Effect of Hydrostatic Pressure on the Egg Development of Marine Calanoid
Copepods
海産カラナス目カイアシ類の卵発生に与える圧力の影響
05D5504 吉木
朝子
指導教員
戸田
龍樹
SYNOPSIS
動物プランクトンの分布深度と卵の圧力耐性との関係を調べるために、生活史や産卵深度また、分布深度が異なるカイア
シ類 3 属 5 種の Acartia steueri, Calanus sinicus, Neocalanus cristatus, N. flemingeri, N. plumchrus の卵発生に与える圧力の影響
を調べた。まず実験室で圧力環境をつくるため、(1)プランクトンが現場で経験するような、緩やかな圧力上昇が可能で
あり、(2) 圧力容器の内部に 2 重構造を有することで、海水による圧力容器の腐食、また金属による生物への影響をなく
し、(3)動物プランクトンの卵から成体までの幅広いサイズ(100-10000μm)の試料を観察することができる、圧力装置を開
発した。この装置を用い、異なる圧力、水温条件下においてカイアシ類の卵発生速度、卵の孵化率を調べ、卵発生に与え
る圧力の影響を見た。また、圧力の変化が卵発生に及ぼす影響を見るために、圧力上昇速度を急激に上昇させた場合と、
緩やかに上昇させた場合における孵化率、奇形率の違いを観察した。卵発生速度はすべての種において、温度の変化に依
存し、圧力条件間における違いは見られなかった。孵化率においては、A. steueri では 1atm と 10atm で有意差が見られな
かったのに対し、C. sinicus の卵は圧力条件の上昇と共に孵化率が低下する様子が観察された。また C. sinicus では、10atm
まで圧力を徐々に上昇させた場合、急激に上昇させた場合よりも、孵化率が顕著に低い結果となり、緩やかな圧力変化で
よりダメージを受けやすいことが明らかとなった。Neocalanus 属 3 種の卵の孵化率は、全ての実験条件において 1atm
における孵化率と有意な差はなく、1atm から 100atm までの広い範囲の圧力に耐性を持っていることが明らかとなった。
孵化した幼生において、A. steueri と Neocalanus 属では奇形個体は観察されなかったのに対して、C. sinicus では奇形個体
が観察された。Neocalanus 属と C. sinicus はどちらも生活史を通して、表層から深度 1000m 以深までの生活史に伴う鉛直移動
OVM を行っているが、卵の圧力耐性は異なった。Neocalanus 属カイアシ類は深層 1000m 以深で産卵された後に、孵化したノープ
リウスは表層まで上昇するため、初期生活史において 0～1000m の幅広い圧力範囲を経験する。それに対して、 C. sinicus の卵は
表層付近で産卵され沈降し、深度 100m よりも浅い深度で孵化し、ノープリウス幼生は表層へと上昇する。そのため、C. sinicus の
卵からノープリウス幼生の時期は 0m から深くても 100m までの圧力しか経験しない。このような Neocalanus と C. sinicus の初期生
活史において経験する圧力範囲の違いが、卵の圧力耐性と関係があると考えられた。
Key words: hydrostatic pressure, egg development, egg hatching success, vertical distribution, Acartia steueri,
Calanus sinicus, Neocalanus cristatus, N. plumchrus, N. flemingeri
relation to spawning in zooplankton. (1) Adult is usually
distributed and spawns at the surface layer. Spawned eggs
sink and hatch in the water column. Hatched nauplii ascend
to feed and develop at the surface. Nauplii grow to copepodid
V stage (CV) near the surface layer. CV copepods descend to
deeper depth and molt to adult. Adult copepods return to the
surface layer to feed and reproduce. (2) Adult is distributed at
deep layers, and they spawn at the depth. Spawned eggs float
and hatched nauplii ascend to the surface. They develop to
copepodid V stage at the surface and descend to the depth
again. Thus, copepods experience wide pressure range from
surface to deep by undergoing vertical migration, and they
reproduce eggs at specific depth range by according to
species.
Eggs spawned into the water column float at certain
depth or sink to deeper waters. These eggs experience
cleavage and environmental changes during sinking and/or
ascending. In the cleavage process, different blastomere
stages appear and they are subsequently exposed to different
environmental factors such as variation in temperature and
pressure in the water column. Egg development time of krill
was examined in various pressure increasing conditions. The
cleavage process was accelerated under moderate pressure of
1 to 5-20 atm than at 1 atm (George 1984). On the other hand,
abruptly increasing pressure from 1 to 40-60 atm inhibited
the cleavage process of krill eggs (George and Stromberg
1985). The pressurizing rate, abruptly or gradually, is
considered to have some influence on the development of
embryos. Also, the sensitivity of embryos to environmental
factors may be variable among blastomere stages.
