Hatching controlled by the circatidal clock, and the roleof the medulla terminalis in the optic peduncle of the eyestalk, in an estuarine crab Sesarma haematocheir
Laboratory of Behavior and Evolution, Graduate School of Natural Science and Technology, Okayama University, Tsushima 3-1-1, Okayama 700-8530, Japan
(e-mail: saigusa{at}ccmail.cc.okayama-u.ac.jp)
Accepted 12 August 2002
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Summary |
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Key words: circatidal pacemaker, estuarine crab, gentle-release behavior, hatching synchrony, medulla terminalis, optic peduncle, eyestalk, neuronal pathway, vigorous-release behavior, Sesarma haematocheir
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Introduction |
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Aquatic crustaceans also exhibit circadian rhythms in a variety of
behavioral and physiological events; e.g. hormone level
(Keller, 1981), serotonin
content (Castañón-Cervantes
et al., 1999
), retinal structure
(Arikawa et al., 1987
),
electroretinogram (ERG) amplitude
(Aréchiga and Wiersma,
1969
; Larimer and Smith,
1980
; Barlow, 1983
;
Aréchiga et al., 1993
)
and locomotor activity (Fanjul-Moles et
al., 1996
). In the horseshoe crab Limulus polyphemus,
many physiological events are modulated by retinal function. These events in
the retina are generated by the efferent input from a circadian pacemaker
located in the brain (Barlow et al.,
1977
,
1987
). In the locomotor
activity of crayfish Procambarus clarkii, the circadian pacemaker may
be located in the supraesophageal ganglion
(Page and Larimer, 1975
).
However, in the ERG circadian rhythm, the pacemaker is suggested to be present
in the eyestalkprotocerebrum complex
(Barrera-Mera and Block,
1990
).
Marine crustaceans also show rhythmic activity patterns in locomotion,
swimming and reproduction. However, the tidal cycle, as well as the day/night
cycle, affects the timing of the activity in the marine environment.
Accordingly, the activity of marine crustaceans is often synchronized not only
with the day/night cycle but also, more or less, with the tidal cycle and
demonstrates complex patterns (e.g. Saigusa,
1981,
2001
;
Saigusa et al., 2002
). Much
work on the timing systems has been conducted on the locomotor activity of
intertidal and estuarine crabs (see
Palmer, 1995
). However, very
few specimens show well-demarcated rhythmic patterns in constant conditions in
the laboratory (e.g. Honegger,
1976
). The lack of clear endogenous rhythmicity in these crabs has
interrupted elaborate experimental studies in the laboratory. Most
experimental results obtained from a number of crab locomotor activities were
not reproducible. Therefore, hypotheses proposed on the timing systems of the
circatidal rhythms are not acceptable. For example, the circatidal rhythms
have been explained in terms of an interaction between circadian and
circatidal clocks (see Palmer,
1995
). However, no clear evidence supports this hypothesis (see
Saigusa, 1986
,
1988
). Information on the
anatomical location of the pacemaker controlling circatidal rhythms is also
conspicuously lacking.
Well-demarcated synchrony with the tidal cycle has been observed in the
larval-release activity of a number of intertidal and estuarine crabs
(Saigusa, 1981,
1982
;
Christy, 1986
;
Paula, 1989
;
Queiroga et al., 1994
). In
contrast to the locomotor activity, a free-running tidal rhythm of the
larval-release activity is very clear in constant darkness or in 24 h
light:dark (L:D) cycles in the laboratory (Saigusa,
1986
,
1992a
;
Saigusa and Kawagoye, 1997
).
This suggests that the larval-release activity is clearly controlled by an
internal clock (circatidal clock) or a pacemaker (circatidal pacemaker). The
larval-release rhythm is well suited for experimental analysis of the timing
system in marine organisms.
In an estuarine terrestrial crab Sesarma haematocheir, each female
incubates 20 000-50 000 embryos in her incubation chamber. Hatching occurs
prior to the larval release; all of the embryos hatch within 1 h in the
female's incubation chamber. Hatched zoea larvae are immediately liberated
into the water by the vigorous fanning behavior of the abdomen, which lasts
for only 4-5 s (Saigusa,
1982). The circatidal system of hatching in S.
haematocheir is characterized by (1) highly synchronous hatching of
embryos attached to the female and (2) synchrony of hatching with nocturnal
high tide. These events are controlled by the circatidal clock of the female
(Saigusa, 1992b
,
1993
).
The present study investigated whether the circatidal pacemaker controlling
hatching and hatching synchrony is present in the optic peduncle of the female
eyestalk and, if so, where it is located within the optic peduncle. Ablation
of both eyestalks can easily answer this question (e.g.
Page and Larimer, 1975).
However, ablation results in serious damage to the animals, especially in
terrestrial crabs, which may affect the interpretation of the results.
Furthermore, the optic peduncle of the eyestalk contains the X organ and the
sinus gland, i.e. the major neuroendocrine system in crustaceans, and the
expectation is that the tidal rhythm is generated through the neuroendocrine
system. In view of this possibility, it is important to maintain at least
blood circulation after surgery. In the present study, surgery on the optic
peduncle was performed either from an incision at the tip of the eyestalk,
i.e. to remove the region of the optic peduncle from the compound
eyeretina complex to the medulla interna (MI), or from a triangle
`window' opened on the eyestalk exoskeleton, i.e. to create lesions on the
medulla terminalis (MT). This study indicates that synchronous hatching among
embryos and synchrony with the tide are lost when the ventral half of the MT
is damaged.
