The Horseshoe crab, Limulus polyphemus is well known amongst physiologists as a model system for investigating visual photochemistry and reception. Many experiments have been focused on the Limulus eye because of the easy access to nerves and photoreceptors. Barlow and his colleagues (Barlow et. al., 1985) have provided a large body of evidence supporting the idea that Limulus have robust circadian rhythms of visual sensitivity. In particular, lateral eye sensitivity shows a marked circadian rhythm with an increase in sensitivity at night (Powers and Barlow, 1985). This effect has been shown to be arbitrated by the efferent neural output of a circadian clock located in the brain (Barlow, 1983). While this aspect of their physiology is clearly organized temporally according to the time of day, field locomotor behavior appears to be strongly dependant on tidal rhythms.
Tidal rhythms of mating behavior have long been known to exist in Limulus although published reports detailing the phenomenon are sparse. Adult horseshoe crabs migrate into the intertidal zone in the late Spring in order to attempt to mate (Rudloe, 1979). During this period, mating behavior can be observed, especially during high tide up and down the East coast. Barlow et al. (1986) showed that horseshoe crabs migrate to mating areas one hour before high tide and return to deeper waters about two hours after high tide during the two high tides per day.
While mating activity is clearly synchronized to high tides, time of day appears to play an important role as well. In the field, Rudloe (1980) found that nocturnal peaks of mating activity are apparent in adults in Apalachee Bay, FL. This contrasts sharply with a study done on nearby (150km) Seashore Key, FL where peaks of activity occurred during the diurnal high tide (Cohen and Brockmann, 1983). Similar increased activity during the day has also been seen in juvenile Limulus in the field (Rudloe 1978, 1979b). These studies further contrast with a study conducted at Woods Hole, MA, where Limulus actively mated during both day and night high tides (Barlow et al., 1986). Interestingly, after the mating season is over, they appear to become dormant in deeper waters (Shuster, 1979).
Several other marine species
however have been shown to exhibit tidally rhythmic behaviors and some
of these behavioral rhythms have been shown to be endogenous. Clear
endogenously controlled tidal rhythms have been demonstrated in fiddler
crabs in constant conditions (Bennet et al., 1957; Palmer, 1963; Lehman
et al., 1975) and in the shore crab?? (Naylor, 1958). Honegger (1973)
found that Uca crenulata, the California fiddler crab, has
an endogenous rhythm with a phase close to that of the tidal cycle that
may be coordinated by either light or tidal cues. While Limulus
exhibit clear behavioral tidal rhythms during the summer it is
unclear if these rhythms 1) persist in DD; 2) can occur in the lab or
3) can occur at other times of year. Two short reports indicate that
Limulus are primarily nocturnally active (Casterlin and Reynolds,
1979; Borst and Barlow, 2002) and that these rhythms are endogenous
(Borst and Barlow, 2002). That Limulus are nocturnal is bolstered
by a wealth of physiological data (Barlow, 2001). Interestingly, neither
the physiological nor the behavioral studies make mention of any circatidal
component of activity. The purpose of this series of experiments was
to determine if locomotor activity in Limulus polyphemus has
circatidal and/or circadian components. Another purpose was to determine
if Limulus are nocturnally or diurnally active and establish
whether or not these rhythms persist under constant darkness, constant
light or both.
Experimental Animals and Environmental Conditions:
Horseshoe crabs, Limulus polyphemus (2 females, 519, 676g; 4 males197-310g) were caught in lobster traps in Great Bay, NH in October, 2002. The animals were not fed after being caught and were always singly housed in activity chambers. The sea water that was used in these experiments was collected from the UNH Jackson Estuarine Lab, Durham, NH.
Behavioral Activity :
Locomotor
activity was measured in activity chambers in two separate tanks (Jewel
Industries Inc., Chicago IL, Model Oceanic-55) by dividing each tank
into four approximately equal areas (39cm wide X 32cm long) using ceiling
light grating (1 cm X 1cm openings). A "ceiling" (9cm high)
was used to prevent the animals from flipping over and becoming stuck.
The ceiling was held rigidly in place and supported using cable ties
and PVC plastic posts (1 cm diameter) in the corners. In addition, three
bricks were placed on the ceiling to weigh down the ceiling and to create
a shielded, darker area over approximately half of each activity chamber.
Immediately after being brought to the lab in Plymouth, NH, the animals
had magnets affixed to their dorsal surfaces between the lateral eyes
using cyanoacrylate glue and duct tape. A plastic practice golf ball
was placed approximately 1.5 inches on to the tail and secured with
cyanoacrylate and duct tape in order to prevent their telsons from becoming
stuck in the chamber partitions. A magnetic reed switch to detect Limulus
movement was located on the ceiling to one side of each of the
activity chambers and was attached to a CPU-based data collection system
(Drosophila Activity Monitor IV, Trikinetics Inc. Waltham, MA). Activity
was recorded in five minutes intervals.
