Larval molting and growth of the Japanese spiny lobster Panulirus japonicus under laboratory conditions Hirokazu MATSUDA* AND Taisuke TAKENOUCHI Fisheries Research Division, Mie Prefect
Trang 1FISHERIES SCIENCE 2006; 72: 767–773
*Corresponding author: Tel: 81-5-9953-0130
Fax: 81-5-9953-2225 Email: matsuh07@pref.mie.jp
Received 24 October 2005 Accepted 26 January 2006.
Larval molting and growth of the Japanese spiny lobster
Panulirus japonicus under laboratory conditions
Hirokazu MATSUDA* AND Taisuke TAKENOUCHI
Fisheries Research Division, Mie Prefectural Science and Technology Promotion Center, 3564-3 Hamajima, Shima Mie 517-0404, Japan
monitor body length (BL) and intermolt period, and 2000 were cultured in groups to sample specimens for measurement of body weight Phyllosoma were fed with Artemia and mussel gonad; the culture
seawater temperature was 24–26°C The individually cultured phyllosoma showed an increment in body length by the first molt of approximately 0.5 mm, and the molt increment increased to
approx-imately 1 mm at 5 mm BL; it was constant to 15 mm BL Thereafter, the molt increment increased
exponentially The duration of the first instar was 6–7 days Instar duration increased with development
up to approximately 2 weeks at the 20th instar (∼16 mm BL) and then became constant Of the 10
lar-vae reared individually, five metamorphosed to the puerulus stage The entire phyllosoma life ranged from 245–326 days (mean 289.0 days), and the number of instars ranged from 22–29 (mean 26.2) Body length in the final instar ranged from 28.50–33.10 mm (mean 30.280 mm) For the phyllosoma
cultured in groups, relationships between BL and wet/dry body weights (WW/DW, mg) were expressed as exponential equations: WW = 0.0686BL2.2023 and DW = 0.0209BL2.1905
INTRODUCTION
The Japanese spiny lobster Panulirus japonicus
occurs in shallow rocky areas of the north-western
Pacific.1 In Japan, the lobster plays an important
role in coastal fisheries because of its high
eco-nomic value.2 Hence, the ecology, stock
manage-ment and aquaculture technology have long
been studied, and some measures to manage
and enhance the natural population have been
proposed.2,3
Panulirus japonicus has a pelagic larval stage
(phyllosoma) in its early life history as do scyllarids
and other palinurids The phyllosoma stage has
been the target of research because an
understand-ing of the variation in recruitment to the fishunderstand-ing
ground is crucial for establishment of
stock-man-agement strategies for the lobster.4–6 The number of
phyllosoma that had been caught in the ocean,
however, was too small, and little information
was available on their development, distribution
and subsequent recruitment Recently, Yoshimura
et al.7 successfully collected many later-stage phyl-losoma off the south coast of Kyusyu Island and demonstrated that the late-stage larvae were dis-persed widely in and south of the Kuroshio Cur-rent In addition, Sekiguchi8 and Sekiguchi and Inoue9 advanced hypotheses on larval recruitment processes by re-examining plankton samples col-lected to survey the distribution of ichthyoplank-ton These studies provide important information
to elucidate the recruitment processes
Research on larval culture of P japonicus has
been conducted both to produce a large number of juveniles for use in aquaculture, and to ascertain ecologic and ethological aspects of larvae.10–12 Thus far, outlines of phyllosomal development have been reported from larval cultures in the labora-tory Under laboratory conditions, the entire length
of the phyllosoma stage ranged from 231–417 days
(n = 136, 24–27°C) and the total number of molts from hatch to the puerulus stage ranged from 20–
31.11 The molt increment was constant at about
1 mm up to approximately 18 mm body length
(BL), and then this increment increased
exponen-tially.10 However, exhaustive observations on larval development throughout the phyllosoma life have not yet been made; a better understanding of larval development is necessary to interpret behavioral
Trang 2traits in the ocean and to determine adequate
culturing conditions, which would alter as larvae
develop
In the present study, phyllosoma of P japonicus
were cultured individually from hatch to the
puer-ulus stage in order to monitor their growth
Phyl-losoma were also mass-cultured to obtain samples
for measurements of body weight The present
paper reports the growth and the change in body
weight under laboratory conditions for P japonicus
phyllosoma, from hatch to the puerulus stage
MATERIALS AND METHODS
Individual culture
An ovigerous female of P japonicus was collected
off Shima (34°17′Ν, 136°49′E), Mie Prefecture, on 5
June 1997 using tangle nets On the day of capture,
the lobster was transferred to Mie Prefectural
Science and Technology Promotion Center It was
maintained at ambient temperature (20–24°C) in a
flow-through tank until hatching occurred
Seawa-ter was supplied afSeawa-ter it was filSeawa-tered through sand
The lobster was fed daily with the mussel Mytilus
galloprovincialis and frozen krill.
