The Science of Nature (2020) 107: 25

https://doi.org/10.1007/s00114-020-01681-2

ORIGINAL PAPER

Timing of male territorial flight and foraging of the large carpenter

bee Xylocopa appendiculata related to serotonin in the brain
Ken Sasaki 1,2 & Kenta Goto 1 & Ken-ichi Harano 2

Received: 2 April 2020 / Revised: 10 May 2020 / Accepted: 15 May 2020 / Published online: 3 June 2020
# Springer-Verlag GmbH Germany, part of Springer Nature 2020

Abstract
To determine how males of the large carpenter bee, Xylocopa appendiculata, maximize access to females while minimizing
energy cost and acquiring energy for territorial flights, we investigated the times of territorial flights by males and foraging by
males and females. Males were present continuously in territories from 8:00 to 12:00. They approached, chased, and excluded
conspecific males from their territories. In the laboratory, males showed higher locomotor and flight activities in the morning and
lower activities in the afternoon. Both males and females visited flowers from 8:00 to 16:00, but the most frequent visits were
earlier in females (10:00–12:00) than in males (12:00–13:00). Relative body weights in territorial males often increased. These
results indicate that the males time their territorial flights to maximize contact with females and obtain nectar as fuel between and
after the territorial flights. The time-related territorial flight in males might be based on a time-keeping system in the brain. Brain
levels of serotonin and its precursor tryptophan were significantly higher in males collected at 16:00 than at 11:00, suggesting a
relation between time-related territorial flight and serotonin synthesis in the brain.

