Smearing behavior of Apis cerana japonica in response only to attacks by Vespa mandarinia: First report on the use of diverse organisms as smeared materials by worker bees in emergency situations

Abstract

　Vespa mandarinia, is a natural predator known for its collective assaults on the colonies of Apis cerana japonica, often leading to their annihilation during the autumn season. It is recognized that A. c. japonica form heat balls around scouting hornets that venture close to the hive, effectively killing them with heat and preventing subsequent catastrophic group attacks. Through the authors' research since 2011, it has been demonstrated for the first time that after attacks of scouting hornets, worker bees of A. c. japonica actively engage in the behavior of vigorously smearing plant fragments such as leaves and buds from various plant species around the entrance of their hives and it was also observed that they perform “emergency dance” during this behavior. These behaviors not occur with other hornet species (Fujiwara et al. 2016, 2018).

　Additionally, during the investigation in the autumn of 2011, numerous smeared materials believed to be of non-plant biological origin were observed, with such observations consistently made during the same period in subsequent years. To verify these behaviors, from 2011 to 2015, the smearing behavior of worker bees was documented using video cameras and other devices, capturing both videos and images. In 2015, the materials smeared were observed under a microscope to verify the contents contained within. During this examination, it was observed that not only plant fragments but also multiple insect species and unidentified materials of biological origin were present.

　In the autumn of 2016, field investigations and marking studies of individual worker bees confirmed that worker bees gnaw on individuals of mushroom sp. and used them as materials for smearing after attacks by V. mandarinia. In the autumn of 2017, it was discovered for the first time that worker bees were carrying larvae such as moths and Diptera back to the hive, clamped in their mandibles, engaging in smearing behavior. Upon observing these insects immediately after they were smeared by the worker bees, it was noted that many of the insect individuals were in a fresh state, suggesting they had been alive just before, with several individuals also found to be oozing body fluids. These evidences strongly suggested that at least a portion of the insects used as smearing materials were captured in a living state by worker bees and subsequently applied to the hive as a means of V. mandarinia.

　The authors' continuous behavioral research on worker bees since 2011 has demonstrated the use of smearing materials not only from plants but also from mushrooms and insects. Furthermore, it has been suggested that a broader array of organisms was used in their smearing behavior. On the other hand, until now, there have been no studies conducted that have analyzed the DNA of smearing materials to determine their origins from various organisms. Therefore, to investigate the smearing materials through DNA analysis, in the autumn of 2021, smeared materials were collected from a total of six colonies of A. c. japonica reared at three different research sites with varying surrounding environmental conditions. An attempt was made to assess the biotic composition of these materials through amplicon sequencing targeting the plant, fungal, insect, mammalian, and avian (birds) communities. The analysis revealed the detection of 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. Furthermore, preliminary DNA analysis also detected algae, mosses, fish, crustaceans, and other organisms.

　In this study, we have for the first time discovered and demonstrated that A. c. japonica used a variety of organisms as smearing materials in response to attacks by their natural predator, V. mandarinia, through both behavioral investigation of worker bees and DNA analysis. From the smear samples collected in the surveyed areas of biodiverse Satoyama landscapes, rare species such as the mountain hawk-eagle (Nisaetus nipalensis), which have been officially designated as nationally rare wild plant and animal species by the Ministry of the Environment, were detected. Additionally, other wild animals inhabiting Satoyama and forests were found in higher abundance compared to other surveyed areas. In this way, distinctive use patterns of specific organisms reflecting the differences in surrounding natural environments and biotic compositions have been identified. Furthermore, this study demonstrated the potential contribution to the identification of the habitat of rare species and wild organisms that are difficult to confirm visually by analyzing the smearing materials of A. c. japonica. The understanding of the diverse use of various organisms through the analysis of smeared materials is considered to be intriguing, as it can demonstrate the connection between A. c. japonica and the local natural ecosystems, indicating the interplay between them.