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Materials and methods |
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The 24 h L:D cycle is critical for maintaining the phase of the circatidal
rhythm of S. haematocheir (see Saigusa,
1986,
1992a
). In the field, the time
of sunset shifts from 19:20 h to 18:15 h, and that of sunrise shifts from
05:00 h to 05:45 h, from early July to mid-September, respectively. So, the
photoperiod in the field is 14.3 h:9.7 h L:D in early July and 12.5 h:11.5 h
L:D in mid-September. In the laboratory, we employed similar photoperiods and
phases to those observed in the field, i.e. a 15 h:9 h L:D cycle (light off at
20:00 h and on at 05:00 h) or a 14 h:10 h L:D cycle (light off at 19:00 h and
on at 05:00 h). The intensity of illumination on the floor was 700-1200 lux in
the light phase and <0.05 lux in the dark phase. Temperature was constant
at 24±1°C. In these conditions, the larval-release activity of the
population clearly shows the free-running tidal rhythm, the phase of which
roughly coincides with the time of nocturnal high tide in the field for at
least one month after collection.
Monitoring hatching and assessment of hatching synchrony
Hatching of the embryos (embryos attached to the female and detached
embryos) was monitored by either of the following two methods.
Water-exchange method
Ovigerous females were individually placed in perforated plastic cages (7
cmx14 cm, width x height), with small holes in the sides. Each
cage was then suspended by a fine wire from the rim of a 11 glass beaker
containing 400 ml of diluted, clean seawater (salinity 10)
(Fig. 1A). Only the bottom of
the plastic cage was immersed in the water. The water was strongly aerated
with an air stone. At intervals of 30 min, the plastic cage was transferred to
another beaker containing the same quantity of diluted (10
) seawater.
Exchange of the beaker was carried out under a red light in the dark phase of
the L:D cycle (Saigusa, 1993
).
The original beaker was removed from the experimental room, and the number of
hatched zoeas was counted with the aid of a pipette. This method detected the
temporal distribution of the number of zoeas hatched. Moreover, if the embryos
attached to the female hatch synchronously, zoeas are released by the vigorous
fanning behavior of the abdomen, which lasts for 4-5 s (`vigorous-release
behavior'). As the water was exchanged every 30 min, it was easy to judge when
this behavior occurred.
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Photoelectric-switch method
Ovigerous females were individually placed in a perforated plastic cage
(the same cage as shown in Fig.
1A). The cage was suspended from the rim of a glass beaker
containing 400ml of diluted (10
) seawater, and the bottom of the
cage was immersed in the water (to approximately 1
cm depth). The beaker
was set in the recording apparatus used to detect larval release, which
consisted of a sensor unit (infrared source and receiver; E3S-2E4, Omron Co.
Ltd, Japan) placed on both sides of the beaker and a controlling unit
(S3S-A-10, Omron Co. Ltd, Japan). The infrared beam passed through under the
plastic cage. When the zoeas were released by the female, the beam was
partially interrupted by swimming zoeas, and this interruption was detected by
the controlling unit placed outside the experimental room. Furthermore, the
output of the controlling unit was monitored by an event recorded (R17-H12T,
Fuji Electric Co. Ltd., Japan) (for a figure of this apparatus, see
Saigusa, 1992a
). This method
was effective when hatching occurred synchronously and zoeas were liberated by
vigorous-release behavior. It was also less labor-intensive than the
water-exchange method. When hatching synchrony deteriorated and hatch time was
prolonged, zoeas were liberated by a gentle pumping movement of the abdomen
(`gentle-release behavior). Time of hatching associated with gentle-release
behavior was not accurately detected by the photoelectric-switch method,
because larval release often continued for hours. Thus, hatching associated
with gentle-release behavior was monitored by the water-exchange method.
Hatching of detached embryos
A female S. haematocheir has four pairs of abdominal appendages,
each of which bears one plumose seta and one ovigerous seta. Embryos are
attached to many ovigerous hairs arranged along the ovigerous seta
(Saigusa, 1994). One ovigerous
seta with its attached embryos was cut at its base, and bleeding was stopped
using a soldering iron. The embryo cluster was then tied by a cotton thread to
the nylon thread in the center of a small plastic, perforated container (8
cmx6 cm diameter x depth) with small holes in the sides. This
container was placed in a 500 ml glass beaker containing 200 ml of diluted sea
water (salinity 10
), and the water was strongly aerated with an air
stone (Fig. 1B). At intervals
of 30 min (or 1 h), the plastic cage was transferred to another beaker with
the same quantity of diluted sea water, and the number of hatched zoeas was
counted with the aid of a pipette. Exchange of the beaker was carried out
under a red light in the dark phase of the L:D cycle.
Discrimination of mature zoeas from immature zoeas
Hatching (breakage of the egg envelope) occurs just before releasing zoeas.