Keywords Biogenic amine . Hymenoptera . Mating . Serotonin . Territory

Introduction Gerling 1980; Gerling et al. 1989). Males defend territories

The defense of territory is one of the reproductive strategies or
tactics used by males to monopolize mating with females.
Males can advertise their presence to attract virgin females
in the territory and exclude rival males from the territory.
This territorial behavior can produce a high mating success
for males, and, therefore, it is spread widely among species in
vertebrates and invertebrates.
In the carpenter bee Xylocopa, males maintain territory
during the mating season (Anzenberger 1977; Velthuis and
Communicated by: John A. Byers
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00114-020-01681-2) contains supplementary
material, which is available to authorized users.
* Ken Sasaki
sasakik@agr.tamagawa.ac.jp
1
Department of Bioresource Science, Tamagawa University,
Machida, Tokyo 194-8610, Japan
2
Honeybee Science Research Center, Tamagawa University,
Machida, Tokyo 194-8610, Japan
that contain resources for females in several species, whereas
in others, males defend small non-resource territories (Paxton
2005; Leys and Hogendoorn 2008). These territorial behav-
iors are considered to be implemented under conditions of low
female and male density (Paxton 2005).
Territorial male Xylocopa hovers above their territory for a
long time and may expend a large cost in energy because
energy consumption by hovering flight is positively correlated
with the duration of the flight (Wittmann and Scholz 1989).
Therefore, if males have limited energy to invest in territorial
behaviors, to save energy, they need to engage in these activ-
ities more often when females are most likely to visit terri-
tories. In fact, it has been reported that male territorial activi-
ties in Xylocopa varipuncta were correlated with female ac-
tivities (Alcock 1996), suggesting under limited energy, males
time their mate-attracting behavior to maximize contact with
receptive females.
To maximize mating success, obtaining sufficient energy
for territorial behavior is also important. Males fuel them-
selves by foraging nectar at flowers. For foraging, males
might have to leave their territory or abandon territorial be-
havior. Therefore, to maintain territorial efficiency, males
need to allocate time for territorial defense and for obtaining
25 Page 2 of 9
an energy supply. However, how males time their foraging
activities in relation to their territorial behavior is still
unknown.
The time allocation for territorial defense and foraging ac-
tivity in males is an important issue in behavioral biology.
This issue includes a trade-off between energy-consuming
reproduction and energy acquisition in order for males to op-
timize their behavior by switching these different activities
based on a neural time-keeping system. Research on this time
allocation system might help to understand the adaptations of
territorial males. To explore the reproductive strategy of males
comprehensively from behavior to physiology, the large car-
penter bee is an appropriate animal because of its abundance,
conspicuous territorial behavior, and ease of handling in the
laboratory.
In the present study, we investigate territorial and foraging
behaviors in males of the large carpenter bee Xylocopa
appendiculata. The males in this species defend their territory
during the mating season in Japan, occurring in April through
June. Since males hover above the territory located near nectar
and pollen source or available resting sites but not containing
these, they may defend an area where females pass through
frequently to foraging or resting sites. Therefore, we hypoth-
esized that males engage in territorial activities when females
are most actively passing through territories and then males
obtain their energy supply at a different time than when
defending their territory. To confirm this hypothesis, we in-
vestigated the following: (1) timing of territorial behavior in
males; (2) potential activity levels of locomotion and flight in
males; (3) timing of foraging in males and females; and (4)
changes of body weight during territorial behavior in males.
We also explored a possible physiological mechanism involv-
ing brain serotonin and other biogenic amine levels that may
underlie territorial behavior in males.
Materials and methods
Field observation of territorial behavior in males
Territorial flight behaviors in males of X. appendiculata were
observed during the mating season (April–June) in a field in
Machida, Tokyo, Japan. Since males do not perform territorial
flights on rainy days, the observations were carried out only
on sunny days from 8:00 to 14:00. The observation time was
determined on the basis of our preliminary observation that
males initiated the territorial flight between 7:00 and 8:00, and
then they moved from the territory before about 14:00 and did
not return. To identify individual males, they were caught in
the territory, marked on the thorax with a paint (paint marker,
Mitsubishi Pencil, Tokyo, Japan) without anesthesia, and re-
leased in the territory immediately after marking.
Replacements of the territorial male by other males by this
Sci Nat (2020) 107: 25
treatment have never been observed. We recorded the duration
of the territorial flight by males in their territory every hour
from 8:00 to 14:00 and their territorial behaviors, including
approaching and chasing conspecific males.
Measurements of locomotor and flight activities in
the laboratory
To investigate the potential activity levels of locomotion
and flight in males, the males hovering around the terri-
tory were collected from the field in the morning from
April to June. If males allocate time for territorial flights
and foraging, the males may maintain high behavioral
activity outside the territorial behavior period. The col-
lected males were individually placed inside plastic cups
(height 55 mm, inner diameter 75 mm) with a filter paper
in a dark box and transported to the laboratory. In the
laboratory, each male was individually transferred into a
test chamber (outer diameter 150 mm; inner diameter 90
mm; height 30 mm, Fig. S1a) with a transparent cover at
25 °C until behavioral experiments. Behavioral experi-
ments followed Akasaka et al. (2010) and Sasaki and
Nagao (2013). The transparent cover of the test chamber
was separated into four divisions by crosshairs. The spon-
taneous locomotor activity of each bee was recorded by a
digital video for 10 min every hour. From the recording
data, the number of times the males crossed the crosshairs
was counted.