Hatching occurred on 20 July 1997 Of several
hundred thousand newly hatched phyllosoma, 10
were used for individual culture The culture
meth-ods were similar to those used for larval Panulirus
longipes.13 Each larva was kept in a 120-mL glass
cup until 100 days after hatching, after which each
was cultured in a 400-mL glass cup The first to
fourth instar larvae were fed Artemia nauplii
(∼0.6 mm BL) exclusively From the fifth instar
onward, they were fed Artemia cultured with the
diatom Phaeodactylum tricornutum and finely
minced mussel gonads The size of Artemia given
gradually increased up to approximately 6 mm BL
as the phyllosoma developed; accordingly, the
density of Artemia decreased from 2–0.3
individ-ual/mL The size of the pieces of mussel gonad
given also increased (from ∼1–4 mm3) as the larvae
grew About 10 pieces of mussel gonad were placed
in each culturing vessel Artemia and mussel gonad
were replaced daily during culture
The seawater temperature was maintained at
26.0°C until 130 days after hatching; it was
gradu-ally decreased to 24.0°C over 2 weeks and then
maintained at 24.0°C until the end of the culture,
according to Matsuda and Yamakawa.10 The larvae
were exposed to a constant artificial light–dark
cycle (lighting from 08:30–20:30 hours) by
full-spectrum fluorescent bulbs equipped with an
elec-tric timer The light intensity during the light phase
was about 5 μE/m2 per s
During the individual culture, the intermolt
period and BL were monitored regularly for each
phyllosoma and puerulus Body length of the phyl-losoma refers to the length from the anterior mar-gin of the cephalic shield between the eyestalks to the posterior end of the abdomen (Fig 1) Body length of the puerulus was measured from between the supraorbital horns to the posterior
margin of the telson Measurements of BL were made every 2–7 days after molting To measure BL,
each larva was transferred from the culture vessel
to a 12-cm diameter laboratory dish with a small amount of seawater using a small ladle Body length was then measured with a profile projector (V-12A, Nikon, Tokyo, Japan) after the sea water had been poured out by tipping the dish Finally, the larva was returned to the same vessel During the measurement process, special care was taken
Fig 1 Body length (BL) of Panulirus japonicus
phyllo-soma, from the anterior margin of the cephalic shield between the eyestalks to the posterior end of the abdomen.
Trang 3to avoid damaging the larva The larval culture
continued until all of the larvae had either died or
metamorphosed to the puerulus stage
Mass culture
A total of 2000 phyllosoma hatched on 27 July 1998
from a female lobster that had been captured on 8
June 1998 off Shima were used in the mass culture
They were placed in two 40-L specialized acrylic
hemispherical tanks11 and cultured in a
flow-through system at a water flow rate of 20–80 L/h
The seawater temperature was controlled at a
sim-ilar temperature as for the individual culture using
a unit to supply constant-temperature seawater
(Aquatron-portable APS-206A, Koito Industries,
Yokohama, Japan) Food items were also similar to
those used in the individual culture The density of
Artemia fed was lower in the mass culture than in
the individual culture: 1.0 individual/mL with
nau-plii and 0.1 individual/mL with larger Artemia.