The hydrostatic pressure appears to be an important
Introduction
Copepods are one of the most prime important animals in
marine ecosystems because of their large biomass and trophic
importance as prey organisms of other animals such as fishes
and whales at higher trophic levels. In order to understand
population dynamics and biomass of copepods in nature, it is
necessary to investigate recruitment rate of copepods,
Recruitment of copepod is determined by the egg production
rate, egg hatching success and egg mortality rate. On the
recruitment process, spawned eggs sink or ascend, and thus
these eggs are exposed to variation in temperature and pressure.
Although effect of temperature on egg hatching success and/or
egg mortality has been studied quite a lot, pressure effect on
eggs almost has never been considered in the recruitment.
Because there are not many pressure apparatus which can
mimic in situ pressure environment for zooplankton. A system
producing in situ pressure conditions is essential for examining
the effect of pressure on eggs and recruitment rate.
Copepods occur from surface to sea bottom and they are
exposed to various pressures throughout their lifetime. Most of
copepods undergo diel vertical migration (DVM) and/or
ontogenetic vertical migration (OVM). DVM is characteristic
for later stages of copepod, probably for females (Conover
1988). Many calanoid copepods usually spawn eggs at night
time when they migrate to the surface (e.g. Uye et al. 1990,
Pagano et al. 2004). OVM is called as seasonal vertical
migration, because large migration of zooplankton is related to
boreal- or arctic- area where seasonal variations in temperature
and phytoplankton production. Zooplankton has been evolved
OVM as a survival strategy to get over seasonal environmental
variations (Sekiguchi 1975). There are two patterns of OVM in
1
different areas from shallow coastal waters to open waters of
West Pacific Ocean near Japan. These species differ in body
size, habitat depth range, life cycle and spawning depth. A.
steueri is dominant in near shore waters such as embayment
(c.a. 5 ~ 20 m depth). C. sinicus and Neocalanus copepods
are distributed from surface layer to deeper layer than 1000
m. All five species undergo DVM. C. sinicus and Neocalanus
copepods also migrate ontogenetically from surface to deeper
than 1000m. C. sinicus belong to OVM pattern (1) mentioned
above. While Neocalanus copepods spawn at the depth, and
thus these species belongs to OVM pattern (2).
factor in egg development of marine animals, whereas pressure
effect on eggs has not been understood well yet. The purposes
of the present study were firstly (1) to develop new hydrostatic
pressure apparatus which can increase pressure gradually for
marine zooplankton egg study, (2) to select species which have
different habitat and spawning depth range and to examine the
effects of hydrostatic pressure and also pressurizing rate on
eggs of selected zooplankton, and (3) to examine the effects of
hydrostatic pressure between different egg development stages
which change during egg sinking and/or ascending in the water
column.
Materials and Methods
Study 1. Development of hydrostatic pressure apparatus
The hydrostatic pressure apparatus developed in the
present study can function at pressures of up to 200 atm and at
temperatures ranging from -20 to 80 oC (Yoshiki et al. 2006).
The pressure apparatus system is composed of a plunger pump,
a pressure gauge, a pressure chamber, a specimen holder which
is placed in the pressure chamber, a temperature-controlled bath,
a CCD camera, a monitor and a video-recorder.
Depth (m)
0
ri
Whole life cycle
Female distribution
Spawning depth
500
1500
(Modified from Shimode 2003; Kobari and Ikeda 1999, 2000 a,b)
Fig. 2. Distribution ranges of Acartia steueri, Calanus sinicus,
Neocalanus spp. in Sagami Bay and Oyashio region. Spawning is
conducted at female distributed depth.
Pressure gauge
◆Sampling
Acartia steueri
Acartia steueri were collected in Manazuru Port (35◦
09’49” N, 139◦ 10’ 33” E; depth, 5.5 m) where is located at
near the north coast of Sagami Bay, Japan in November,
December 2006, April, November 2007. Samplings were
carried out by oblique tows using a 180 μm mesh net from
the bottom to the surface. Adult females were individually
incubated in a 10 ml beaker filled with a suspension of
105-106 cells mL-1 of Thalassiosira weissflogii mixed with
Isochrysis galbana as food at 15.0 ◦C. Eggs spawned by the
females were collected and used for pressure experiments.
Calanus sinicus
Adult females of C. sinicus were collected on board R.
