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Bull Mar Sci. 91(1):33–43. 2015
http://dx.doi.org/10.5343/bms.2014.1055
33
Bulletin of Marine Science
© 2015 Rosenstiel School of Marine & Atmospheric Science of
the University of Miami
Spawning behavior and paternal egg care in a circular
structure constructed by puersh, Torquigener
albomaculosus (Pisces: Tetraodontidae)
Hisoshi Kawase
1 *
Yoji Okata
2
Kimiaki Ito
3
Akihiko Ida
4
ABSTRACT.—Males of the pufferfish, Torquigener
albomaculosus Matsuura, 2014, are known to construct
a nest surrounded by radially aligned peaks and valleys on
sandy seabeds, which plays an important role in female mate
choice. Here, we report the spawning and parental egg care
behavior by the pufferfish based on underwater observations.
Pufferfish spawning occurred from spring to summer.
Although semi-lunar mating and hatching were observed
in the latter period of the spawning season, their periodicity
was not consistent throughout the spawning season. Females
visited males’ nests on the sandy bottom, and males courted
them by stirring up sand in the nest and performing a rush
and retreat behavior. e females and males mated in pairs,
releasing gametes on the nest within approximately 1 s. ey
mated repeatedly until females left the nest. e males mated
with other females on the same day. After matings, males
stayed at the nest site and cared for the eggs deposited on the
sandy bottom until hatching; males flapped their fins to stir
the eggs on the bottom, and drove away fishes that passed
close to the nest site. Hatching occurred synchronously with
the male flapping behavior around sunset. e reproductive
ecology of T. albomaculosus is unique in its paternal egg care
and mating system of male-territory-visiting polygamy, and
this is the first record of this behavior in marine pufferfishes.
Pufferfishes, family Tetraodontidae, are distributed in tropical, subtropical, and
temperate regions of the Atlantic, Indian, and Pacific oceans, and contain 19 genera
with about 130 species (Nelson 2006, Nakabo 2013). e pufferfishes inhabit chiefly
marine waters, and also occur in brackish and fresh waters (Nelson 2006). As marine
pufferfishes contain some commercially important species, such as Takifugu rubripes
(Temminck and Schlegel, 1850), decades of studies focusing on fishery resources
and aquaculture have been conducted (e.g., Fujita and Ueno 1956, Kusakabe 1962,
Takami et al. 1974, Fujita1988). Behavioral research has also focused on reproductive
behavior; for example, field observations revealed that Takifugu niphobles (Jordan
and Snyder, 1901) groups consisting of a female and scores of males migrate to an
intertidal beach, where they come out of the water to release gametes on boulders
1
Coastal Branch of Natural
History Museum and Institute,
Chiba, 123 Yoshio, Katsuura,
Chiba 299-5242, Japan.
2
Higashi-Komagata, Sumida-ku,
Tokyo 130-0005, Japan.
3
Marine Station Amami,
Yadorihama, Setouchi, Oshima,
Kagoshima 894-1523, Japan.
4
NHK Enterprises, Inc. 5-20
Kamiyama-cho, Shibuya-ku,
Tokyo 150-0047, Japan.
*
Corresponding author email:
<kawase@chiba-muse.or.jp>.
Date Submitted: 13 August, 2014.
Date Accepted: 6 November, 2014.
Available Online: 11 December, 2014.
research paper
O A
Open access content
Bulletin of Marine Science. Vol 91, No 1. 201534
(e.g., Uno 1955, Katayama et al. 1964, Honma et al. 1980, Yamahira 1994). Males of
Canthigaster punctatissima (Günther, 1870), Canthigaster valentini (Bleeker, 1853),
and Canthigaster rostrata (Bloch, 1786) establish a territory, in which several females
defend smaller territories (Kobayashi 1986, Gladstone 1987a,b, Sikkel 1990), and the
territorial males and females mate in pairs and deposit demersal eggs on algae, which
are not tended by either parent (Gladstone 1987a, Sikkel 1990).
We have reported that males of the pufferfish, Torquigener albomaculosus
Matsuura, 2014, recently reported to be a new species (Matsuura 2014), constructed
a large (approximately 2 m in diameter) geometric circular structure on the seabed
(Kawase et al. 2013). e male dug valleys at various angles in a radial direction, con-
structing a nest surrounded by radially aligned peaks and valleys, which appeared to
play an important role in the female mate choice, although definite factors affecting
this choice remain unknown. Furthermore, males created an irregular pattern com-
prising fine sand particles in the nest before spawning (Kawase et al. 2013). Here, we
report the spawning season and periodicity, mating behavior, and parental egg care
in T. albomaculosus, based on underwater observations. ese aspects are compared
with those of other tetraodontids and related species, and the characteristics of T.
albomaculosus are highlighted.