Experimental Procedure :
Experiment 1 : The animals were first exposed to a 12:12 light-dark (LD) cycle. Light intensity during the day was approximately 150 lux (LunaPro Light Meter, Gossen, Germany) at water level and was provided by a broad spectrum fluorescent bulb (CoralLife 10,000K SuperDaylight). Light levels were 0 lux during D. Any procedures during D were performed using an infrared viewer (Model 6100, Electrophysics, Nutley, NJ). Water temperatures were monitored continuously using temperature data loggers (Hobo, Onset Corporation, Pocassett, MA) and ranged between 11 and 14°C but did not vary with time of day. The salinity ranged from 24 to 26 parts per thousand. Activity data were collected from November 21, 2002 thru December 5, 2002.
Experiment 2 : In an effort to stimulate activity levels, and hence the clarity of any activity patterns, the light-dark cycle was changed to 14:10 and the temperature increased to range between 17 and 21°C. Salinity ranged from 22 to 26 parts per thousand. Activity data were collected from December 6, 2002 thru January 3, 2003. The photoperiod change went into effect at 8 pm the night of December 6, 2002.
Experiment 3 : In order to determine if the rhythmic timing of activity was endogenous, the light-dark cycle was changed to constant darkness (DD) at 8 pm on January 3, 2003 and continued thru January 14, 2003. Temperature was maintained between 17 and 21°C while salinity was kept between 25-27 parts per thousand.
Experiment 4 : From January 14 -February 28, 2003 the animals were exposed to constant light (LL) in order to ascertain the effects of this treatment on any circadian timing system. Constant light began at 3:40 pm on January 14, 2003. Temperature was kept between 17 and 21°C. Salinity was kept between 24-27 parts per thousand.
The activity data were organized using Microsoft Excel and analyzed by a graphical/statistical program (Clocklab, Actimetrics Evanston, IL) that created actograms and calculated periodograms. Significance of rhythmicity was determined both visually and by Chi-square periodogram analysis (p<0.01; Sokolove and Bushell, 1975). The period (Tau) in the circadian range for each individual during each experiment was determined by recording the highest significant peak on the periodogram between 22 and 26 hours. Similarly, circatidal rhythm periods were determined by recording the highest significant peak on the periodogram between 10 and 14 hours. To determine whether more activity occurred during L or D, the amount of activity during L and D for each day for each animal was calculated. These L and D values for each animal were compared using paired Student's t-test (p<0.05; Statview , Abacus Concepts, Berkeley CA). Student's t-test or ANOVA were also used to compare other means (p<0.05).
Our
results are the first to demonstrate endogenous circatidal locomotor
rhythms in Limulus . Other marine species have also been shown
to exhibit circatidal rhythms. Honegger (1973) found that Uca crenulata
has an endogenous rhythm with a period close to the environmental
tidal cycle that can be coordinated by light as well as by tidal cues.
Naylor (1958) found a locomotor activity component of tidal frequency
(ca.12.4 hr.), with peaks at the time of high tide in Carcinus maenas
(L.). Field studies and a wealth of anecdotal observations have
long cited Limulus ' propensity to mate during high tides (Barlow
et al., 1986; Shuster, 2001). Our results indicate that the control
of the timing for mating behavior may, at least in part, be endogenous.
We also show that the locomotor activity of adult Limulus can be synchronized to an LD cycle with 24 h periods and the activity will persist rhythmically in constant conditions. This is in agreement with a previous short report showing that juvenile Limulus exhibit significant circadian rhythms of locomotion (Borst and Barlow, 2002). An interesting difference in the results is that while all of our animals exhibited significant circadian rhythms in both DD and LL only 40% (2/5) of their animals did. It is not surprising that Limulus would exhibit circadian rhythms of locomotion - circadian modulation of visual sensitivity has been very well documented by Barlow and his colleagues (Barlow et al., 2001). In Limulus , the temporal patterns of activity seem to also be modulated by other factors as well: in field experiments, Rudloe (1980) found evidence of tidal breeding activity patterns with nocturnal peaks of activity.