V. “Tachi-bana” of Yokohama National University, at a fixed
location off the coast of the Manazuru Peninsula
(35°09’55”N; 139°10’78”E, depth 300m), Sagami Bay, Japan.
Samplings were carried out from June to November 2004. C.
sinicus was obtained by gently towing a vertical zooplankton
net with 330 μm mesh opening fitted with a 2 L cod-end from
depth of 200 m to surface. Females of C. sinicus sorted with
a wide-mouth pipette and individually placed in incubation
chambers filled with suspension of 105-106 cells mL-1 of
Thalassiosira weissflogii to obtain fresh eggs. The beakers
were placed at 14 °C in temperature controlled incubators.
Eggs were checked every morning and harvested for pressure
experiments.
Neocalanus copepods
Adult females of N. cristatis, N. flemingeri, N.
plumchrus were collected on board the R. V. "Hokko-maru"
of Hokkaido National Fisheries Research Institute, in the
Oyashio region, Western Subarctic Pacific, in January 2006
and January 2007. Sampling was conducted by vertical hauls
of a ring net with 330 μm mesh opening fitted with a 2 L
cod-end from depth of 1500 m to surface. Females of
Neocalanus copepods were sorted and transferred in to 1-L or
250ml bottles filled with filtered seawater. Bottles were kept
Pressure chamber
Distilled water
CCD
Light
Specimen
holder
Monitor & video recorder
icus . cristatus . plumchrus . fleminge
N
N
C. sin
N
1000
Plunger pump
camera
eri
A. steu
Temperature
Fig. 1 Structure of hydrostatic controlled
pressurebath
apparatus.
Hydrostatic pressure in the chamber is controlled by
sending distilled water into the chamber with a plunger pump
(AC-L9, Shimadzu). The increasing velocity of pressure in the
chamber is controlled by the flow rate of distilled water from
the plunger pump (0.001 – 9.999 ml/min). The flow rate is
adjusted so as to maintain a constant increase in pressure. The
pressure chamber enables us to control the pressure increasing
rate by adjusting a plunger pump which changes the flow rate
of water into the pressure chamber. The pressure inside the
pressure chamber is monitored by a pressure gauge (1~200 atm
pressure range). A CCD camera (STC-R640, Sensor
Technology Corporation) is placed in front of an observation
window of the chamber and a light source is placed at the
opposite side of the chamber. Specimen of image is displayed
on a monitor and recorded by a video-recorder connected to the
CCD camera.
Study 2. Effects of hydrostatic pressure on copepods eggs
◆Selected species
In the present study, clanoid copepods: Acartia steueri,
Calanus sinicus, Neocalanus cristatis, N. plumchrus and N.
flemingeri were selected. These species are the dominant in
2
at 4 °C in cold room and incubator. Bottles were checked every
morning and spawned eggs were used to pressure experiments.
◆Pressure experiments
For A. steueri, experiments were conducted at two
pressure conditions, 1 and 10 atm and five temperatures, 4, 10,
15, 20 and 25 °C, for A. steueri. At 10 atm, two pressurizing
patterns, abruptly (A) at 10 atm/min (10atm(A)) and gradually
(G) at 0.1 atm/min to 10 atm (10 atm(G)), were used. In the
gradual increasing experiment, the pressure was increased in a
stepwise fashion during the first 100 min of the experiment and
then maintained at constant during the remainder of the
experiment.
For C. sinicus, experiment was conducted at four
pressures, 1, 10, 50 and 100 atm, and six temperatures, 5, 8, 10,
15, 20 and 25 °C. At 10 atm, two pressurizing patterns, 10 atm
(A) and 10 atm (G), were used as mentioned above.
For Neocalanus copepods, experiments were conducted
at four pressures, 1, 10, 50 and 100 atm at 4 °C. At 10, 50 and
100 atm, two pressurizing patterns, abruptly and gradually,
were used.
Egg development times, egg hatching successes and
deformation frequencies at each condition were examined.
same as that at 1 atm between different pressure conditions
and pressurizing rates, at all temperature conditions. These
egg development times were changed depending on
temperature which has been thought the most important
factor for the egg development time. On the basis of the
difference of the life histories and the spawning depths of
examined copepods, probably it is thought that the egg
development time of calanoid copepods is not influenced by
the hydrostatic pressure. Egg development time of Euphausia
superba is changed by pressure increasing rates (George
1984, George and Stromberg 1985). To clarify the difference
related to taxa, copepods and krill, examining the response of
egg development to hydrostatic pressure between copepods
and E. superba in detail is needed.