After measuring locomotor activities, the duration of flight
initiation was measured in a net cage (45 cm × 60 cm × 45 cm,
Fig. S1b) at 28 °C, following Mezawa et al. (2013) and Sasaki
and Nagao (2013). The test chamber with a male was covered
with a red plastic sheet and transferred into the net cage placed
in an illuminated room (light intensity 820–940 lx, tempera-
ture 25 °C). Then, the red plastic sheet was removed in the net
cage to allow the male to fly spontaneously. The duration until
initiation of flight was recorded. Males that flew within 60 s of
removing the red plastic sheet in the net cage were evaluated
as “motivated males for flying” and counted. After the behav-
ioral experiments for locomotor and flight activities, the males
were transferred back into the plastic cups with 15% sucrose
solution in the dark box and used repetitively. To evaluate the
influence of collection time in the field on the locomotor and
flight activities, the locomotor and flight activities of one
group of males was measured at 12:00, 14:00, and 16:00,
and measured at 8:00, 9:00, 10:00, and 11:00 on the next
day (PM–AM males). The locomotor and flight activities of
the other group of males were measured the next day of the
collection at 8:00, 9:00, 10:00, 11:00, 12:00, 14:00, and 16:00
(AM–PM males). Since the results of locomotor and flight
activities were similar between PM–AM and AM–PM males
(see the “Results” section, Figs. S2 and S3), we pooled the
data of both males for statistical analyses.
Sci Nat (2020) 107: 25
Field observation of flower visiting by males and
females
To investigate when males and females collect nectar to sup-
ply energy, the numbers of individuals visiting flowers (cherry
blossom Cerasus × yedoensis “Somei-yoshino” and Japanese
wisteria Wisteria floribunda) were counted on four sunny
days from late April to early May. The flowers were in full
bloom when the observation was carried out and the large
carpenter bees visited the flowers in this field frequently dur-
ing this period. The bee counts around these flowers were
done continuously for consecutive 10 min periods for 3 h
10 min to 7 h in a period from 8:00 to 16:00 each day of
observation.
Repetitive measurements of body weight in territorial
males
To confirm whether the territorial males forage on flowers
and resupply their energy between the territorial flights,
the body weight of marked territorial males was repeated-
ly measured in the morning (7:50–11:45). If the males
visit flowers and intake nectar, their body weight will
increase. Alternatively, if the males release feces or re-
spire energy (nectar) during hovering in the territory with-
out a supply of energy, their body weight will continually
decrease. The males were caught with an insect net and
placed in a plastic container (4.5 cm diameter × 6 cm
height) without anesthesia. Then, the weight of the con-
tainer housing a male was measured by an electric balance
(Mettler Toledo, ML104T, Tokyo, Japan) within 5 min of
capture. The males were then released in their original
territory soon after measurement, whereupon they re-
sumed territorial flight. We confirmed the accuracy of
these measurements by preliminary experiments in which
the same individual was successively weighed five times
without releasing in 5 min. The standard errors for the
repetitive measurements were quite small (range, 0.1–0.2
mg, less than 0.1% of mean male weight, N = 3), indicat-
ing the consistency of the measurements.
Measurement of biogenic amines in the brain and
thoracic ganglion
To explore physiological mechanisms underlying time-related
territorial flight by males, we focused on the behavioral
changes from territorial flight to foraging behaviors based on
circadian rhythm. Biogenic amines can be candidate sub-
stances to regulate the behavioral changes with a circadian
clock (Page 1987; Flecke and Stengl 2009; Hirsh et al.
2010). Therefore, the amounts of biogenic amines in the brain
and meso-metathoracic ganglion were quantified and com-
pared between males collected in the morning and afternoon.
Page 3 of 9 25
All males were collected during hovering in the morning and
individually transferred into plastic cups with a filter paper
and transported to the laboratory. Some males were eutha-
nized by liquid nitrogen at 11:00 (AM male), and the others
were kept in a dark box with 15% sucrose solution and eutha-
nized by liquid nitrogen at 16:00 (PM male). All collected
samples were kept in liquid nitrogen until analyses. The frozen
brains and meso-metathoracic ganglion were dissected and
analyzed for biogenic amines as shown in the
Supplementary materials.
Statistics
The duration in hours of hovering by males in the field obser-
vation was compared by the Kruskal–Wallis test with Steel–
Dwass multiple comparison test. The number of crossings by
males to evaluate the locomotor activities in the laboratory
experiments was compared in hours by the Kruskal–Wallis test
with the Steel–Dwass multiple comparison test. Flight activity
in the laboratory experiments was examined by Fisher’s exact
test with Bonferroni correction. The proportions of males and
females visiting flowers were compared in hours by a 2 × 8
Pearson’s chi-squared test. Levels of biogenic amines in the
brains or thoracic ganglion were compared between AM and
PM males by the Mann–Whitney U test.
Results
Territorial behavior in males
Males of X. appendiculata performed territorial behaviors,
including hovering, chasing, and attacking intruders from
8:00 to 12:00 in their territory. After 12:00, they did not
hover in the territory. The duration of hovering by males
each hour was a range of 44–54 min per hour on average
from 8:00 to11:00 and 13 min per hour from 11:00 to
12:00 (6 males, Fig. 1a). The durations of hovering were
significantly different between each session (Kruskal–
Wallis test, H = 29.075, P < 0.001) and were longer from
8:00 to 11:00 than from 11:00 to 14:00 (Steel–Dwass test,
P < 0.05).
The territorial males usually hovered at a particular aerial
position in the territory. They approached or chased intruders
away a few meters from the position, probably ending the
chase near their territories border, and then the male returned
to the hovering position again. The intruders excluded by the
territorial males were not only conspecific males but also other
flying insects and birds (Fig. 1b). In relation to birds, the males
only approached but did not chase them. The males responded
to the flying animals even though they were significantly larg-
er than conspecific males. Males showed the following three
types of territorial behavior against conspecific intruders: (1)
25 Page 4 of 9 Sci Nat (2020) 107: 25