The egg case of S. haematocheir abruptly cracks, and the dorsal
thorax of the zoea appears (Saigusa and
Terajima, 2000). The zoea vigorously bends and stretches its body,
with strong vibration of the appendages. Via this process, the larva can shed
the sticky embryonic exuvia and is transformed to a mature zoea. In our
experiments, some of the mature zoeas swam, but others did not. These zoeas
were submerged at the bottom of the beaker. Furthermore, immature zoeas (the
larvae that were still folded by the embryonic exuviae) were sometimes
released. They could easily be discriminated from mature zoeas under a
stereomicroscope. In this study, zoeas were divided into three types:
`immature zoea', `mature zoea that cannot swim' and `swimming mature
zoea'.
Assessment of the degree of hatching synchrony
In the water-exchange method, the degree of hatching synchrony was assessed
using a `synchrony index' (SI). This index was defined as the maximum value
(%) of hatching divided by the `number' of bars arranged at intervals of 30
min that contain 47.5% above and below the median value, respectively. For
example, in Fig. 4A (top
panel), the maximum value is 87.7%, and the number of divisions containing 95%
hatching (enclosed by the broken line) is 2. So, SI is 43.9 (87.7 divided by
2).
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Surgery on the optic peduncle of both eyestalks
Eyestalk ablation
One of the eyestalks or both eyestalks were removed with a knife, and
bleeding was stopped with a small soldering iron. Hatching was monitored
either by the water-exchange method or the photoelectric-switch method.
Removal of the compound eyeretina complex, the medulla externa
(ME) and the medulla interna (MI)
Ovigerous females were individually buried in an appropriately sized
crushed-ice bed, with the head facing up so as to expose both eyestalks. A
knife blade (100 µm thick) was made by splitting the edge of a razor blade
and was attached to the knife holder (Handaya Co., Tokyo, Japan). A new blade
was used for each incision.
Fig. 2A shows a schematic representation of the optic peduncle of S. haematocheir. First, the cornea (Fig. 2B) was cut along the edge of the exoskeleton of the eyestalk and removed. Next, the compound eyeretina complex was carefully removed using a paper towel. The lamina ganglionalisME complex was visible after the removal of the compound eye. These ganglia were cut and removed under the stereomicroscope. Removal up to the MI was also carried out in the same way (see Table 3). After surgery, bleeding was stopped using a small soldering iron (Fig. 2C).
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Lesions of the medulla terminalis (MT)
Surgery towards the MT was difficult to perform from the tip of the
eyestalk, so a right-angled triangle was cut into the exoskeleton of both
eyestalks under the stereomicroscope (Fig.
2D). A cluster of neurosecretory cells
(Fig. 2F) was readily visible
as a white spot from the triangle `window'
(Fig. 2E). After determining
the position of this cluster of neurosecretory cells (i.e. white spot), it was
possible to locate the correct position to make an incision. The MT was cut
three different ways (Fig. 3):
cutting transversely from the dorsal part to the ventral part along the
hypotenuse (transverse cut), cutting the upper half of the MT perpendicular to
the optic peduncle (dorsal-half cut) and cutting the ventral half of the MT
perpendicular to the optic peduncle (ventral-half cut; see
Table 5). After each operation,
the triangular exoskeleton was returned to the original site, and the wound
was sealed using the soldering iron. Under these conditions, the optic
peduncle remained alive throughout the experiment (compare
Fig. 2G with
Fig. 2B).
In this study, surgery on the optic peduncle was performed while ovigerous females were individually buried in a crushed-ice bed. However, it is possible that contact with ice might have an effect on the phase of the circatidal rhythm. For the control experiment, 26 females were immersed in ice water for 10 min at various times on a given day, and the timing of hatching was compared with that of the control females that had not been immersed in ice water.
Assessment of the success of surgery on the MT
After the conclusion of each experiment, both eyestalks were separated
(Fig. 2G), the optic peduncle
was excised, with care being taken not to damage it, and the success of the
operation was assessed under the stereomicroscope
(Fig. 2H). Although detailed
histological studies may be required to specify the region of tissues of the
MT that was damaged by the surgery, inspection of the optic peduncle under the
stereomicroscope could roughly determine what region of the peduncle was cut
by the surgery.
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Results |
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The hatching of embryos attached to Female 2 (Fig. 4B, upper panel) peaked at 02:00-02:30 h (median 02:15 h) on 1 September (87.5%). Larvae were liberated by vigorous-release behavior. The remaining 12.5% appeared in the water at 02:30-03:00 h on 1 September. (A small quantity of zoeas often remains after vigorous-release behavior. Such zoeas are often liberated during a second episode of vigorous-release behavior.) An embryo cluster that had detached at 13:35 h on 31 August all hatched (middle panel), with hatching peaking at 03:45 h on 1 September (SI=7.0). An embryo cluster that had detached at 17:35 h on 30 August all hatched on 1 September, with all zoeas swimming (bottom panel). However, their hatching was delayed and peaked at 05:45 h on 1 September (SI=3.6). As shown in these two females, the embryos that detached at least 1 day before larval release all hatched and swam. In contrast, no embryo cluster that had detached more than 2 days before larval release hatched in aerated water (not shown).
