J. Exp. Biol. (1969), 51, 715^735
With 7 text-figures
Printed in Great Britain
715
SYNCHRONIZATION OF THE LOCOMOTOR TIDAL
RHYTHM OF CARCINUS
BY BARBARA G. WILLIAMS* AND E. NAYLOR
Department of Zoology, University College of Swansea
(Received 30 April 1969)
INTRODUCTION
During the summer months in Britain Carcinus mamas (L.) collected from the shore
exhibits tidal and circadian locomotor rhythmicity which persists overtly for 5-6 days
in constant laboratory conditions (Naylor, 1958). The tidal rhythm has been shown to
be controlled by an approximately tidal endogenous component (Williams & Naylor,
1967; Naylor & Williams, 1968) but, so far, little is known of the mechanism of environmental phasing of the rhythm. Previous work has suggested that temperature
changes associated with tidal rise and fall may be important in synchronization (Naylor,
1963, 1964) and the present paper considers the effects of temperature cycles and other
variables of tidal frequency upon the tidal rhythm.
MATERIALS AND METHODS
Two types of Carcinus were used in the experiments, some showing normal tidal
rhythmicity which were collected from between tidemarks and others showing nocturnal activity which were collected from a non-tidal dock (Naylor, i960). Movement
of animals kept moist in air was recorded in mechanical actographs (Naylor, 1958),
or, if the animals were immersed in running sea water, in an inverted form of that
actograph. Activity was expressed as the number of actograph tilts per unit time and it
was assumed that such a measure of activity bore a constant relationship to total
activity. In experiments involving eyestalkless crabs, eyes were removed following the
method described earlier (Naylor & Williams, 1968).
Laboratory experiments were concerned with the effect of cyclic temperature changes, alternate periods of immersion in sea water and exposure to air at the same
temperature, and combinations of these two variables, all at tidal (12-4 hr.) frequency.
Temperature cycles were achieved by transferring the crabs in their actographs from
one constant-temperature room to another, all in continuous dim light, for alternate
periods of 6-2 hr. In experiments involving cycles of immersion and exposure to air,
with water and air at the same temperature (190 C), the crabs were maintained in
constant dim light. In this type of experiment the actographs were flooded and covered
with sea water or drained and exposed to air for alternate 6-2 hr. periods during the
treatment period. In other experiments animals were subjected to fluctuating temperatures combined with alternate periods of immersion and exposure to air. In all
experiments activity was recorded in constant conditions, in air, for a period of about
• Present address: Department of Zoology, University of Otago, Dunedin, New Zealand.
716
BARBARA G. WILLIAMS AND E. NAYLOR
3 days after treatment and, where possible, activity was also recorded during the treatment period.
Field experiments involved transferring crabs from non-tidal docks to a tidal environment for varying lengths of time. Some were placed in a cage at mid-tide level
on a boat slip where they were subjected to tidal variables including exposure to air,
and others were placed in a weighted creel below tidemarks where they were subjected
to the effects of tidal rise and fall but were never exposed to air. After the treatment
period crabs were placed in actographs in constant conditions in the laboratory and
their activity was recorded for about 3 days.
FIELD EXPERIMENTS
Figures 1 and 2 illustrate the results of field experiments in which crabs from nontidal docks were kept in cages between and below tidemarks. Experiments were carried
Aa.
Time (hr.)
Fig. 1. Mean hourly activity of crabs collected in early summer from a non-tidal dock and treated
as follows: (a) 8 crabs after 11 dayB in a creel below tidemarks; (b) 8 crabs after 11 days in a creel
at mid-tide level; (c) 12 freshly collected controls. (Activity expressed as mean actograph tilts/
hr.; vertical dotted lines indicate 'expected' times of high tide; M, midnight.)