M M
Underwater observations of T. albomaculosus and circular structures were con-
ducted using scuba off Seisui (28°07′N, 129°19′E) and Katetsu (28°08′N, 129°20′E),
southern Amami-Oshima Island, Japan. e two research sites have calm condi-
tions, and are situated in embayments just west and east of Kurosaki Cape.
Presence/absence of the circular structures was recorded in the same area (ap-
proximately 100 × 100 m width, 10–30 m water depth) of Seisui and Katetsu 1–18
times a month throughout the year during 2011–2013 to determine the spawning
season of the pufferfish. e circular structures were categorized into three stages:
under construction, without irregular patterns on the nest (stage 1, corresponding
to early and middle stage in Kawase et al. 2013); completion, with irregular patterns
on the nest (stage 2, corresponding to final stage); and collapse after mating (stage 3,
corresponding to the duration of egg care). Water temperatures were measured near
the bottom of the research sites with a dive computer.
Intensive surveys were conducted at Seisui and Katetsu from 11 June to 13 July,
2012. An observation area of approximately 50 × 30 m was set up on the sandy bottom
at water depths of 23–28 m and 18–23 m, near Seisui and Katetu, respectively. Up to
three underwater observations (approximately 30–40 min per dive, at various times
between 05:00 and 21:00) were conducted daily at each site, except during inclement
weather. Most observations were made from a place on the perimeter of the circular
structures, and male and female behavior was recorded on digital still cameras (e.g.,
Olympus TG-1) and a video camera (Sony HDR-TG5V) for analysis of behavioral
patterns. In addition to underwater observations, two types of video cameras (GoPro
Hero and custom-made remote control camera) were also set unattended on the pe-
rimeter of the circular structures to record behavior over a longer period of time
from the same angle. e latter camera was controllable from the research boat via a
200-m cable, allowing the behavior of pufferfishes to be watched through a monitor
to control the timing of underwater observations. e videos recorded from 06:00 to
Kawase et al.: Reproductive ecology of the puersh 35
07:49 on 29 June at Seisui and from 05:47 to 05:58 on 13 July at Katetsu were used to
clarify mating behavior. e videos that were recorded at Seisui between17:00 and
19:05 on 17 June, at Katetsu between 19:10 and 19:36 on 5 July, and between 19:19 and
19:48 on 6 July were used to elucidate parental egg care and hatching.
Eggs were collected from the nests on 18 June (4 d after mating) and 5 July (5 d after
mating), 2012, to examine external features and egg diameters under a microscope.
As the July eggs hatched soon after collection, larvae were fixed in formalin and the
total length of each larva was measured. e eggs and larvae have been registered
and maintained in the Coastal Branch of Natural History Museum and Institute,
Chiba (CMNH-ZF17587-17590).
H Kawase observed parental egg care behavior and hatching of the pufferfishes,
and analyzed field observation data and videos. Y Okata observed courtship and
spawning behavior of the pufferfishes. K Ito monitored circular structures and puff-
erfishes throughout the year. A Ida designed a photography plan using unattended
video cameras during the 2012 research.
R
S S P.—Circular structures of T. albomaculosus
were observed on the seabed from 17 May to 25 August, 2011, 19 April to 8 August,
2012, and 24 March to 20 September, 2013, off Amami-Oshima Island, Japan (Fig.
1). Usually one to two, and at most seven, circular structures were concurrently ob-
served, and a total of four to 18 circular structures were observed in one spawn-
ing season on the seabeds at the Seisui and the Katetsu observation sites (Table 1).
Construction stages of the circular structures were well synchronized within and
between the two sites. e circular structures of stage 2 were observed around spring
tides after mid-June (Fig. 1, black bars). One reproductive cycle from the beginning
of construction of the circular structure to hatching (stages 1–3) continued for more
than 26 d in April (e.g., Fig. 1, Katetsu B in 2013), whereas it concluded within 12 d
during June–July (e.g., Fig. 1, Katetsu E in 2012). A total of four to nine reproductive
cycles were observed in a spawning season (Table 1).
Even when circular structures were found, their owners were not always found in
or around the structures. As the pufferfish were small and body color was similar to
that of the sandy bottom, it was difficult to detect the pufferfish that were away from
the circular structures.
Water temperature ranged 20–29 °C throughout the year at the observation sites:
it was around 22 °C from March to May at the beginning of the spawning season; it
rose dramatically between June and July, reaching 29 °C in August at the end of the
season.