Table 1 summarizes hatching synchrony of the embryos attached to the female and that of detached embryos. In attached embryos, the SI of all females was >25. Hatching was always associated with vigorous-release behavior. In contrast, hatching synchrony of detached embryos deteriorated. Deterioration increased for the embryos detached one day before larval release. The difference in SI between the embryos detached on the day of larval release and those detached one day before larval release was significant at the 1% level (t-test; P=0.0017, N=23).
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Synchrony of hatching with nocturnal high tides
Ovigerous females (73 specimens) were collected on 16 August 1988 and were
randomly separated into two groups. In one group (47 females), hatching of the
embryos attached to the female was monitored by the photoelectric-switch
method (Fig. 5A, open circles).
Hatching of all embryos occurred around the time of nocturnal high tide. The
mean shift (± S.D.) of hatching from nocturnal high tide was
0.91±0.61 h. Clear correlation of hatching with the nocturnal high tide
lasted for at least 3 weeks in non-tidal laboratory conditions.
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A second group (26 females) was immersed in ice water for 10 min at various times on 20 August. Zoeas were always released by vigorous-release behavior, so their hatching was monitored by the photoelectric-switch method (Fig. 5A, filled triangles). Hatching of these females also coincided with the nocturnal high tide. The mean shift (± S.D.) of the time of release from the nocturnal high tide was 1.05±0.83 h, indicating that the timing of hatching was not disturbed by ice water.
Ablation of the eyestalk
Experiment I-1: ablation of one eyestalk
Fifty ovigerous females were collected in July-August 2000. One of the
eyestalks was cut, and hatching of most females was monitored by the
photoelectric-switch method, while that of others was monitored by the
water-exchange method. Hatching of all females in which the eyestalk had been
cut did not differ from that of intact females. Zoeas were liberated by
vigorous-release behavior and swam (Table
2).
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Experiment I-2: ablation of both eyestalks
Ovigerous females (110 specimens) were collected on 23 August 1987 and were
randomly separated into two groups. The eyestalks of one group (50 females)
were left intact (control experiment) while those of the second group (60
females) were ablated on 25 August. Hatching was monitored by the
photoelectric-switch method.
In the control group, all females released larvae within 2 weeks of collection (Table 2). For the group with ablated eyestalks, locomotor activity decreased remarkably and their appetites became voracious. Embryos of 14 females hatched within 2 days of ablation. Embryos of these females had already started the hatching program before eyestalk ablation, so their embryos all hatched despite ablation of both eyestalks. Hatched zoeas were liberated by the gentle-release behavior. The results of 46 other females are shown in Table 2. More than half of the females died in the course of the experiments. Five females molted with living embryos, and six females discarded embryos without hatching. A small number of zoeas hatched from only two females until 25 September (marked with an asterisk in Table 2).
Surgery from the tip of the eyestalk
Experiment II-1: Removal of the compound eyeretina
complex
Seventeen ovigerous females were collected on 25 June 2000 and the compound
eye and retina were removed. Hatching was monitored by the water-exchange
method (Table 3). No females
died after surgery. Five females had already started the hatching program
before surgery (see Table 6).
Hatching of 12 other females occurred within 1 h, and zoeas were liberated
into the water by vigorous-release behavior. The mean shift (± S.D.) of
hatching from the nocturnal high tide was 1.1±0.7 h, indicating that
the timing of hatching is not affected by the surgery.
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Eighteen ovigerous females were collected on 16 August 1988, and the same surgery was performed on 20 August (data not shown). Hatching was monitored by the photoelectric-switch method (Fig. 5B). No females died after surgery. Hatching of the females coincided with nocturnal high tides. Thus, the pattern of the larval release activity was the same as that of control groups (Fig. 5A), indicating that the circatidal rhythm of these females is maintained.
Experiment II-2: Removal up to the medulla externa
Six ovigerous females were collected on 25 July 2000, and both optic
peduncles were removed up to the ME on 28 July. No females died after surgery.
Hatching was monitored by the water-exchange method. No females had started
the hatching program when the operation was made. Hatching of each female
occurred within 1 h, associated with vigorous-release behavior
(Fig. 5C). The mean deviation
(± S.D.) of hatching from nocturnal high tide was 0.8±0.4 h
(Table 3), indicating that the
timing of hatching is not affected by the operation.
Experiment II-3: Removal up to the medulla interna
Ten ovigerous females were collected on 25 July 2000, and both optic
peduncles were removed up to the MI on 28 July (five females) and on 9 August
(five females), respectively. Hatching was monitored by the water-exchange
method. No females died after surgery. Four females had already started the
hatching program before surgery (Table
6). Hatching of six other females occurred within 5 days, and the
larvae were liberated by vigorous-release behavior (data not shown). The mean
deviation (± S.D.) of hatching from nocturnal high tide was
1.1±1.2 h (Table 3),
indicating that the circatidal rhythm of these females is still
maintained.
