“Smearing behavior”of Apis cerana japonica only in response to attacks by Vespa mandarinia : First report on diverse organisms used by worker bees in emergency

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 Apis cerana 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 immediately following the arrival of scouting hornets, worker bees of Apis cerana 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 nests 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, 2017).

  Additionally, during the investigation in the autumn of 2011, numerous smeared substances 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 substances smeared were observed under a microscope to verify the components contained within. During this examination, various insect species and unidentified substances of biological origin were found intermixed with plant fragments.  In the autumn of 2016, field investigations and marking studies of individual worker bees confirmed that worker bees gnaw on individuals of fungal species and use them as substances for smearing during attacks by Vespa mandarinia. In the autumn of 2017, a novel behavior was documented for the first time, wherein worker bees were observed carrying insect larvae of moths, in their mandibles back to their hives and then smearing them to their hive. According to these results, it is thought that worker bees hunt (or collect) these insects in the wild as a means of defending their hives against predators, particularly the Vespa mandarinia.

  The authors' ongoing behavioral research on worker bees since 2011 has suggested that a diverse range of organisms, not just plants but also fungi and insects, among others, are being utilized in their smearing behavior. However, there have been no studies conducting DNA analysis on smeared substances to determine the origin of the various organisms involved. In autumn 2021, smeared substances were collected from a total of six colonies of Apis cerana japonica reared at three different research sites with varying surrounding environmental conditions. An attempt was made to assess the biotic composition of these substances through amplicon sequencing targeting the plant community, fungal community, insect community, and mammalian community (including birds). The analysis revealed the detection of 39 genera from 30 families of plants, 31 genera from 25 families of insects, 38 genera from 28 families of fungi (basidiomycota), 15 genera from 11 families of mammals, and 13 genera from 11 families of birds. Furthermore, preliminary DNA analysis also detected algae, mosses, fish, crustaceans, and other organisms. From these results, it has been demonstrated for the first time that a variety of organisms are utilized by worker bees during smearing behavior, particularly in emergency situations such as attacks by the predatory hornet Vespa mandarinia.

  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 utilization patterns of specific organisms reflecting the differences in surrounding natural environments and biotic compositions have been identified. The understanding of the diverse utilization of various organisms through the analysis of smeared substances is considered to be intriguing, as it can demonstrate the connection between Apis cerana japonica and the local natural ecosystems, indicating the interplay between them.