from the hive, we collected fresh materials as soon as possible after they were smeared, rather than materials that had accumulated. Furthermore, we collected smearing materials directly from returning worker bee individuals. The DNA analyses were outsourced to Bioengineering Lab Co., Ltd. The analysis revealed 30 families and 39 genera of plants, 25 families and 31 genera of insects, 28 families and 38 genera of fungi (Agaricomycetes), 11 families and 15 genera of mammals, and 11 families and 13 genera of birds. In addition to this study, diverse organisms including algae, bryophytes, fish, and crustaceans have been detected through DNA analysis of materials conducted separately since 2015.

　In this study, we discovered and demonstrated for the first time—through the use of multiple methods, including behavioral observations of worker bees, examination of smearing materials, and DNA analysis—that A. c. japonica utilize a variety of organisms as smearing materials in response to attacks by the predator hornet V. mandarinia. Furthermore, we were able to grasp how organisms were collected by worker bees and the process through which they were smeared onto the hive, which cannot be fully understood through DNA analysis alone.

　Through comparing materials collected from three different regions, characteristic utilization trends of diverse organisms reflecting variations in natural environments and biota were observed. Furthermore, rare organisms such as Nisaetus nipalensis, a nationally scarce species of wild fauna and flora in Japan, were also detected. These findings suggest that the DNA analysis of smeared materials can help understand rare species and wildlife that are challenging to detect visually. Moreover, this approach may serve as a new tool to understand the connection between local biota, natural ecosystems, and the A. c. japonica.