Approximately 50–200 pieces of minced mussel
gonad were prepared and fed once daily for each
group tank Uneaten foods were removed before
feeding Artemia were removed by changing
screens with 0.25-mm mesh attached around a
drainpipe, to those with 1- or 3-mm mesh that
ensured escape of Artemia Uneaten mussel
gonads were siphoned The cultures were
trans-ferred to clean tanks twice per week
Until the sixth instar, all larvae molted within 2–
4 days and distinct morphological changes were
observed with ecdyses This enabled sampling,
from the first to fifth instar, of larvae for
measure-ments of body weight 2–4 times within each instar
For the first to fifth instar, 10–50 larvae were
col-lected in triplicate at each sampling, then the total
wet weight of each group was measured and
con-verted to individual weight Beyond the fifth instar
it was impossible to determine the number of the
instar for each larva because of the large individual
differences in BL, intermolt period and
morphol-ogy, and larvae were consequently collected once
or twice every 2 weeks From the sixth instar
onward, larval weights were measured individually
The larvae were placed in sea water without food
for 2 h prior to measurement; the larvae were then
placed on filter paper and rinsed with 3.5%
ammo-nium formate to remove saline matter.14 They were
transferred to a preweighed aluminum sheet with
tweezers, and the wet body weight was measured
Dry body weight was measured after the samples
were dried at 60°C for 24 h and placed in a
desic-cator at an ambient temperature for 1–2 h Wet and
dry body weights were measured with an
autobal-ance (1712MP8, Sartorius, Göttingen, Germany)
As the wet and dry weights increased linearly with increasing days after hatching during the first to fifth phyllosoma instar, the relationships
between days after hatching (D) and wet/dry body weights (WW/DW, mg) could be described by:
WW or DW = aD + b where a and b are constants However, because the difference in BL between individuals became
larger beyond the fifth instar and it was no longer appropriate to describe the relationships between days after hatching and body weights, increases in
body weights were expressed relative to BL by the
following power function:
WW or DW = c × BL d
where c and d are constants The constants a, b,
c and d can be estimated by the least-squares
method using MS Excel (Microsoft, Redmond, WA, USA)
The survival rate (Su) in the mass culture was
calculated by excluding the number of larvae sam-pled for measurements of body weight as follows:
where S i represents the survival rate from the ith to the (i + 1)th sampling.
RESULTS Individual culture
Of the 10 phyllosoma cultured, five died in the course of the culture and five reached the puerulus stage Mortalities were found on days 75, 126, 129,
239 and 318 after hatch (Fig 2) The causes of
mor-tality were bacterial disease (n = 3), a symptom of which was cloudiness of the mid-gut gland and
hind-gut, and complications in molting (n = 2) The five phyllosoma that reached the puerulus stage metamorphosed after 22–29 molts (mean 26.2 molts) Their phyllosoma lifetimes ranged from
245–326 days (mean 289.0 days), and BL in the final
phyllosoma instar ranged from 28.50–33.10 mm (mean 30.280 mm)
(mean ± SD, n = 10) for the first instar It then
increased linearly with moltings: 7.69 ± 0.39 mm
for the 10th instar (n = 10), 16.30 ± 2.54 mm for the
20th instar (n = 7) and 20.95 ± 3.16 mm for the 25th instar (n = 4) (Fig 3) There were three phases in
the relationship between BL before a molt and increment in BL by the next molt (phases A, B and
C, Fig 4) The molt increment increased gradually
i
n
=
-’ 0 1
Trang 4from approximately 0.5 mm (at ∼1.5 mm BL at
hatch) to about 1 mm (at ∼5 mm BL, phase A), then
it was constant at approximately 1 mm (until
∼15 mm BL, phase B) Beyond approximately
15 mm BL, it increased exponentially and at the
same time the variability in molt increment became larger as the larvae grew (phase C) The
growth index of increase in BL from one instar (n)
to the next (n + 1), calculated as BL (n+1) /BL n, ranged
initially from 1.29 to 1.41 (mean 1.348, n = 10); it decreased with development up to approximately
15 mm BL, and then increased gradually (Fig 5).
The instar duration increased from approximately 1–2 weeks until the 20th instar; thereafter it became constant at 2 weeks (Fig 6)
Figure 7 shows the relationships between BL
and days after hatching for the two animals with
Fig 2 Survival of Panulirus japonicus phyllosoma in
the individual culture with a still-water system.
0
20
40
60
80
100
0 50 100 150 200 250 300 350
Days after hatching
Metamorphoses to the puerulus stage
Fig 3 Changes in body length with development of
Panulirus japonicus phyllosoma cultured individually in
a still-water system Means ( 䊉) and ranges (vertical bars)
of body length are shown.
0
5
10
15
20
25
30
35
40
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29
Instar
Fig 4 Relationship between body length (BL) before a
molt and increment in BL by the next molt for Panulirus
japonicus phyllosoma cultured individually in a
still-water system Three phases (A, B and C) were
recog-nized Solid lines are drawn through the data points as
visual aids.
0
1
2
3
4
5
6
0 5 10 15 20 25 30 35
Body length (mm)
Phase A
Phase B
Phase C
Fig 5 Growth index of the increase in body length from
one instar (n) to the next (n + 1) for Panulirus japonicus
phyllosoma cultured individually in a still-water system Solid lines are drawn through the data points as visual aids.