Locomotor tidal rhythm of Carcinus
717
|put in summer when air temperatures were higher than sea temperatures and, in the
Wo sets of experiments illustrated, crabs were exposed in each situation for 11 days
(Fig. 1) and 2 days (Fig. 2). Activity records in constant laboratory conditions after
treatment show that 11 days exposure was almost equally effective in entraining a
normal persistent tidal rhythm whether crabs had been between tidemarks (Fig. 1 b) or
4 -
2 -
Fig. 2. Mean hourly activity of crabs collected in early summer from a non-tidal dock: (a)
4 crabs after 2 dayB in a creel below tidemarks; (b) 5 crabs after 2 days in a creel at mid-tide level;
(c) 12 freshly collected control* (symbols as in Fig. 1).
below (Fig. 1 a). However, the 2-day exposure was much more effective in entraining a
persistent tidal rhythm in crabs kept between tidemarks (Fig. 26) than in crabs kept
below low watermark (Fig. za). Evidently the rhythm is most effectively entrained
when crabs are periodically exposed to air, suggesting that cycles of immersion and
perhaps also the associated temperature changes are important in phasing the rhythm.
Subsequent laboratory experiments reported in this paper were designed to investigate
the effects of these tidal variables. It should be noted that present field and laboratory
experiments suggest that other tidally varying factors such as hydrostatic pressure
7i8
BARBARA G. WILLIAMS AND E. NAYLOR
change and water movement may be important, but an experimental analysis of thesa
factors is not attempted here.
LABORATORY EXPERIMENTS
Temperature cycles
In experiments with crabs kept continuously moist in air and subjected to cyclical
temperature changes of tidal periodicity, the activity of dock and shore crabs was
recorded during and after 5 days of treatment. Fig. 3 illustrates the results of such
Treatment
20
20
Post treatment
I
i/'/c-i'
J
3 -
2-
JlkA.
1 -
5- 0
I I I I I I I I I I I I I
i t i i i l i i i i i i i i i i l i l l I l i ll
(b)
3-1
2 -
1 -
U
w
A
ri
0
i i i i i i i i i
25
0
Time (hr.)
Fig. 3. Mean and range of hourly activity values presented as 25 hr. form estimates during a
5-day treatment period and a 3-day post-treatment period in constant conditions. Each experiment used 5 freshly collected shore crabs subjected to alternate half-tidal (6-a hr.) periods:
(a) at 20 and 130 C; (6) at 24 and 130 C, with (a') and (b') indicating subsequent activity at
13° C. (Arrows in (a) and (b) indicate times of high tide; shaded regions in (a') and (b") indicate
'expected low temperature' periods.)
experiments on shore crabs which, on collection, possessed the normal type of tidal
and circadian rhythmicity and for which the imposed temperature cycles were so
arranged that the middle of the cold period coincided with their period of inactivity.
A temperature differential of n ° C, from 13 to 240 C (Fig. 3A), quickly resulted in
rephased rhythmicity during treatment, with peak activity occurring at the times of
Locomotor tidal rhythm of Carcinus
719
low temperature. Entrained peaks were therefore 6 hr. out of phase with those of the
normal rhythm. After 5 days of such treatment the locomotor activity of the crabs was
recorded at 13° C over a period of 3 days (Fig. 36'), during which time the rhythm
impressed by the fluctuating temperatures persisted with reasonable accuracy. After
temperature variations were withheld activity in 'expected high temperature' periods
Post treatment
Treatment
6-
4-
2-
•«•
M l i r i l l l l T I l l l l l
24
24
M
i
l M
M
M M
M i l
M M )
I M
< in^lT^f
W.ZlM 24 | °C
8-
6-
4-
2-
ii
i i i i i i i i i i i i i i i ii
25
0
Time (hr)
Mil
I I I I I I I I I I 1 \
2S
Fig. 4. Mean and range of hourly activity values (as 25 hr. form estimates) during a 5-day treatment period and a subsequent 3-day period in constant conditions (130 C). Each experiment
used 6 freshly collected dock crabs subjected to alternate half-tidal (6-a hr.) periods (a) at
17 and 130 C, (6) at 24 and 130 C, with (ar) and (60 indicating subsequent activity at 130 C.
