To investigate the seasonality of smeared plant materials, we observed patches (>0.5 mm in diameter) around
the entrances of a total of 20 colonies by eye and by video
recording at least once a month during the annual
A. cerana active season (April–October) from 2011 to
2015. Dark-coloured stains, presumably from the
discoloured, smeared plant materials, were much more
frequent in September and October (autumn). Most stains
were concentrated within 15 cm of the hive entrance, and
the number of stains increased with time during autumn.
To simulate Vespa attack, we divided six honeybee
colonies into three pairs and subjected each pair to experiments in 2015 with one of the three hornet species:
V. mandarinia , the suspected inducers of entrance
smearing, along with V. simillima xanthoptera and
V. analis, which are sympatric with A. cerana and
sporadically attack honeybee hives (Ono et al. 1987).
We carried out attack simulation experiments using each
Vespa species to simulate the scouting behaviour of the
respective species. In the V. mandarinia experiments, we
forced the experimental hornet fastened by its slender
petiole to a fine wire (Tan et al. 2012) to fly around the
bee hive for 30 s and then to walk around the hive
entrance for 30 s. Each treatment consisted of five cycles
of hornet flight/walking, and the treatment cycles were
repeated four times for each bee colony: twice on July 21
and once each on July 22 and 23. In the V. simillima
xanthoptera or V. analis experiments, the hornets were
forced to hover near the entrance of the hive, which was
achieved by allowing them to flap their wings freely for
1 min. As above, bee colonies were subjected to four
replicates of five treatment cycles with these hornet
Figure 1. A worker gnawing P. nepalensis near the hive. a Multiple bite marks on the leaves. b Ventral view of a
worker bee holding plant material between its mandibles and proboscis. c Smearing the gnawed material on the hive
entrance.
Table I. The results of a series of attack simulation experiments performed from July 21 to July 23 with three hornet
species.
Vespa species Apiary Colony
species on the same dates as the V. mandarinia experiments. We checked the presence or absence of entrance
smearing and staining 30 min and 3 days after each
treatment via naked-eye observations and video. Fivesix days after the attack simulation experiments, i.e. on
July 28–29, all experimental bee colonies were subjected
to an additional attack simulation experiment using
V. mandarinia , also comprising five cycles.
Before and after the attack simulation experiments
with V. mandarinia , we randomly collected 100 workers
with a butterfly net from each of the two colonies to
determine if they had ingested plant materials. These
experiments were conducted twice on 28–29 July. We
used Fisher’s exact probability test to detect significant
differences in the percentages of workers that brought
back collected plant materials before and after the attack
simulation experiments by V. mandarinia .
Only V. mandarinia attack induced entrance
smearing in the experimental bee colonies. Smearing
started 15–30 min after attack simulation began,
irrespective of the exposure date and time. Neither
plant-collecting nor plant-smearing responses were
observed when the other colonies were exposed to
simulated attack from V. simillima xanthoptera or
V. analis (Table I). The number of workers that
brought plant materials increased after the experimental exposure to V. mandarinia (Fisher’s exact
probability test P < 0.01). In colonies in which no
entrance smearing was observed in the first experiment with V. simillima xanthoptera and V. analis,
smearing was induced by additional exposure to
V. mandarinia scouting (Table I).
Overall, our study revealed a close relationship between the previously reported leaf-gnawing behaviour of
A. cerena (Yokoi 2005, 2015) and the occurrence of
dark-coloured stains around the entrances to the hives
of these bees (Okada 1997; Sasaki 1999). The purpose of
this behaviour is presumably to prevent mass attack by
giant hornets. How the smeared materials counteract
V. mandarinia attack remains unknown, but they may
alter the activity or recognition of the hornet ’s forage
site-marking pheromone (Ono et al. 1995). A. cerana did
not practise entrance smearing against V. simillima
xanthoptera and V. analis, presumably because these
species are less powerful enemies (Tan et al. 2013) and
do not use recruitment pheromones.
Similar entrance smearing has been described for
A. cerana in Vietnam and Korea, where V. mandarinia
and its different subspecies are sympatric (Sasaki
and Fujiwara unpublished data). Therefore, among
Asian honeybees, this type of entrance smearing
seems to be a common counteraction against Asian
hornets of the genus Vespa .
ACKNOWLEDGMENTS
We thank G. Chisaka (Kubokawa Ihatov Nature Restoration Project) for making the study area available to
us. We are grateful to M. Tanaka with respect to his
expert opinion about plant-gnawing behaviour and
Vespa attack. We are grateful to R. Sato, M. Tamukai,
S. Fujiwara and Y. Fujiwara (Japanese Original Honeybee Organization) with respect to the hornet observation
and experiment. This work was supported by Grant-inAid for JSPS Fellows Grant Number 15J07621 and by
JSPS KAKENHI Grant Number 26292181.
Note scientifique sur un comportement de l’abeille
japonaise, Apis cerana , déclenché par la première
approche d’un frelon asiatique Vespa mandarinia et
consistant à appliquer autour de l’entrée de la ruche
une Bbouillie végétale^
Eine wisenschaftliche Notiz zum Beschmieren des
Stockeingangs mit Pflanzenmaterial bei Apis cerana
in Japan, ausgelöst durch Kundschafter der asiatischen
Riesenhornisse Vespa mandarinia
REFERENCES