1.0 1.1 1.2 1.3 1.4 1.5
Body length (BLn, mm)
Fig 6 Changes in intermolt period with development
of Panulirus japonicus phyllosoma cultured individually
in a still-water system Means ( 䊉) and ranges (vertical bars) of body length are shown.
0 5 10 15 20 25
Instar
Trang 5the shortest and longest phyllosoma life,
respec-tively, among the five larvae that reached the
puer-ulus stage Their growth rates were similar to each
other until approximately 180 days after hatching,
at which point the difference in BL between the
two larvae widened, resulting in a disparity of
3 months in larval life length
Mass culture
The phyllosoma cultured in groups suffered from a
disease that showed necrosis on the pereiopod and
antennule from 2 weeks after hatching; the disease
continued until the end of the culture Hence,
the survival rate decreased gradually to 45% at
100 days and 35% at 200 days after hatching
(Fig 8) In the mass culture, two phyllosoma
meta-morphosed to the puerulus stage
For the first to fifth instar larvae, WW and DW
increased linearly as the days after hatching
pro-gressed (Fig 9) The relationships between WW or
DW and days after hatching (D) can be expressed
by the following linear equations:
WW = 0.0213D + 0.1310 (R2= 0.9861)
DW = 0.0076D + 0.0259 (R2= 0.9807) From the data throughout the entire phyllosoma
phase, WW and DW increased exponentially as BL increased (Fig 10) The relationships between WW
or DW and BL were expressed as the exponential
equations below:
WW = 0.0686BL2.2023 (R2= 0.9966)
DW = 0.0209BL2.1905 (R2= 0.9946)
The moisture content of WW (%), calculated from the equation (WW − DW)/WW × 100, was
approxi-Fig 7 Growth of the two larval Panulirus japonicus
with the shortest (black line) and the longest (gray line)
larval life among the five larvae that reached the
pueru-lus stage.
0
5
10
15
20
25
30
35
1 50 100 150 200 250 300
Days after hatching
Metamorphoses to the puerulus
stage
Body length of the puerulus stage
Fig 8 Survival of Panulirus japonicus phyllosoma in
the mass culture for obtaining samples for
measure-ments of body weight.
0
20
40
60
80
100
Days after hatching
Fig 9 Relationships between days after hatching and (䊊) dry (DW), and (䊉) wet body weight (WW) for
Panu-lirus japonicus phyllosoma from the first to fifth instar
under laboratory conditions.
DW = 0.0076D + 0.0259
R2 = 0.9807
WW = 0.0213D + 0.1310
R2 = 0.9861
0.0 0.2 0.4 0.6 0.8 1.0 1.2
Days after hatching (D)
1st instar 2nd instar 3rd instar 4th instar
5th instar
Fig 10 Relationships between body length and ( 䊊) dry
(DW), and ( 䊉) wet body weight (WW) throughout the entire phyllosomal life of Panulirus japonicus under
laboratory conditions.
0 20 40 60 80 100 120
Body length (BL; mm)
Trang 6mately 80% for newly hatched larvae It decreased
to 63–70% at approximately 5 mm BL and then
increased to 68–75% at approximately 10 mm BL.