(Symbols as in Fig. 3.)
averaged 0-23 tilts/hr. (S.D. ±0-28) and that in 'expected low temperature' intervals
0-63 tilts/hr. (S.D.+0-51). Comparing these two sets of values (unequal variances)
gives P < o-ooi, indicating that activity was significantly greater at the times of
'expected low temperatures'.
When freshly collected shore crabs were subjected to a temperature differential of
0
7 C (20-13° Q (Fig. 3 a), peak activity at first corresponded with the times of high
water which crabs would have experienced on the shore, regardless of the higher
temperature at these times during treatment. Later during the 5-day treatment period,
activity began to coincide with the low temperature parts of the imposed temperature
BARBARA G. WILLIAMS AND E. NAYLOR
720
cycle and was inhibited at the higher temperature. This change in the amount of
activity during the 200 C periods explains the very wide range of values observed at
those times in Fig. 3 a. In the 3-day period when temperature cycles were withheld
(Fig. 3 a') results indicate that entrainment had occurred. Activity values were significantly greater (P < o-oi)at' expected low temperature'times (mean 0-57, S.D. ±0-52)
than at 'expected high temperature' times (mean = 0-29, S.D. ±0-32).
Treatment
Post treatment
19°C
10-
5-
\ r
*A
Time (hr.)
Fig. 5. Mean and range of hourly activity values of 5 crabs during and after treatment with
tidal cycles of exposure to air and immersion in sea water, all at 190 C. Results plotted as
25 hr. form estimates: (a) during the 5-day treatment period and (a") during the 3-day posttreatment period when kept moist in air. (Shaded and stippled periods indicate immersion and
'expected immersion', respectively.)
Crabs from non-tidal docks again responded very readily to temperature cycles
during the treatment periods. An n ° C differential (13-240 C) set up a marked
rhythm during treatment (Fig. 4J) which persisted after temperature cycles were
withheld (Fig. 4ft'). Activity values were significantly higher (P < o-ooi) in 'expected
low temperature' periods (mean 2-16, S.D. + 1*36) than in 'expected high temperature'
periods (mean 0-87, S.D. ±0-77). A 40 C differential (13-170 C) resulted in a rhythm
during treatment (Fig. 4a) but this did not persist in constant conditions (Fig. 4a').
In Fig. 4a' 'expected low temperature' values (mean 1-43, S.D. ±070) were not
significantly different (P > 0-5) from 'expected high temperature' values (mean 1*34,
S.D. ±0-83).
Cycles of immersion and exposure to air
Figure 5 shows the results of an experiment in which shore crabs in dim light were
alternately immersed in sea water at 190 C and exposed to air at the same temperature
for alternate periods of 6-2 hr. During the treatment period of 5 days (Fig. 5 a) most
Locomotor tidal rhythm of Carcinus
721
activity clearly occurred at the times of immersion, but in the subsequent 3 days in
constant conditions (Fig. 5 a') only slight evidence of rhythmicity was apparent.
Activity values in the periods of 'expected immersion' (mean I-IO, S.D. + 0-75) compared with values in the periods of 'expected exposure to air' (mean o-68, S.D. ± 0-57)
gives 0-05 > P > 0-02. In other experiments with dock crabs kept in the light/dark
regime of the laboratory a cycle of immersion and exposure to air at 200 C had little
effect on the normal nocturnal rhythm of the animals after 7 days.
Combined temperature and immersion cycles
In these experiments dock crabs were subjected to temperature fluctuations combined with alternate periods of immersion and exposure to air, the periods of immersion coinciding with the change to low temperature. Five-day treatment periods
were given with temperatures varying from 24 0 C i n a i r t o i 3 ° C i n water (Fig. 6 b) and
170 C in air to 13° C in water (Fig. 6a). The figure shows the activity pattern during
Post treatment
^
1-
24
24
JTl": I 13: :\: -1 24~! °C
4-
3-
= 2-
1-
tik
1 1 1 1 1 1 1 1
25
Time(hr.)