18 ovigerous females were collected on 2 September 1988, and both optic peduncles were removed up to the MI. No females died after surgery. The hatching of 14 females was normal, although the hatching of two other females was extremely delayed (release at 20:40 h on 22 September and at 23:50 h on 24 September). The time of hatching of these 14 females could be monitored by the photoelectric-switch method (Fig. 5D). The mean deviation (± S.D.) of hatching from nocturnal high tide was 0.9±0.6 h, indicating that neither hatching nor hatching synchrony of these females are affected by the removal of the optic peduncle up to the MI and that the circatidal rhythm of these females is still maintained. However, for two other females, mature zoeas hatched every night from 20 September to 27 September (data not shown). Very few zoeas could swim. The hatching synchrony of these two females was obviously affected by the surgery.
Lesions of the medulla terminalis (MT)
The surgery on the MT through the triangle window of the eyestalk
exoskeleton (Fig. 2E) was
performed on 50 females. Hatching was monitored by the water-exchange method.
As shown in Table 4, the
effects of this operation could be classified into four types (types 1-4).
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Experiment III-2: transverse cuts of the MT
The surgical transverse cuts performed through the triangle window
(Fig. 2E) were made in 14
females. In 5 females (collected on 25 July 2000) surgery was performed on 31
July, while in the remaining nine females (collected on 10-12 August) surgery
was performed on 13 August and 27 August. No females died after surgery. Three
females had already started the hatching program when the cuts were made (see
Table 6).
Hatching of three out of the remaining 11 females (Fig. 6A-C) was not different from that of the control group (type 1). The SI was >99.0, suggesting that neither hatching nor hatching synchrony in these females is affected by the surgery. Mean deviation (± S.D.) from the nocturnal high tide was 0.8±0.2 h, indicating that the circatidal rhythm of these females is maintained. However, hatching synchrony in the other eight females was affected to varying degrees (Table 5; types 2, 3 and 4). Zoeas hatched from these females were liberated by gentle-release behavior.
Hatching synchrony of one female (Fig. 6D) deteriorated but occurred periodically for 4 days (19-22 August; type 2). None of the embryos hatched from this female. Hatching of another female (Fig. 6E) started at night on 20 August, and a few embryos hatched over the next 4 days. This female showed type 3 hatching; characterized by a loss of rhythmicity during the first 2 days that sometimes reappears in the last 2 days. In a third female (Fig. 6F), hatching started on 5 August and lasted for 4 days. Hatching was arrhythmic for the first 3 days (type 3), and a huge number of zoeas that hatched at midnight on 8 August were released by gentle-release behavior. Immature embryos also hatched.
Hatching of five other females was suppressed (type 4). These females carried their embryos for >10 days after surgery. The hatching of two of these females is shown in Fig. 6G,H. Very few mature zoeas were sporadically released and submerged in the beaker. It was not clear whether the pattern of hatching in these five females was arrhythmic or persistent. Most embryos that remained attached to the females died during the 2-week experiment or slipped from the female without hatching (Fig. 6H).
Experiment III-3: legions of the dorsal-half of the MT
Females were collected on 25 July and 10-12 August 2000, and the
dorsal-half of the MT was cut on 2 August (seven females) and on 10-13
September (five females), respectively. Seven females had started the hatching
program before surgery (Table
6). No females died after surgery. In all cases, the hatching
pattern was classified as type 1. Zoeas were always liberated by
vigorous-release behavior and almost all of the zoeas swam. The mean deviation
(± S.D.) of hatching from the nocturnal high tide was 1.7±0.9 h,
suggesting that the circatidal rhythm of these females is still
maintained.
Experiment III-4: legions of the ventral half of the MT
Females were collected on 25 July and 10-12 August 2000, and the ventral
half of the MT was cut on 16 August (10 females). One female died after
surgery, and three females had already started the hatching program before
incision (Table 6). Hatching of
two females (Fig. 7A,B) was not
different from that of the control group (type 1). However, one of the females
(Fig. 7B) took 18 days to
release her larvae.
As shown in Fig. 7C, hatching synchrony of one female deteriorated and was periodic for 4 days (type 2). The pattern of eight other females was classified as type 4; five of these patterns are shown in Fig. 6D-H. A few zoeas hatched only sporadically from these females. Some females dropped their embryos without hatching, during 3-8 September, while others still carried their embryos almost one month after surgery. The pattern of hatching in three other females (data not shown) was similar to those in Fig. 6D-H.
Hatching of embryos that had started the hatching program before
surgery
The day of hatching is difficult to predict in S. haematocheir;
the only indication that hatching is imminent is the brownish-green color of
the embryos. Thus, some females had started the hatching program before the
surgical cuts were made, and embryos of those females were all destined to
hatch irrespective of surgery (Table
6). Hatching from the females in experiments II-1 (five females)
and II-3 (four females) all occurred within 1.5 h, indicating that hatching
and hatching synchrony were hardly affected by the removal of the optic
peduncle up to the MI. Hatching in experiment III-3 (seven females) was also
highly synchronous [period of hatching (t) 1.0 h], indicating
that dorsal-half cuts of the MT do not affect either hatching synchrony or
timing of hatching. Zoea larvae were liberated by vigorous-release
behavior.