a Locomotor and flight activities in males

60
a
a
Duration of hovering

50 a Locomotor activities in males collected from their territory

differed significantly between each hour of duration during
(min / hour)

40
the day (Kruskal–Wallis test, total data: H = 63.777, P <
30
0.001, 18 males, Fig. 2a; PM–AM males: H = 22.876, P <
20 b 0.001, seven males, Fig. S2a; AM–PM males: H = 45.536, P <
10 0.001, 11 males, Fig. S2b) and showed higher levels from
b 8:00 to 13:00 (Steel–Dwass test, P < 0.05, Fig. 2a) in the
0
laboratory. The active time for locomotion was longer than
that for territorial flight in the field.
Males flew within 60 s in the net cage from 8:00 to 17:00
(total data: 18 males, Fig. 2b; PM–AM males: seven males,
Fig. S3a; AM–PM males: 11 males, Fig. S3b). The flight
b
activity levels differed significantly among the hour time-
300
Others
Numbers of individuals

periods (Fisher’s exact test, total data: P < 0.001, PM–AM

excluded by males

250 males: P < 0.01; AM–PM males: P < 0.001) and were higher
Conspecific males
200 from 8:00 to 13:00 than from 16:00 to 17:00 (Bonferroni
150
correction, P < 0.05, Fig. 2b). The graph (Fig. 2b) shows that
the active time for flight in the laboratory was longer than that
100
for territorial flight in the field.
50

0 Visits to flowers by males and females

Males visited the flowers the most frequently from 12:00 to

13:00 and continued foraging less frequently thereafter until
16:00 (Fig. 3a). Males were also observed on the flowers
c between 8:00 and 11:00, but the frequency was relatively
160 low. Several males that were chased by territorial males es-
Chase and raise caped into the flower area outside of the territory in the morn-
Numbers of territorial

140
behaviors by males

120 Approach and chase
100 Approach
80
60
40
20
0 which was earlier than the times that males visited (Fig. 3b).
Fig. 1 Territorial behaviors in males of Xylocopa appendiculata. (a)
Duration of hovering in the territory (mean + SE). (b) Total number of
intruders excluded by males, and c: total number of territorial behaviors
by males against conspecific males. Six territorial males were observed
from 8:00 to 14:00. Different letters above the bars indicate significant
differences (Steel–Dwass test, P < 0.05)
approached an intruder but returned immediately; (2)
approached and then chased an intruder; and (3) chased an
intruder and then rose higher into the air parallel with the
intruder (Fig. 1c). These behaviors were frequently observed
from 8:00 to 11:00. No pheromonal marking behavior by
territorial males was observed.
ing and then foraged. Most males were unmarked and it is
unknown whether they were maintaining a territory or not,
but at least, two marked territorial males visited the flowers
in the morning.
In contrast to the visiting times by males, females visited
the flowers the most frequently between 10:00 and 12:00,
The proportion of males and females visiting flowers gradu-
ally changed over time from 8:00 to 13:00 toward male bias
and were significantly different among each hourly duration
(2 × 8 Pearson’s chi-squared test, χ2 = 29.506, df = 7, P <
0.001, Fig. 3c).
Several males kept territories around the flower area and
caught females in the air as they departed from the flowers.
One male that caught a female landed on a branch of a tree in
the territory and then mated successfully with the female.
Such mating by males within their territories was observed
three times between 9:00 and 10:00 (Fig. 3b, three arrows).
Body weight in males
The territorial males marked on the thorax hovered only in the
morning as well as the previous observation of males in the
Sci Nat (2020) 107: 25 Page 5 of 9 25

a Locomotor activity
70
Numbers of crossings

60 a a
50
a a
40
30 ab
20
10 bc c
0

b Flight activity
a
Proportion of individuals

1 a
ab ab ab
0.8

0.6
flying

bc
0.4
c
0.2

Fig. 2 Locomotor and flight activities of males of Xylocopa

appendiculata in the laboratory. (a) The locomotor activities (mean +
SE, n = 18). (b) The flight activities (n = 18). Different letters above the
bars indicate significant differences (a, Steel–Dwass test, P < 0.05; b,
Bonferroni test, P < 0.05)