Fig. 6. Mean and range of hourly activity values (as 25 hr. form estimates) during 3 days in air
at 13° C after 5 days treatment with tidal cycles of high temperature combined with exposure to
air alternating with low temperatures combined with immersion in sea water: (a) 5 crabs immersed at 13° C and exposed to air at 170 C; (6) 4 crabs immersed at 130 C and exposed to air
at 24° C. (Stippled areas indicate 'expected immersion/low temperature' periods.)
BARBARA G. WILLIAMS AND E. NAYLOR
722
3 days in constant conditions after treatment, and greatest activity clearly occurred
during the ' expected' periods of low temperature/immersion, indicating in each case
an experimentally induced rhythm. In Fig. 6 a activity values at times of 'expected'
low temperature/immersion (mean o-8o, S.D. ±0-67) compared with 'expected' high
temperature/air (mean 0-36, s.D. ±0-4) give 0-005 > P > 0001. In Fig. 6b activity
values at times of 'expected' low temperature/immersion (mean 1-74, s.D. ±1-26)
compared with' expected' high temperature/air (mean 0-49, s.D. + 070) give P < o-ooi.
When comparing the results in Fig. 6a with those in Fig. \a! and Fig. 5 a' it seems
clear that combined temperature and immersion cycles were more effective in entraining a persistent rhythm than either factor alone.
19°C
20
Duration of experiment (hr.)
30
Fig. 7. Average hourly activity of batches of 6 eyestalkless crabs over a period of 31 hr. and
subjected to successive 6-z hr. periods of (a) 20 and 13 0 C in air; (6) 24 and 13 0 C in air; (c)
immersion in sea water and exposure to air, always at 19 0 C. (Hatched periods indicate low
temperatures, black periods indicate immersion.)
Effects on eyestalkless crabs
Previous work has suggested that eyestalks play an important role in the internal
control of rhythmicity, and that eyestalkless crabs are incapable of exhibiting rhythmic locomotor activity in constant conditions (Naylor & Williams, 1968). Experiments
were therefore carried out in which eyestalkless crabs were subjected to temperatures of
Locomotor tidal rhythm of Carcinus
723
0
20 and 13 C for alternate 6-2 hr. periods (Fig. ja), and others to temperatures of
24 and 130 C for similar periods (Fig. 76). Unlike normal animals, eyestalkless specimens did not show maximum activity at the lower temperature. Indeed, there was an
indication that activity values were lowest at the lower temperatures. When comparing
the 200 C activity values (mean 2-40, s.D. ±0-98) with the 130 C values (mean 0-75,
S.D.+ 0-82) (Fig. ya), and 240 C values (mean 1-59, s.D. ±091) with i3°C values
(mean 0-71, s.D. + 0-56) (Fig. jb), both comparisons give highly significant differences
with P < o-ooi in each case.
Finally, eyestalkless crabs were exposed to alternate 6-2 hr. period of immersion and
exposure to air, when it was found that they responded in a manner similar to normal
crabs, activity being greatest when in water (Fig. yc). Clearly the increased activity of
normal crabs when immersed does not wholly depend upon the presence of the eyestalks. This observation is of interest when recalling that cycles of immersion and
exposure to air without temperature changes had little effect in entraining a persistent
rhythm in normal crabs. All these results emphasize the importance of the eyestalks in
mediating the effects of fluctuating temperatures on normal crabs.
DISCUSSION
The results of the experiments involving temperature fluctuations of tidal frequency
agree with earlier results on the effects of sporadic temperature fluctuations (Naylor,
1963) and provide experimental support for the suggestion that regular changes in
temperature associated with tidal rise and fall are partly involved in the phasing of
tidal rhythmicity. A temperature cycle involving a very high differential of 11° C can
establish a persistent rhythm after 10 cycles. However, a differential of 40 C, an order
of magnitude similar to changes experienced on the shore, failed to establish any
persistent rhythmicity in the same number of cycles. Nevertheless, such a pattern of
temperature change would probably establish a persistent rhythm if it was continued
for a longer period, since during the experiments activity soon came to occur at the
times of low temperature, even though the pattern did not persist subsequently in
constant conditions.