S B M P.—Torquigener albomaculosus
males maintained the outer structure of the nest and irregular patterns in the nest,
comprising fine sand particles, before mating. When a male found a female ap-
proaching his nest, he stirred up the fine sand particles in the nest with his fins (Fig.
2A). When a female came into the nest, the male moved to the outer edge of the nest
and repeated a rush and retreat directed at the female (Fig. 2B). If the female decided
to spawn, she slowly went down to the seafloor in the nest (Fig. 2C), and the male
then approached the female. e female and male vibrated their bodies and released
Bulletin of Marine Science. Vol 91, No 1. 201536
gametes on the bottom of the nest within approximately 1 s. During this process, the
male kept biting the female on the rearmost outer part of her mouth (Fig. 2D).
A series of mating behaviors of T. albomaculosus were observed in a total of five
cases on 14, 29, 30 June and 1, 13 July, 2012, between 05:40 and 10:30. Based on the
observations on 29 June (Fig. 3A), the first female appeared in the nest of the male
at 06:36 (sunrise: 05:27). e male then stirred up fine sand particles and then per-
formed the rush and retreat behavior; however, the female soon left the nest site. A
second female (either the same individual or a new individual) appeared at 06:58, and
again the male performed the stirring up behavior and the rush and retreat behavior.
Next the female and the male mated in the nest four times in succession by 07:00.
After the matings, though the female temporarily went out of the circular structure,
she remained on the perimeter of the structure, and she entered the nest again. In
this way, the same female repeatedly visited the same nest 33 times, and the female
laid eggs one to four (mean: 1.30) times during one visit, a total of 43 times in 38
min by 07:37 (Fig. 3A, black bars except the first one). Male rush and retreat behavior
was observed zero to five (mean: 0.88) times, a total of 29 times in 16 out of 33 visits,
whereas behavior incorporating stirring up of fine sand particles was never observed
except at the first visit at 06:58. e irregular pattern on the inside of the structure
Figure 1. Lunar cycles and stages of circular structures constructed by puffershes, Torquigener
albomaculosus. Each rectangle surrounded by a black line indicates a separate circular structure.
Dark gray bars = stage 1, under construction without irregular patterns on the nest; black bars =
stage 2, completion with irregular patterns on the nest; light gray bars = stage 3, collapse after
mating; blank = no observations. Circular structures of Seisui (D–I) and Katetsu (E–H) in 2012
are equivalent to those of S1–S6 and K1–K4 shown by Kawase et al. (2013).
Table 1. Numbers of circular structures constructed by Torquigener albomaculosus discovered at
two observation sites off Amami-Oshima Island. For more information, see Figure 1.
Number of circular structure,
per one reproductive cycle
Number of reproductive
cycles
Total number of circular
structures
Year Seisui Katetsu Seisui Katetsu Seisui Katetsu
2011 1 1–2 4 6 4 8
2012 1–3 1–2 6 6 12 9
2013 1–7 1–6 8 9 18 16
Kawase et al.: Reproductive ecology of the puersh 37
Figure 2. Mating behavior of Torquigener albomaculosus. (A) Male (black arrow) stirring up of
ne sand particles, creating irregular pattern on the nest, after detecting a female (white arrow);
(B) male (left) rush and retreat directed at the female (right) that enters the nest; (C) female (right)
descending to the bottom of the nest just before spawning; (D) releasing of gametes by a pair at
the bottom (female = left). Photo by YO.
Bulletin of Marine Science. Vol 91, No 1. 201538
had been maintained in proper condition at the beginning of the observation; how-
ever, it gradually collapsed during consecutive matings in the nest.
Based on the observation on 13 July (Fig. 3B), mating had already begun at 05:47
(sunrise: 05:34). No stirring up of fine sand particles or rush and retreat behaviors
were observed. e first female mated three times in 4 min, and the second female
drove away the first female and mated once with the male. e irregular pattern in
the nest had already collapsed at the beginning of the observation.
P E C H.—After mating, T. albomaculosus females
left the nest site, whereas males stayed there to care for the eggs. Males flapped their
fins (Fig. 4A), and they often sucked the sand into their mouths and spat it out on the
bottom of the nest. us, males cared for the eggs by stirring the sand and accompa-
nying attached eggs. Males also cared for the eggs by removing debris that washed
into the nest (Fig. 4B) and by driving away fishes that passed close to the nest. Based
on the observation of a parental male on 17 June, 2012, 3 d after mating, the male
stayed in the nest for 38.9% of the time (91 min observation), whereas he was out of
the nest, performing no parental egg care, for the rest of the time. e frequencies of
parental care behaviors (in bouts per 10 min) were 10.66 (flapping), 0.66 (sucking and
spitting), 0 (removing), and 0.44 (driving away).