territory and were weighed repetitively in the morning (7:50–

11:45) (Fig. 4). When the first body weight was set at 0, the
relative weight of five out of nine males increased between 8.0
to 69.3 mg at the second measurement (black symbol) and the
relative weight of four of the nine decreased by 14.9 to Fig. 3 Flower visits by males and females of Xylocopa appendiculata.
Mean number (+SE) of individuals visiting flowers in each hour by males
86.7 mg (white symbol). With regard to the latter four males (a) and females (b). Numbers in parentheses indicate numbers of counts.
whose weights initially decreased, the weight of all of them Mating behaviors were observed at three times (arrows). The proportion
eventually increased by 1.4 to 82.6 mg by the third or fourth of males and females visiting flowers in each hour (c)
measurements in time (Fig. 4).

Biogenic amine levels in the brain and thoracic
ganglion
Brain serotonin levels were significantly greater in PM males
than in AM males (Mann–Whitney U test, z = − 2.34, P <
0.05, NAM = NPM = 9, Fig. 5a), but the levels of other assumed
functional amines (dopamine, octopamine, and tyramine) did
not differ between AM and PM males (dopamine: z = − 1.457,
P = 0.145, Fig. 5b; octopamine: z = − 0.309, P = 0.757, Fig.
5c; tyramine: z = − 0.574, P = 0.566, Fig. 5d). The levels of
tryptophan, a precursor of serotonin, were significantly greater
in PM males than AM males (z = − 3.135, P < 0.01, Fig. 5e),
whereas the levels of N-acetylserotonin, a metabolite of sero-
tonin, did not significantly differ between AM and PM males
(z = − 0.397, P = 0.691, Fig. 5f). The levels of serotonin and
tryptophan in the brains were positively correlated
(Spearman’s ranked correlation, rs = 0.692, tau = 3.839, P <
0.01, n = 18).
25 Page 6 of 9 Sci Nat (2020) 107: 25

Fig. 4 Repetitive measurements

of body weight in marked
territorial males of Xylocopa
appendiculata (7:50 to 11:45
AM). Body weight at first
measurement was set as 0 (gray
symbol). Males with increased
body weight and with decreased
body weight are indicated as
black and white symbols,
respectively

In the meso-metathoracic ganglion, however, the levels of of serotonin relative substances (tryptophan and N-
all four functional amines that we examined did not differ acetylserotonin) also did not differ between AM and PM
significantly between AM and PM males (Table 1). The levels males (Table 1).

Fig. 5 Amounts of biogenic a b

amines (mean + SE) in the brain Serotonin Dopamine
of territorial males of Xylocopa 60 1000
appendiculata collected at 11:00 P < 0.05 P = 0.145
(AM) and 16:00 (PM). (a) 50

(pmol / protein mg)

(pmol / protein mg)

800

Amount of amine
Amount of amine

Serotonin, (b) dopamine, (c)

40
octopamine, (d) tyramine, (e) 600
tryptophan, and (f) N- 30
acetylserotonin. Differences be- 400
20
tween groups were assessed by
the Mann–Whitney U test (NAM = 10 200
NPM = 9)
0 0
AM PM AM PM

c d
Octopamine Tyramine
140 30
P = 0.757 P = 0.566
120 25
(pmol / protein mg)

(pmol / protein mg)

Amount of amine

Amount of amine

100
20
80
15
60
10
40

20 5

0 0
AM PM AM PM

e f
Tryptophan N-acetylserotonin
2500 30
P < 0.01 P = 0.691
Amount of amino acid
(pmol / protein mg)

(pmol / protein mg)