Thereafter, it was constant at approximately 70%
DISCUSSION
Several reports on the length of the phyllosoma
lifetime of P japonicus grown in the laboratory
have been published Yamakawa et al.15
success-fully obtained a puerulus from about 1000 newly
hatched larvae and reported that the puerulus had
a phyllosoma stage that lasted 307 days (24–26°C)
Kittaka and Kimura16 also noted that two
phyllo-soma metamorphosed to the puerulus stage 340
and 391 days after hatching (24–28°C) Sekine
et al.11 produced 325 pueruli in the laboratory for
several years and reported that the phyllosoma
319.4 days) (24–27°C) In the individual culture
of the present study, five larvae reached the
puer-ulus stage 245–326 days after hatching (mean
289.0 days), with shorter phyllosoma phases than
those reported in other studies This is probably
related to the high survival rate in the present
study The survival rate from hatch to puerulus
stage was 50% in the present study, while other
studies reported survival rates under 10% The high
survival rate in this study indicates that the
indi-vidual culture using small glass cups provided
better conditions for the larvae than other studies
did, resulting in their fast growth
P japonicus phyllosoma reached the puerulus
stage after 22–29 molts (n = 5, mean 26.2 molts) in
the present study The variability in the number of
instars as well as in the length of larval life was
caused mainly by differences in molt increment
and BL of the final instar between individuals The
animal that possessed the smallest number of
phyllosoma instars (22) gained a 4.95-mm molt
increment at 23.50 mm BL, and it metamorphosed
to the puerulus stage from the final phyllosoma
instar of 28.45 mm BL by the next molt The animal
that had the greatest number of phyllosoma instars
(29) had molt increments of 2.40 and 2.60 mm
at BL of 25.50 and 27.90 mm, respectively, and
reached the puerulus stage from the final
phyllo-soma instar of 33.05 mm The fact that all larvae
were cultured individually with the same methods
suggested that these variabilities were induced by
the growth potential of each larva Once a larva
showed a small molt increment, in many cases it
grew with small molt increments for several
subse-quent instars Although it is not clear what
deter-mines the growth potential, variation in the length
of life and the number of instars could be
charac-teristic of P japonicus phyllosoma This finding is
indicated by the fact that wild pueruli can be col-lected on the coast of Japan from April to Decem-ber, which is a long period compared with the relatively short period (from July to August) when hatching occurs.17,18
During the entire phyllosoma life of P japonicus,
some changes in the behavior and optimal growth
conditions have been recognized Matsuda et al.12
observed diel timing of molting in P japonicus
phyllosoma and reported that the timing of molt-ing varied with individuals until around 1 month
after hatch (4–5 mm BL), at which point individual
differences became small and all larvae molted synchronously around dawn The larval response
to light changes at approximately 4–5 mm BL.
Newly hatched larvae display strong positive pho-totaxis, which gradually disappears with
develop-ment, and beyond 4–5 mm BL they generally show
negative phototaxis.19 The rearing temperature for
high survival and rapid growth of P japonicus
phyl-losoma shifted from 26 to 24°C at 15 mm BL.9
Moreover, molt death occurs more frequently in
larger (BL > ∼18 mm) than in smaller larvae.20
These changes occur at around 4–5 and 15–18 mm
BL In the present study, the trend in the
relation-ship between BL before a molt and the increment
in BL by the next molt changed at approximately 5 and 15 mm BL, and the trend in the growth index of increase in BL from one instar to the next also altered at approximately 15 mm BL, indicating that
the boundaries at 5 and 15 mm appear to have
bio-logical implications for P japonicus phyllosoma.
Accordingly, the three phases (partitioned at 5 and
15 mm BL) can be defined as early, middle and late
phases in the long phyllosoma life Optimal culture conditions should be determined for each stage Body weight has been measured for larvae of several decapod crustaceans.14,21–23 Larvae of many decapod crustaceans have unique and complex body forms, and the body forms change drastically with molts, which make it impossible to measure
BL throughout the entire larval stage in the same
way Therefore, the increase of BL was generally examined not in relation to changes in BL but
instead with increasing days after hatch or number
of instars The length of phyllosoma life of
P japonicus, however, was extremely long (245–
326 days in the present study), and the number of instars was large (22–29) Also, differences between individuals were large These reasons prevented expressing the relationship between body weight and days after hatching or number of instar, except for the early stages Unlike other decapod crusta-ceans, the body form of the phyllosoma underwent
no change throughout the entire phase, and BL
increased linearly with molts (Fig 3), suggesting
Trang 7that it is appropriate to express increases in body
weight in relation to BL The relationships between
dry/wet body weights and BL could be well
described using exponential equations with high
coefficients of determination (R2 = 0.9966 and
0.9946, respectively) The exponential equations
have exponents of about 2 (2.2023 and 2.1905,
respectively), indicating that phyllosoma growth is
directed not toward an increase in body volume
but toward spreading the surface of their
dorsoven-trally flattened body The flattened body form of
the phyllosoma is considered to be advantageous
for passive horizontal transport with currents.24 It
is likely that a body surface that increases with
development is greatly beneficial for oceanic larval
dispersal
ACKNOWLEDGMENTS
We thank Professor M Tanaka, Kyoto University,
and Associate Professor T Yamakawa, University of
Tokyo, for helpful and constructive criticism
dur-ing the preparation of our original manuscript
Thanks to the staff of our laboratory for help during
larval culture This research was financially
sup-ported in part by a grant from the Ministry of
Agriculture, Forestry and Fisheries of Japan
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