Hatching of T. albomaculosus occurred in the evening around sunset after 5 d
of mating in July 2012, at which time parental males stayed in their nests for 96.3%
of the time (40 min 50 s observation). ey performed the flapping behavior at a
significantly higher frequency (27.43 bouts per 10 min) than on the observation day
without hatching (Mann–Whitney U test: P = 0.003, z = 2.962). Many larvae hatched
synchronously with the male flapping behavior (Fig. 4C, Supplementary Video 1).
Hatching of larvae began at 19:10 (sunset: 19:24) and 19:36 (sunset: 19:23) on 5 and
6 July, respectively.
Eggs of T. albomaculosus were adhesive and nearly spherical in shape, measuring
0.92 (SD 0.03) mm (n = 11) in diameter. Newly hatched larvae measured 2.23 (SD
0.10) mm (n = 8) in total length.
Figure 3. Mating patterns of Torquigene r albomaculosus on 29 June (A) and 13 July (B), 2012.
Gray and black bars show presence of males and females, respectively. “F” means male stirring
up of ne sand particles, creating irregular pattern on the nest; numbers are bouts of male rush
and retreat behaviors directed at the female; “S” means spawning.
Kawase et al.: Reproductive ecology of the puersh 39
D
Lunar and semilunar spawning cycles have been reported in a variety of reef fishes
(e.g., resher 1984). Tetraodontiform examples include the grass pufferfish, T. ni-
phobles, which spawns for several days around the new and full moon in spring and
summer (e.g., Uno 1955, Katayama et al. 1964, Honma et al. 1980, Yamahira 1994).
e yellow margin triggerfish, Pseudobalistes flavimarginatus (Rüpell, 1829), has a
semi-lunar mating and hatching system (Gladstone 1994). In T. albomaculosus, a
series of stages within the reproductive cycle, namely the construction of a circular
structure, matings, and parental egg care, were concluded within a semi-lunar cycle,
with matings performed around neap tide after mid-June. As most males were not
identified, it is unknown if the same males repeated reproduction throughout the
spawning season. Kawase et al. (2013) reported that two identified males repeated re-
production at least twice. At the beginning of the spawning season, the reproductive
cycle continued through a lunar cycle (Fig. 1); the reproductive cycle was thus not of
consistent duration throughout the spawning season. e difference in reproductive
cycle duration within T. albomaculosus arose from the days required for circular
structure construction (stage 1) and for parental egg care (stage 3), apparently caused
by the change in water temperature throughout the spawning season: in many fishes
Figure 4. Paternal egg care and hatching of Torquigener albomaculous. Parental male cares for
the eggs deposited on the nest by apping his ns (A) and by removing debris (B). Parental males
promote hatching around sunset; white dot-like gures are newly hatched larvae (C). Photo by
HK.
Bulletin of Marine Science. Vol 91, No 1. 201540
hatching is delayed at low water temperatures (e.g., Nakamura, 1958). us, males
were forced to continue parental egg care over a longer period in the beginning of the
spawning season. However, it is unclear why it took so many more days for circular
structure construction.
Torquigener albomaculosus males exhibited two types of courtship behaviors: stir-
ring up of fine sand particles in the nest; and rush and retreat. ese two courtship
behaviors were observed only on the day of spawning and the day before, when the
circular structure was completed (Kawase et al. 2013). e stirring up behavior was
observed only when nest-holding males found a visiting female outside the circu-
lar structure; thus, the behavior seems to be important in determining the female’s
decision whether to inspect the nest site for spawning. It appears that, through the
stirring-up behavior, the males may be demonstrating to the females that the nest
contains a sufficient quantity and/or quality of fine sand particles, which were ef-
ficiently gathered and deposited in the nest site by the radially-aligned structure and
male behavior of passing through the valleys (Kawase et al. 2013).
e same males and females of T. albomaculosus mated repeatedly in pairs on
the same day. Although the release of gametes was not visible to the naked eye, the
abdomens of the females deflated gradually over the repeated matings. erefore,
we can be fairly certain that the male-female pairs released gametes when they vi-
brated their bodies at the bottom of the nest. Repeated mating is well known in other
fish groups. For example, damselfish (Pomacentridae) males establish a territory on
the bottom substrate, such as reefs and boulders, attract females passing the ter-
ritory, and mate with them. Females deposit demersal eggs on the substrate, and
males fertilize the eggs soon after that. e males and females repeat matings until
the females leave the male territory (e.g., resher 1984). On the other hand, the
repeated mating of females has never been reported in tetraodontidae nor related
species; females released all eggs at one time, and they never mated again on the
same day (e.g., Gladstone 1987a, Kawase and Nakazono 1995, 1996, Kawase 2003).
us, the repeated mating in female T. albomaculosus is the first such record from
the Tetraodontiformes species.