2000
Amount of amine

20
1500

1000
10
500

0 0
AM PM AM PM
Sci Nat (2020) 107: 25 Page 7 of 9 25

Table 1. Amounts of biogenic

amines (pmol/protein mg) in the AM (mean ± S.E.) PM (mean ± S.E.) U testa
meso-metathoracic ganglia in
males of Xylocopa appendiculata Serotonin 51.758 ± 6.283 54.847 ± 3.150 P = 0.085, z = −1.722
collected at 11:00 (AM) and Dopamine 113.498 ± 6.805 128.451 ± 19.989 P = 0.757, z = −0.309
16:00 (PM) Octopamine 124.257 ± 11.050 113.559 ± 14.088 P = 0.402, z = −0.839
Tyramine 50.462 ± 7.371 60.957 ± 22.667 P = 0.102, z = −1.634
Tryptophan 813.328 ± 116.090 815.356 ± 69.769 P = 0.627, z = −0.486
N-acetylserotonin 15.858 ± 2.096 20.432 ± 3.440 P = 0.310, z = −1.015
a
Levels of biogenic amines in the thoracic ganglion were compared between AM and PM males by the Mann–
Whitney U test

Discussion during 9:00–10:00. Therefore, territorial males advertise their

In the present study, we demonstrated for the first time the
time-related territorial flight of male X. appendiculata mostly
in the morning before noon and its energy acquisition between
or mainly after the territorial flights. The time of male territo-
rial flight corresponded with the time of frequent foraging by
females, supporting our hypothesis that males engage in terri-
torial activities more often at times when females are most
likely to visit or pass through territories. Our data also impli-
cate that a neuroendocrine mechanism involving serotonin in
the brain underlies time-related behavior by males.
Time-related territorial flight
The territorial flights by male X. appendiculata were recorded
only in the morning. During the territorial flight, males hov-
ered within each territory, and approached, chased, and
attacked the intruders of conspecific males. Pheromonal mark-
ing behavior as described in other species (Gerling et al. 1989;
Vinson and Frankie 1990) was not observed in this species.
The purpose of a territory seems to be for mating with females
because several males were observed catching females in the
air and mating with them within their territory. The mean
duration of the territorial flight was 44–54 min per hour before
noon, indicating males spent most of their time in the territory
and likely expend a large amount of energy in territorial flight.
We, therefore, expect an optimized time allocation for territo-
rial flight and energy supply in males.
The results of laboratory experiments and field observa-
tions indicated that locomotor and flight activities in males
were relatively higher in the morning, but the active time
was longer in the laboratory experiments than that for territo-
rial flight in the field. The activities in the elongated time in
males may be explained by foraging to collect nectar after
territorial flight.
Time-related territorial flight in males overlaps the time of
frequent flower visits by females. The time of the most fre-
quent flower visits by females was 11:00–12:00, which was
earlier than that by males (12:00–13:00). It was also observed
that three territorial males succeeded in mating with females
presence to encounter females in the morning. Since males
necessarily respire and metabolize energy during the territorial
flight, males may hover only in the morning to reduce energy
costs. However, males also hovered during 8:00–9:00 when
females did not frequently visit the flowers. This might be to
encounter virgin females as early as possible. The time of
mating observed during 9:00–10:00 was not at the peak of
flower visiting by females, implying females might accept
males to mate before females begin active foraging.
It has been reported that male territorial activities in
X. varipuncta correlated with female activities (Alcock
1996). This result suggests that males time their mate-
attracting behavior to maximize contacts with receptive fe-
males. However, in X. varipuncta, the time of male hovering
fluctuates day-to-day (Alcock 1996). In other species of
Xylocopa, the time of territorial hovering appears uniform
throughout most of the day (Anzenberger 1977; Velthuis
and Gerling 1980), suggesting the property of time-related
hovering in X. appendiculata may not necessarily be common
among Xylocopa. In the bumblebee Bombus, the flight activity
for patrolling in males follows a constant daily rhythm: the
activity is higher in the morning than in the afternoon and
subsides during daytime (Svensson 1979; Bertsch 1984;
Surholt et al. 1988). These bumblebee patterns of activity
are similar to that in X. appendiculata.
Timing of energy supply
Males visited flowers most frequently between 12:00–13:00
when many males had finished their territorial flight. This
peak of flower visits in males did not correspond with the peak
in females, which occurs between 10:00–12:00. The foraging
by males after territorial flights lasted until 16:00, and it is
expected that the nectar collected in the afternoon might be
used for hovering the next day. This is the case in bumblebees,
where males exhibit a constant pattern of daily food intake at
the end of patrolling flight time and store the nectar in the
digestive tract for energy the next morning to use in flight