Cycles of immersion in water and exposure to air without accompanying temperature
changes appear to be much less effective than temperature cycles alone. However,
whereas a temperature cycle of 40 C change is ineffective in inducing a persistent
rhythm in 5 days, such a temperature change coupled with a cycle of immersion is
relatively very effective in entraining a rhythm. One must conclude therefore that a
combination of these two environmental variables is more effective in phasing a
rhythm than the variables considered separately. Moreover, the field experiments on
Carcinus emphasize that crabs below tidemarks will entrain to a tidal rhythm, suggesting that other tidal variables, apart from temperature change and immersion, are
also important. Periods of as little as 2 days below tidemarks, where temperature
changes would have been minimal, had some effect in entraining tidal rhythmicity,
while 11 days in the same situation was sufficient to entrain a persistent tidal rhythm in
previously arrhythmic crabs. In view of demonstrations that mechanical agitation in
water and changes in hydrostatic pressure synchronize the rhythm of some species
(Enright, 1963, 1965; Jones & Naylor, 1969) these factors should be investigated in
46
Exp. BioL 51, 3
724
BARBARA G. WILLIAMS AND E. NAYLOR
Carcinus. It should be noted, however, that those species which have so far been
shown to be entrained by mechanical agitation are sand-beach organisms for which
tidal rise and fall agitates the sandy substratum into which they burrow. On a rocky
shore this factor is likely to be a less important synchronizer than changing hydrostatic
pressure. Carcinus megalopa larvae are highly pressure-sensitive (Knight-Jones &
Qasim, 1967), so there are good a priori reasons to suggest that pressure cycles can
induce rhythmicity in adults. It would appear therefore that the tidal rhythm of
locomotor activity in Carcinus in its natural environment is phased by tidal changes in
temperature and to a lesser extent by tidal cycles of immersion and exposure to air.
Other factors probably also play a part in synchronization and, though it has not yet
been confirmed experimentally, variations in hydrostatic pressure seem likely to be
important.
The effects of temperature changes on the rhythmicity of Carcinus are of particular
interest, especially when considering the responses of eyestalkless crabs to fluctuating
temperatures. The interest arises in view of earlier work suggesting that locomotor
rhythmicity is mediated via an inhibitory hormonal mechanism in the eyestalk (Naylor
& Williams, 1968). It has been suggested that temperature could affect the production
or release of the inhibitor, so that low temperatures, for example, would reduce the
output of inhibitor and thus increase the level of activity. Results with eyestalkless
animals confirm this suggestion since animals fail to show increased activity at low
temperatures. In normal crabs temperature changes may therefore establish overt
locomotor rhythmicity by imposing a rhythm on the amount of inhibitory substance
available which could then continue for a short time in constant conditions. Also,
since there is some suggestion that activity actually increases at higher temperatures in
eyestalkless crabs (Fig. 7 a, b), further investigation is necessary to consider whether
this indicates the existence of a mechanism which promotes activity and complements
the eyestalk inhibitory mechanism.
SUMMARY
1. A period of 11 days between tidemarks or below tidemarks entrains a persistent
tidal rhythm in Carcinus. Two days exposure is effective between tidemarks but much
less so below.
2. Laboratory temperature cycles of tidal periodicity for 5 days will impose a persistent rhythm on crabs kept moist in air when the temperature differential is 11° C but
not when it is 40 C. A 4° C differential is effective if crabs are immersed in sea water
at the low temperature parts of the cycle.
3. A combination of tidal variables therefore appears to act as synchronizers for the
locomotor rhythm and, though not investigated, it is suggested that hydrostatic pressure change is another possible synchronizer.
4. Eyestalkless crabs show increased activity at high temperatures and decreased
activity at low temperatures. These responses are the reverse of those occurring in
normal crabs and are consistent with the view that eyestalks are the site of an inhibitory
neuroendocrine mechanism.
We are grateful to Professor E. W. Knight-Jones for the provision of laboratory
facilities and to the Science Research Council for financial support.
Locomotor tided rhythm of Carcmus
725
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