In contrast, in experiment III-2 (three females), hatching synchrony of all females deteriorated. In one female (Fig. 8A), hatching lasted for one night. Almost all embryos were mature but could not swim. The SI was 2.3. In the second female (Fig. 8B), hatching was still synchronized among most zoeas, but the SI decreased to 8.9. In the third female (Fig. 8C), the SI was 5.4. The SI of these three females was similar to those of detached embryos (Table 1). Zoea larvae were released by gentle-release behavior. In experiment III-4 (three females), hatching synchrony of one female (data not shown) was maintained and zoeas were released by vigorous-release behavior. The SI of the second female was 5.6 (Fig. 8D) and hatching of the embryos attached to the female was delayed as much as 6 h from that of the embryo cluster detached on 5 August (data not shown). In the third female (data not shown), hatching lasted through the night. Females 2 and 3 liberated zoeas by gentle-release behavior.
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Inspection of the success of surgical cuts under the
stereomicroscope
After the experiments (at times indicated by asterisks in Figs
6,
7), both eyestalks were removed
from the female, and the optic peduncles were carefully excised to determine
the success of the surgery (Fig.
2H,I). This inspection was made for three experimental groups
where surgical lesions were made to the MT (i.e. experiments III-2, III-3 and
III-4; Table 5).
Experiment III-2 (11 females; data presented in
Fig. 6)
Fig. 6A. The right
peduncle was completely cut between MI and MT. The left peduncle was also cut
in the lower half of the MT. Fig.
6B. The upper half of the MT in the right peduncle was cut at
the middle region (see Fig.
3A), but the lower half seemed to be undamaged. In the left
peduncle, the lower half of the MT was cut at the posterior region
(Fig. 3A).
Fig. 6C. No
inspection. Fig. 6D.
The right peduncle was almost cut at the middle region of the MT, leaving a
portion of the upper half. Both MI and ME were lost in the left peduncle, but
the anterior region of the MT seemed to be slightly damaged.
Fig. 6E. In the right
peduncle, the middle region of the MT was cut, leaving a portion of the upper
half. In the left peduncle, the MT was cut at the middle region, but a portion
of the lower half seemed to remain.
Fig. 6F. The MT of the
right peduncle was almost all cut at the middle region; only a portion of the
upper half remained. The left peduncle was partially cut at the middle region;
only the lower part was slightly damaged.
Fig. 6G. In the right
peduncle, the MT was completely cut at the middle region. The base of the
optic peduncle (Fig. 3) was
completely cut in the left peduncle.
Fig. 6H. In the right
peduncle, more than half of the MT was cut at the middle region; only a
portion of the upper part remained. The base of the optic peduncle was
completely cut in the left peduncle.
Experiment III-3 (five females; data presented in
Table 5)
The impression of the cuts clearly remained on the dorsal half of the MT.
Female 1. In the right peduncle, the dorsal half was cut up to the
middle region of the MT, and a dark white lump appeared at the dorso-lateral
region. In the left peduncle, the dorsal half of the MT was largely cut up to
the middle region. Female 2. In both optic peduncles, the dorsal half
of the MT was cut up to the middle region. The optic peduncles of the other
three females were not inspected.
Experiment III-4 (11 females; data presented in
Table 5 and
Fig. 7)
Fig. 7A. The MT of
the right peduncle was partially damaged from the middle to the posterior
region of the ventral half. The MT of the right peduncle was cut in the middle
region of the ventral half. Fig.
7B. Damage towards the MT was not clearly visible in the
right peduncle (the middle region of the ventral half may have been slightly
damaged). The middle region of the ventral half was cut in the left peduncle.
Fig. 7C. In the right
peduncle, the ventral half of the MT was cut, but damage was limited to the
frontal region of the MT (see Fig.
3B). A white spot (assemblage of neurosecretory cells) was seen
from the opening of the tissue, and the bundle of axons reached upwards. In
the left peduncle, only slight damage was seen in the ventral half of the MT,
just posterior to the retina. Fig.
7D. In the right peduncle, the ventral half of the MT was cut
in the middle to posterior region, and the whole peduncle was small in size.
The left peduncle was completely cut at the base of the optic peduncle.
Fig. 7F. In the right
peduncle, the posterior region of the ventral half of the MT may have been a
little damaged. No serious damage was seen in the MT, at least in appearance.
The left peduncle was cut at the base of the optic peduncle.
Fig. 7G. In the right
peduncle, lesions were seen in the posterior region of the ventral half of the
MT. In the left peduncle, the ventral half of the MT was cut.
Fig. 7H. In both optic
peduncles, the ventral half of the MT was slightly cut in the middle to
posterior regions.
Inspection of the optic peduncles was also carried out in the females that had started the hatching program before surgery (Table 6). Experiment III-2 (three females). Fig. 8A. In the right peduncle, the MT was completely cut in the posterior region. In the left peduncle, lesions were seen in the posterior region of the ventral half of the MT. Fig. 8B. The right peduncle was completely cut in the frontal to middle region of the MT. The left peduncle was also severely damaged in the middle region of the MT, but the ventral half of the MT appeared to partially remain without damage. Fig. 8C. No inspection. Experiment III-3 (seven females). Female 1. The dorsal half of the MT was cut in the middle region in the right peduncle, but no serious damage was seen in the left peduncle. Female 2. The dorsal half of the MT was clearly cut in the middle to posterior regions in both optic peduncles. Female 3. The dorsal half of the MT was clearly cut in the middle to posterior region in both peduncles. Females 4-7. Lesions were much the same as in Females 2 and 3. Experiment III-4 (three females). Female 1 (SI>25). In the right peduncle, the ventral half of the MT was clearly cut in the middle to posterior regions. In the left peduncle, the ventral half of the MT was cut in the posterior region. Female 2 (Fig. 8D). No clear damage was seen in either optic peduncle, at least in appearance. Female 3. No inspection.