Egg hatching successes of A. steueri and C. sinicus at
1 atm were generally higher than 70 and 80 % at all
temperature conditions, respectively (Fig. 3). At 10 atm (A;
abruptly pressure increasing) condition, egg hatching
successes of A. steueri and C. sinicus were not significantly
different to 1 atm. Egg hatching success of A. steueri at 10
atm (G; gradually pressure increasing) condition was not
significantly different to 1 atm. On the other hand, at 10 atm
(G) condition, egg hatching successes of C. sinicus were
significantly lower than 1 atm at all temperature conditions.
Although A. steueri habitat is shallow coastal waters where
depth is around 5-20 m, egg hatching successes were high
even at 10 atm (G) which is as high as pressure of 100 m
depth.
Study 3. Hydrostatic pressure effect on egg development
stages of C. sinicus
Effects of pressure between blastomere stages were
examined on C. sinicus eggs. Sampling method was same as
Study 2. Samplings were conducted on December 2006, April,
May, June and July 2007. The 1-, 2-, 4-, 8-, 16-32 cell, blastula
and limb-bud stages were collected respectively. The eggs were
divided to two aliquots. One aliquot was used in the pressure
experiment and the other was used in the control experiment
where pressure was kept constant at 1 atm. Two pressure
increasing conditions were experimented; abruptly at 10
atm/min (10 atm (A)) and gradually at 0.1 atm/min (10 atm
(G)) to a final pressure of 10 atm.
Hatching success (%)
100
80
40
20
0
Hatching success (%)
100
Results and Discussions
Study 1. Development of hydrostatic pressure apparatus
The hydrostatic pressure apparatus developed in the
present study makes it possible to (1) increase pressure
gradually, (2) prevent corrosion of the pressure chamber by
introducing a specimen holder that separates the pressure
chamber from the surrounding seawater, and (3) observe a wise
size range of zooplankton including eggs (100-10000μm size
range). The present apparatus has made it possible for
examining the effects of hydrostatic pressure on zooplankton,
for instance egg development time and larval behavior by the
response to pressure change.
Hand pump has been previously adopted by most
hydrostatic pressure apparatuses to increase pressure, but this
method exposes zooplankton to abrupt increase in pressure. In
the present study, the rate of pressure increased in the chamber
is controlled by adjusting the flow rate of distilled water from
the plunger pump. This was possible to increase pressure
gradually (0.1 atm/min) and also simulate the pressure in the
field such as that observed when zooplankton migrates
vertically (from 1 to 150 atm).
A. steueri
60
4
10
15
20
25
80
60
1 atm
10 atm (A)
10 atm (G)
50 atm (G)
100 atm (G)
C. sinicus
40
20
0
000
5
00
8
10
15
20
Temperature (ºC)
25
Fig. 3. Egg hatching successes of A. steueri and C. sinicus. A: abrupt
increasing, G; gradual increasing pressure conditions. 0; Hatching
was not observed.
Egg hatching success of C. sinicus decreased with
gradual increasing pressure condition, while that of
Neocalanus copepods have tolerate to wide pressure range
from 1 atm to 100 atm. Both species are exposed to wide
depth range throughout their life time by undergoing OVM.
However spawning depths are different between C. sinicus
and Neocalanus copepods. Adult females of C. sinicus are
distributed from 0 to around 150 m depth and spawn at
surface layer. Spawned eggs sink and hatch around 100 m
depth. Hatched nauplii ascend to surface. Thus, eggs and
nauplii are exposed to narrow pressure range from 1 atm to
10 atm at the most. On the other hand, females of genus
Neocalanus are distributed from 250 m to 2000 m depth and
spawn eggs at the depth. The spawned eggs float at the depth
Study 2. Effects of hydrostatic pressure on copepods eggs
Egg development times of all examined species were
3
or ascend slowly. Also, hatched nauplii ascend to the surface.
Thus, eggs and nauplii of Neocalanus copepod exposed to large
pressure ranges from spawned deeper depth to surface at early
larval stage. The exposing pressure range of their early life
stage of egg and nauplii might be related to the difference of
pressure tolerance between C. sinicus and Neocalanus
copepods.