Our results also suggest that Limulus locomotor activity occurs primarily at night. All of the animals that entrained (3) exhibited significantly more activity during the night than during the day. These findings are consistent with the large body of literature demonstrating greatly increased visual sensitivity at night in Limulus : for why increase visual sensitivity if you are not going to be active? Our findings also agree with two short reports that suggest Limulus are more active at night than during the day (Casterlin and Reynolds, 1975; Borst and Barlow, 2002). Interestingly, some field studies on mating activity provide conflicting data: Rudloe (1980) found a preference of nocturnal mating activity while a diurnal peak of breeding activity was seen 150 km away in a different population by Cohen and Brockmann (1983). These studies differ from a Woods Hole, MA study in which Limulus were found mating both during day and night (Barlow et al., 1986). Some of our animals failed to stably entrain to the LD cycle (Figure 1) but instead exhibited free-running rhythms. This indicates robust internal modulation of activity even in the face of a strong zeitgeber. Overall, our results indicate nocturnal activity preferences for some, but not all, of the Limulus that we tested and suggests that environmental tidal factors and an internal clock(s) may be very important in the temporal organization of locomotor activity in this species.
Barlow, R. B. 1983. Circadian rhythms in the Limulus visual system. J Neurosci. 3: 856-870.
Barlow, R.B., M. K. Powers, H. Howard and L. Kass. 1986. Migration of Limulus for mating: relating to lunar phase, tide height, and sunlight. Biol. Bull. 171: 310-329.
Barlow, R. B., Kaplan, E., Renniger, G.H., and Saito, T. 1985. Efferent Control of Circadian Rhythms in the Limulus Lateral Eye. Neurosci. Res. (Suppl.) 65-78.
Barlow, R.B., J.H. Hitt, and F.A. Dodge. 2001. Limulus vision in the marine environment. Biol. Bull. 200:169-176.
Bennet, M. F., J. Shriner, and R. A. Brown. 1957. Persistent tidal cycles of spontaneous motor activity in the fiddler crab, Uca pugnax . Biol. Bull. 112 :267-75.
Borst, D. and R. Barlow. 2002. Circadian rhythms in locomotor activity of juvenile horseshoe crabs. Biol. Bull. 203 :227-8.
Casterlin, M.E, and W.W. Reynolds. 1979. Diel locomotor activity patter of juvenile Limulus polyphemus Linnaeus. Rev. Can. Biol. 38 :43-44.
Cohen, J.A. and Brockman, H.J. 1983. Breeding Activity and Mate Selection in the Horseshoe Crab, Limulus polyphemus . Bull. Mar. Science. 33 :274-281.
Honegger, H.W. 1973. Rhythmic Motor Activity Responses of the California Fiddler Crab, Uca Crenulata to Artificial Light Conditions. Mar. Biol. 18 :19-31.
Lehmann, U. 1975. Interpretation of entrained and free-running locomotor activity patterns of Uca . pp. 77-92. In P. J. DeCoursey (ed.) Biological Rhythms in the Marine Environment. University of South Carolina Press, Columbia.
Naylor, E. 1958. Tidal and Diurnal Rhythms of Locomotor Activity in Carcinus maenas . J. Exper. Biology. 35 :602-610.
Palmer, J. D. 1973. Tidal rhythms: the clock control of the rhythmic physiology of marine organisms. Biol. Rev. 48 :377-418.
Powers M. K, and R. B. Barlow Jr. 1985. Behavioral correlates of circadian rhythms in the Limulus visual system. Bio Bull. 169: 578-591.
Rudloe A. 1978. Some ecologically significant aspects of the behavior of the horseshoe crab, Limulus polyphemus . Ph.D. Thesis, The Florida State University, Tallahassee, Florida.
Rudloe A. 1979. Limulus polyphemus : A review of the ecologically significant literature. pp.27-35 In Biomedical Applications of the horseshoe crab (Limulidae) . E. Cohen. Ed. Alan Liss, Inc. New York.
Rudloe A. 1980. The breeding behavior and patterns of movement of horseshoe crabs, Limulus polyphemus , in the vicinity of breeding beaches in Apalachee Bay, FL. Estuaries. 3: 177-183
Shuster, C.N. Jr. 1979. Distribution of the American Horsefoot "Crab," Limulus polyphemus (L.) In: Biomedical Applications of the Horseshoe Crab (Limulidae) E.Cohen, ed. pp. 3-26. Alan R. Liss, Inc. New York.
Shuster C.N. Jr. 2001. Two perspectives: Horseshoe Crabs during 420 million years worldwide, and the past 150 years in Delaware Bay. pp 17-40, In : Tanacredi JT, Limulus in the Limelight , Kluwer Academic/Plenum Publishers, New York.
Sokolove P.G., Bushell W.N. 1978. The chi-square periodogram: its utility for analysis of circadian rhythms. J Theor Biol 74 : 131-160.