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Discussion |
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It is clear from Table 7 that the transverse lower-half lesions of the MT caused severe effects on hatching and hatching synchrony. The same results were obtained from females that had started the hatching program before surgery (Table 6). However, these results do not indicate that the circatidal clock is abolished if the hatching synchrony deteriorates (e.g. Figs 6D-F, 7C). A central question arises as to the specific role of the MT in the maintenance of hatching synchrony. Another concern is the mechanism by which hatching and hatching synchrony are accomplished under the control of the circatidal pacemaker.
In this paper, I would first state that the timing of hatching is controlled by a single clock (i.e. a circatidal clock) or not by a circadian rhythm manifested in non-tidal conditions in the laboratory, in order to clearly show the endogenous circatidal rhythm that is the objective in this study. The role of the MT and enhancement of hatching by the circatidal clock are discussed below.
The internal pacemaker controlling hatching: a classic problem of one
or two
The complexity of most circatidal rhythms is synchrony not only with the
tidal cycle but also with the day/night cycle (e.g. Saigusa,
1981,
1982
;
Saigusa and Akiyama, 1995
).
Synchrony with both day/night and tidal cycles has long been interpreted in
terms of an interaction between two internal clocks: i.e. the circadian and
the circatidal clocks (Palmer,
1995
). The circatidal rhythm of S. haematocheir is easily
phase-shifted by a 24 h L:D cycle; one feature is that the magnitude of
phase-shift clearly corresponds to that of the phase-shift of the L:D cycle
(Saigusa, 1986
,
1992a
). It is clear that the
24 h L:D cycle is one of the entraining agents of the circatidal rhythm.
However, this does not imply that the circatidal rhythm is governed by two
kinds of internal clock. The two-clock hypothesis is only an `interpretation'
of the timing system proposed on the basis of manifestation of the activity
patterns monitored in the laboratory. If this hypothesis is tested with
elaborate experiments, we would notice that no positive evidence supports that
two kinds of endogenous rhythm are present simultaneously in individual
animals. A single clock is enough to explain synchrony with the nocturnal high
tide. The property of S. haematocheir circatidal rhythm could be
explained in terms of `oscillators' (
and ß oscillators): the
`subsystem' of the circatidal clock (Saigusa,
1986
,
1988
; see also Pittendrigh,
1960
,
1981
). In brief, the timing
system of the endogenous circatidal rhythm is very similar to that of the
circadian rhythm. The circatidal rhythm responds to tide-correlated cycles or
moonlight cycles, as well as to 24 h L:D cycles, and, accordingly, the role
and the action of each oscillator would be somewhat different from that of the
circadian rhythm (for S. haematocheir, see
Saigusa, 1988
).
A variety of behavioral and physiological events show a circadian rhythm in
the optic peduncle of the eyestalk in many crustaceans; e.g. retinal structure
(Barlow et al., 1977,
1987
;
Arikawa et al., 1987
) and
electroretinogram (ERG) amplitude
(Aréchiga and Wiersma,
1969
; Bryceson,
1986
; Aréchiga et al.,
1993
). Many events in the retina of S. haematocheir may
also be modulated by the circatidal clock. But the circatidal rhythm of S.
haematocheir was not affected by the removal of the optic peduncle from
the compound eyeretina complex to the MI
(Table 3). This may suggest
that the circatidal pacemaker (
and ß oscillators) that controls
both hatching and hatching synchrony is not located in the region of the optic
peduncle from the compound eye to the MI.
Role of the MT in the control of hatching and hatching synchrony
In intact females, highly synchronized hatching was followed by
vigorous-release behavior. However, when the hatching synchrony deteriorated,
zoea larvae were liberated by gentle-release behavior. So, one could speculate
that, although all the embryos actually hatch synchronously, the
vigorous-release behavior is lost as a result of lesions of the MT, causing
deterioration of hatching synchrony. This hypothesis, however, cannot explain
why the hatching is suppressed by lesions of the MT. In addition, if hatching
is highly synchronous, zoea larvae should be released into the water even by
gentle-release behavior, possibly within 1 h. So, lesions of the MT must have
directly caused deterioration of hatching synchrony or suppression of
hatching.
The morphology of the optic peduncle of S. haematocheir is much
the same as that of other crabs; neurosecretory cells are located in four
clusters, at the edge of the ME (Fig.
2F), in the X organ (Fig.
2J), between the ME and the MI, and on the dorsal region of the
MT. Neurons are distributed all over the optic peduncle
(Fig. 2J). They are localized
and form a mass in the MT; one cluster in the frontal part (N1) and another
cluster next to the X organ (N2) (Fig.