Deformation of A. steueri and genus Neocalanus were
not found. However, deformed nauplii hatched under pressure
conditions were observed only on C. sinicus.
northern limit of C. sinicus distribution is the Oyashio front
that an average temperature of annual maximum at 20-30m
of its spawning depth during year 1990-1998 is 13.0±1.0 °C
(Kidachi 1979; Hulsemann 1994; Saito et al. 2002). The egg
development time of C. sinicus at 13 °C is estimated at 1.2
days. Considering the egg sinking rate of 68 m/d, these eggs
will sink to around 100 m depth in 1.2 days. At this depth, the
hydrostatic pressure will cause increasing in the egg hatching
success rate of C. sinicus. Hydrostatic pressure, coupled with
low temperatures in this region, may limit the recruitment of
C. sinicus in the Oyashio region (Yoshiki et al. 2006). In
other words, northern limit of C. sinicus distribution may be
determined by combination of egg sinking, embryonic
development rates and tolerance to hydrostatic pressure.
Study 3. Hydrostatic pressure effect on egg development
stages of C. sinicus
Effects of high pressures on different cleavage stages of
C. sinicus which appears deformation were examined.
Deformity frequencies were from 0 to 54 % between 1-cell and
limb-bud stages at abrupt pressure increasing condition (10
atm/min), and from 8.3 to 91 % at gradual pressurizing
condition (0.1 atm/min) (Fig. 3) (Yoshiki et al. 2008). The
deformity frequency between pressurizing rates on each
blastomere stage was significantly different at the blastula stage
(p < 0.05), while there were no differences between the other
stages. In the result of 2-way ANOVA of deformity frequencies,
there were significant differences among pressurizing
conditions (p < 0.05) and blastomere stages (p < 0.01), though
there was no interaction between them (Fig. 3). This indicates
that the eggs of Calanus sinicus were damaged more easily by
gradual increasing than abrupt increasing pressure. Moreover,
the sensitivity of the blastomere stage increased progressively
with cleavage development, and the blastula stage was the most
delicate in the early phases of egg development.
Control
Deformity frequency
frequency (%)
(%)
Deformity
10 atm A
Implications
Egg of Acartia steueri can tolerate to 10 atm while that
habitat is very shallow coastal area. Why eggs of A. steueri
could tolerate to 10 atm pressure? All examined species are
classified to Calanoida copepods which are supposed to be a
monophyletic group (Park 1986). During the hundreds of
millions of years spanning the evolutionary history of the
subclass Copepoda (Huys and Boxshall 1991), the habitat
shift from benthos to plankton performed by the Calanoida.
This can be viewed as the major and probably earliest
attempt by copepod to colonize the pelagic biome (Ohtsuka
and Huys 2001). On the basis of morphological characters,
Andronov (1974) divide the Calanoida into nine
superfamilies and proposed phylogenetic relations of them.
Calanoids are highly adapted to the planktonic mode of life
and dominate zooplankton communities, though members of
the most primitive families have retained their close
association with the sediment-water interface, the ancestral
hyperbenthic habitat where most copepod orders probably
originated (Huys and Boxshall 1991; Ohtsuka and Huys
2001). That is to say, primitive families of Calanoida
copepods are supposed to tolerate benthic environment which
is including high pressure environment, and to lose the
pressure tolerance after these got into planktonic habitats.
Genus Acartia is classified to primitive group. The pressure
tolerance of A. steueri eggs appears to be related to the
evolutionary history of Calanoida copepods. The relationship
between pressure tolerance of copepods and lineage is clearly
an area that warrants significant research in future.
10 atm
10atm G
100
80
60
40
20
0
00 00
0
0
1
4
8
2
0
16
Blastomere
Blastomere stage
stage
B
0
L
0
0
1
0
2
4
0
0
8
16
0
B
0
L
Blastomere
Blastomere stage
stage
Fig. 4. Deformation frequency between blastomere
stages of Calanus sinicus. A; abrupt pressure increasing, G; gradual
pressure increasing. B; Blastula, L; Limb-bud stage.
The embryonic sensitivity of C. sinicus to hydrostatic
pressure increased with egg development, and eggs were more
fragile to gradual pressure change than abrupt increasing
pressure. This means that egg sinking is supposed to be
detrimental effect for C. sinicus. At higher pressure conditions
at 10, 50 and 100 atm, highly significant decreases in egg
hatching successes of C. sinicus occurred as pressure increased.
These eggs did not have tolerance to even 10 atm, this suggests
that eggs would not survive if they did not hatch within the
upper 100m. Upward spawning behavior of females of C.
sinicus at night in the DVM process has been known. By the
female’s upward behavior, eggs are spawned at lower pressure
and warmer temperature environments in the surface. Harmless
low pressure and warm temperature lead eggs to hatch early
and to recruit.
Egg hatching success rate of C. sinicus decreased with
increasing hydrostatic pressure. This implicated that the egg
pressure tolerance is related to distribution of C. sinicus. The
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