(Bertsch 1984; Surholt et al. 1988).
25 Page 8 of 9
The results of repetitive body weight measurements of
X. appendiculata also demonstrate that males may visit
flowers between territorial flights in the morning. In fact, we
observed that the small number of marked territorial males
foraged during territorial flight in the morning. Such foraging
between territorial flights can be expected because of the in-
field results of territorial flight duration. The duration of terri-
torial flight was 44–54 min per hour, indicating that males
went out of the territory for 6–16 min. Since the time for
foraging might be limited between territorial flights, the ob-
tained nectar mass might be less than 82.6 mg (our largest
male weight increase). In the social carpenter bee Xylocopa
nigrocincta, males minimize their water load by dehydrating
the nectar at the nest entrance before leaving the nest for hov-
ering flights in their territories (Wittmann and Scholz 1989).
Honeybee males use highly concentrated honey as fuel for
mating flights to minimize the weight of the crop honey load
(Hayashi et al. 2016). Thus, male bees can adjust the nectar or
honey fuel load in several ways. Males in X. appendiculata
may carry a minimum weight of nectar fuel and resupply the
fuel between territorial flights when the fuel becomes low.
We marked territorial males, but many unmarked males
were observed on the flowers. These males might (a) forage
from a distant territory, (b) escape from territorial males, or (c)
not form a territory and act as satellite males having alternative
reproductive tactics (Alcock 1997; Barthell and Baird 2004;
Paxton 2005). These possibilities could be tested in the future.
Possible physiological mechanisms underlying time-
related territorial flight
The mechanisms underlying time-related territorial flight in
X. appendiculata do not simply control the locomotor and flight
activities, because the laboratory study showed that these activ-
ities continued after the time of territorial flight. Therefore,
time-related territorial flight might be controlled by a time-
keeping system and the motivation of sexual behavior with
flight. One time-keeping system is a circadian clock in the
brain. Serotonin is known as a brain neurotransmitter that
causes phase-shift of the circadian rhythm of locomotor or neu-
ral activities in the cockroach (Page 1987), crickets (Tomioka
1999), blowfly (Cymborowski 2003), and Drosophila (Yuan
et al. 2005), and the brain levels increase from morning to
afternoon during daytime (Tomioka et al. 1993). In
Drosophila, serotonin also has a role in the selective suppres-
sion of mating behavior in both males and females (Pooryasln
and Flala 2015). Such general roles of serotonin among insects
might be shared in X. appendiculata. The brain levels of sero-
tonin and its precursor tryptophan were significantly higher in
PM males than in AM males, and these levels were positively
correlated with each other. These results suggest that serotonin
was synthesized more actively in the PM with an active supply
of serotonin precursors in the brain. It is possible that increased
Sci Nat (2020) 107: 25
serotonin might inhibit sexual motivation in males in the after-
noon. This possibility remains to be tested.
The thoracic ganglion, especially meso-metathoracic gan-
glion, is associated with the motor systems and central pattern
generators of flight in insects (Burrows 1996). However, the
biogenic amine levels that we examined in the thoracic gan-
glion did not differ between morning and afternoon.
Therefore, time-related territorial flight might be involved in
a higher neural center (brain) but not in a peripheral neural
center (thoracic ganglion).
Conclusions
We investigated the territorial behavior of male X. appendiculata
and the times of territorial flight by males and foraging by males
and females. The results indicate time-related territorial flight and
energy acquisition between or after territorial flights in males.
The time of territorial flight corresponded to the time of frequent
foraging by females, suggesting males engage in territorial ac-
tivities more often at times when females are most likely to visit
territories. The males obtain energy outside this time for territo-
rial behavior. The time-related territorial flight in males may be
based on a time-keeping system in the brain. We suggest that
there is an important relationship between time-related territorial
flight and serotonin synthesis in the brain.
Acknowledgments This work was supported by the Japan Society for the
Promotion of Science (JSPS) KAKENHI grant number JP17K07491 to
KS.
Availability of data and material All relevant data are within the paper
and its Supplementary Materials (data-sasaki et al.xlsx).
Compliance with ethical standards
Conflicts of interest The authors declare that they have no conflict of
interest.
Ethic approval This article does not contain any studies with human
participants. All applicable international, national, and/or institutional
guidelines for the care and use of animals were followed.
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