Marine pufferfishes may utilize a variety of substrates for spawning. Mating of T.
niphobles occurred on the pebbles of the intertidal zone (e.g., Uno 1955, Katayama et
al. 1964, Honma et al. 1980, Yamahira 1994), and the eggs were stranded under peb-
bles of the upper intertidal zone (Yamahira 1996). Eggs of C. valentini and C. rostrata
were deposited on algae at shallow coral reefs (Gladstone 1987a, Sikkel 1990). In the
present study, T. albomaculosus females deposited eggs on the sandy bottom of the
nest (Table 2). e nests are surrounded by radially-aligned peaks and valleys form-
ing geometric circular structures, which may not only influence female mate choice,
but may also hydrodynamically function to gather fine sand particles (important for
female mate choice) in the nest (Kawase et al. 2013). erefore, the spawning sites
of T. albomaculosus would be restricted to sites suitable for the creation of circular
structures that satisfy the following conditions: appropriate granularity of sand par-
ticles for construction of nests; appropriate water currents that facilitate fine sand
particle transport; and calm environments with reduced water movement that could
destroy the circular structures.
e mating system of T. albomaculosus was revealed to be male-territory-visiting
(MTV) polygamy, whereby males mate with multiple females that visit their terri-
tory to spawn (Kuwamura 1988). In other tetraodontid species, promiscuous group
Kawase et al.: Reproductive ecology of the puersh 41
spawning was reported in T. niphobles (e.g., Uno 1955, Katayama et al. 1964, Honma
et al. 1980, Yamahira 1994) and male-dominated harems of territorial females
were reported in C. punctatissima, C. valentini, and C. rostrata (Kobayashi 1986,
Gladstone 1987ab, Sikkel 1990; Table 2). In related groups, Stephanolepis cirrhifer
(Temminck and Schlegel, 1850) (Monacanthidae) males established territories, in
which they mated with resident females that defended smaller territories and non-
resident females that visited the male territory. e two alternative tactics adopted by
females resulted in two mating patterns, haremic polygyny and MTV polygamy, in a
single population of S. cirrhifer (Kawase and Nakazono 1996).
Parental egg care has been reported for several Tetraodon puffer species inhabit-
ing fresh and brackish waters (Cohn 1912, Randow 1934, Feigs 1955, Beller 1958,
Sugiyama et al. 2006). Aquarium observations revealed that Tetraodon turgidus
(Kottelat, 2000) males cared for the eggs deposited on gravel and clay pipe for 6–7 d,
until hatching at a water temperature of 26 °C (Nishimura 2006). Eggs of Tetraodon
palembangensis Bleeker, 1851 are also deposited on gravel and clay pipe in aquaria,
and males care for the eggs by blowing water on them for 9–11 d at 25 °C (Doi 2006).
On the other hand, parental egg care has not been reported in marine puffer species,
except for our report on T. albomaculosus (Table 2). Maternal and biparental egg care
were reported in related groups of monacanthid and balistid fishes (Kawase 2002);
however, paternal care was not performed except under exceptional situations when
the parental female abandoned the eggs (Kawase and Nakazono 1995). Paternal care
of T. albomaculosus seems to be related to the mating system and way of the egg
care. In the mating system, whereby males mate with multiple females that visit their
territory to spawn, paternal care is apt to evolve because parental males can care for
their own broods at one time, together (Kuwamura 1988).
A
We would like to thank H Ida, H Enomoto, T Matsushita, and T Kawasaki of NHK for
field observations at Amami-Oshima Island. We also thank H Doi of Osaka Aquarium
KAIYUKAN for providing us with information on the spawning ecology of freshwater puff-
erfishes. W Gladstone, University of Technology Sydney, gave us helpful comments on the
manuscript. is study was supported by JSPS KAKENHI Grant No. 22570032.
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Table 2. Comparisons of reproductive ecology in marine puffershes. Asterisk indicates the
present study.
Species Mating style
Spawning
substrate Mating system
Parental
egg care
Takifugu niphobles
Group Pebbles Promiscuity No
Canthigaster punctatissima
Pair Algae Haremic polygyny No
Canthigaster valentini
Pair Algae Haremic polygyny No
Canthigaster rostrata
Pair Algae Haremic polygyny No
Torquigener albomaculosus *
Pair Sand MTV polygamy Paternal
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