2K). The major bundle of axons of neurosecretory cells runs from
the X organ at the posterior region of the ventral half of the MT to the
dorsolateral region, towards the sinus gland
(Enami, 1951;
Andrew and Saleuddin, 1978
;
Andrew et al., 1978
;
Jaros, 1978
). Furthermore,
large blood vessels are present on the dorsal surface of the MT and the MI
(Sandeman, 1967
;
Govind, 1992
).
Removal of the MI and dorsal-half cuts of the MT (experiment III-3 in Table 5) may have caused damage to blood vessels, because lesions often caused a large amount of bleeding. Not only the sinus gland but also the cluster of neurosecretory cells at the dorsal region of the MT may have been damaged by this operation. Nevertheless, as hatching was not affected by this operation (Tables 5, 6), we could speculate that hatching is not controlled through the X organsinus gland system.
In both experiments III-2 and III-4, only three out of 14 females had
started the hatching program before surgery was performed
(Table 5). But in experiment
III-3, seven out of 12 females started the hatching program before surgery.
Hatching of five females occurred on the night after surgery, while hatching
of two other females occurred one day after the operation. The possibility
that dorsal-half cuts of the MT advance the date of hatching is suspected.
Hatching is induced through a special developmental process (hatching program)
that lasts 48-49.5 h (Saigusa,
1992b,
1993
). This program would be
initiated around the time of the nocturnal high tide two nights before
hatching. It is not plausible that the interval of hatching program is reduced
to several hours or one day. Females were randomly chosen for each experiment
and, therefore, females that had already started the hatching program would
have been chosen by chance.
Lesions made to the ventral half of the MT (experiment III-4) were observed from the middle to the posterior regions (Fig. 2J, white lines). Neurons are distributed in two areas in the MT. One cluster is localized from the frontal to the lateral region of the ventral half (N1) while the other is localized close to the X organ (N2) (Fig. 2J,K,L). The bundles of neuronal axons are tangled and occupy a large area of the MT. The surgery, especially the lesions made towards the ventral half of the MT, would have cut the bundle of neuronal axons tangled in the ventral half of the MT. It seems that most of the neuronal axons generate from the cluster N1 (see Fig. 2J). So, if the circatidal clock is present in the MT, N1 is the possible location. Suppression of hatching, or only sporadic hatching (Fig. 7D-H), may be caused by completely cutting through the axon bundles related to hatching. The periodic hatching (Fig. 7C) may be caused by incomplete incision of these axon bundles. These speculations suggest that ventral-half cuts would not damage the function of the circatidal pacemaker. In contrast, arrhythmic patterns (especially Fig. 6F) may be evidence to suggest that the clock neurons are actually located in the MT.
In Limulus polyphemus, the circadian pacemaker has also been
suggested to be located in the brain
(Barlow et al., 1977). This
study did not report the effects of lesion of the brain. (Lesions of the brain
severely affected locomotion, which made it difficult to monitor the hatching
itself.) If the circatidal pacemaker is assumed to be located in the brain,
neurons in the MT would only function to induce hatching. The light
information (day/night and moonlight cycles; see
Saigusa, 1988
) would be
transferred to the circatidal pacemaker via the retina or via extra-retinal
photoreceptors (e.g. Hanna et al.,
1988
). If the pacemaker is located in the brain, lesions on the
ventral half of the MT (Table
5) would have cut the bundles of neuronal axons from the
circatidal pacemaker located in the brain to the neurons inducing hatching in
the MT, causing deterioration or suppression of hatching. However, if hatching
and hatching synchrony are generated via the X organsinus gland system,
this possibility may be reasonably supported. However, the present study
supports the possibility that hatching is induced via the neuronal pathway. It
is difficult to answer why hatching is induced by neurons located in the MT.
It seems reasonable to speculate that the circatidal pacemaker is located in
the cluster of neurons located in the MT (possibly N1).
Enhancement of hatching synchrony
When the embryo cluster is detached from the female two or more nights
before larval release and is maintained in the water with aeration, no embryos
hatch (Fig. 4). The critical
period of inducing hatching is 48-49.5 h before larval release (i.e. hatching
of the embryos attached to the female), and this period corresponds to the
time of high tide two nights before larval release
(Saigusa, 1992b). So, I
speculate that the embryos have a special 48-49.5 h developmental process of
hatching called the `hatching program', and that this program is triggered by
the circatidal pacemaker (Saigusa,
1993
). If the embryos are detached from the female after
initiation of this program, they are sure to hatch
(Fig. 4, middle and bottom
panels). On the other hand, no embryos would hatch if they are detached before
initiation of this program (Table
1).
Although hatching of the embryos attached to the female is highly synchronous, hatching synchrony of detached embryos deteriorated (Table 1; Fig. 4). So, the female must enhance the hatching synchrony by an, as yet unidentified, factor that finally determines the time of hatching. Hatching synchrony deteriorated in some females that had already started the hatching program before surgery (Table 6; experiments III-2 and III-4). In these females, the hatching-synchrony-enhancing stimuli may have been lost as a result of the surgery, and the pattern of hatching (Fig. 8) may have been similar to that of detached embryos (Fig. 4). On the other hand, upper-half cuts of the MT (experiment III-3) did not affect the hatching synchrony (Table 6). These results suggest that hatching synchrony is governed by the same pacemaker that induces hatching.
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Acknowledgments |
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