Synchronization to daily changes in light intensity is a prominent feature of most living organisms. Many crustacean species including several species of amphipods (Williams, 1987; Williams, 1988), crayfish (Page and Larimer, 1975; Fingerman and Lago, 1957; Bojsen et al., 1998), crabs (Palmer, 1970; Chatterton and Williams, 1994; Forward et al., 1997), prawns (Atkinson and Naylor, 1976), and lobsters (Fielder, 1965; Branford, 1979; Rimmer and Phillips, 1979; William and Dean, 1989; Nagata and Koike, 1997) have been shown to synchronize behaviorally and/or physiologically to daily light:dark cycles. Physiological changes such as O 2 consumption (Williams, 1988), color changes (Brown et al., 1954) and heart rate (Pollard and Larimer, 1977) have been demonstrated in several species. Several behavioral rhythms have also been described such as swimming (Forward et al., 1997), burrow plugging (de la Iglesia et al., 1994), foraging (Correia, 1998) and overall locomotor activity. Importantly the rhythms of several species have been demonstrated in both laboratory and field situations. Overall, these studies indicate that temporal partitioning of the day is important to crustaceans.
Daily
behavioral rhythms have also been demonstrated in several species of
lobsters. In the laboratory, nocturnal increases in locomotor
activity have been observed in eight species in three genera of lobsters
( Panulirus - Morgan, 1978; Rimmer and Phillips, 1979; Lipscius
and Hernnkind, 1982; Nagata and Koike, 1997), Jasus - Fielder,
1965; William and Dean, 1989) and Homarus - (Cobb, 1969; Ennis,
1973). Field studies also demonstrate a strong preference for
nocturnal locomotor activity in both adult (Smith et al., 1998; Jury,
2000) and larval forms (Rimmer and Phillips, 1979). While these studies
demonstrate that lobsters are strongly synchronized to photoperiod,
the extent of the endogenous control of these rhythms is less well known.
Endogenous control of rhythmicity has been well established in several crustacean species. Circadian control of locomotion has been demonstrated in amphipods (Williams, 1988) and several species of crab (Palmer, 1974; Chatterton and Williams, 1994; Forward et al., 1997) and crayfish (Page and Larimer, 1975; Fingerman and Lago, 1957). In addition, circadian rhythms of oxygen consumption in amphipods and crayfish (Williams, 1988; Fingerman and Lago, 1957), heart rate in crayfish (Pollard and Larimer, 1977), and color changes in crabs (Brown et al., 1954) have also been demonstrated. In lobsters, while some evidence suggests circadian rhythms of locomotion in P. japonicus (Nagata and Koike, 1997), strong evidence exists for circadian control of locomotion for only one species, the New Zealand rock lobster, Jasus edwardii (Williams and Dean, 1989). The purpose of this series of experiments is to determine if locomotor activity in H. americanus is controlled by a circadian clock under both field and laboratory conditions. To address this issue, lobsters were housed in two types of experimental apparatus that allowed continuous monitoring of locomotor activity, a fairly standard circular racetrack and a "running wheel" design more commonly used for measuring activity in rodents.
Adult
Lobsters (n=15; 82-91 mm CL) were maintained in recirculating seawater
tanks at approximately 32 ppt at 16±3 °C at Plymouth State
College, Plymouth, NH. Lobsters were not fed for the duration of the
individual experiments (45-61 d). Light levels for LD and LL conditions
were maintained using 2 broad spectrum, 20W fluorescent lights (Simkar
Corp, Pittsburg, PA) measured at 25-85 lux (LunaPro light meter, Gossen,
Germany). Duration and timing of photoperiod was monitored continuously
using a HOBO Light datalogger (Onset co,).
Running wheels were constructed
from 5 gallon plastic buckets (high-density polyethylene) with a 30
cm inner diameter and inner width of 10 cm (Figure 1B). They
were perforated extensively with > 500 holes (6 mm diameter each)
to allow water circulation and to provide increased traction. The wheel
rotated around a hollow, 1 cm plastic axle fixed to a plastic 
(PVC)
stand. A magnetic reed switch located
on one leg of each stand detected wheel movement when one of two small
magnets attached 180 degrees from each other on the outer surface of
the wheel passed by it. 4-6 lobsters were run simultaneously
by placing individuals into one of 4-6 running wheels in an environmentally
controlled aquarium and allowing them to acclimate to the conditions
while exposed to a 14:10 LD cycle for at least 14 days There was no
effect of an extended initial exposure to LD (28 days, n=4 data not
shown) in the running-wheels on activity or entrainment in LD or subsequent
free-running period in DD (p<0.05) so all data presented below are
for LD of 14 d. After exposure to LD cycles, animals were exposed to
DD for 21-23 days. DD conditions were initiated by unplugging
the light from the timer resulting in 0 lux. For each trial, DD conditions
were followed by exposure of 10-28 days of LL. The intensity of light
during LL ranged from 0.17-1.4 lux.
Activity data from the running wheel experiments were collected in 5 minute bins using a Drosophilia Activity Monitoring System (Trikinetics, Waltham, MA). Actograms were plotted using Ratman (Ratplot, Klemfuss and Clopton, 1993) (Fig. 4). The data were analyzed for significant periodicity (p<0.05) using a periodogram analysis (Ratwave; Klemfuss and Clopton, 1993) and visual inspection. In all cases the best Tau, a measure of free running periodicity, was confirmed visually and these values were used in subsequent analyses. Alpha, the length of the main bout of daily activity, was calculated by drawing objective lines of best-fit along the onsets and ends of activity for each animal in both LD and DD. Phase angle to the LD cycle (the time difference between the onset of activity and the onset of darkness) was calculated by measuring the time difference between the drawn lines of best-fit of onset of activity and the onset of darkness. If neither alpha nor phase angle of entrainment were obvious in LD or DD; these animals were excluded from further analysis of alpha and phase angle (n=4). Amount of activity was calculated by multiplying half of the circumference of the running wheel by the number of events in each 5 minute bin. Significance of the differences between means were calculated using either repeated measures t-test or repeated measures ANOVA (StatView or SuperANOVA, Abacus Concepts, Berkely, CA, p<0.05).
Representative running wheel activity data for two lobsters exposed to LD, DD, and LL (Figure 4) show that when exposed to a 14:10 LD photoperiod the activity was confined primarily to the D portion of the cycle. Periodogram analyses (Figure 5) produced calculated periods of 23.8 and 24.05 for these two lobsters indicating entrainment to the LD cycle. All lobsters (n=15) entrained to the LD cycle in this way with an average Tau of 23.95 ±0.05h. The average phase angle of entrainment to LD was 0.55 ± 0.45h which was not statistically distinguishable from zero (one sample t-test: t(13)=0.78,p=0.45).
When the lobsters were exposed to DD, significant rhythmicity persisted with Taus of 22.83 and 23.85h respectively (Figure 4 and Figure 5). Clear free-running rhythms were seen in 13 out of 15 animals in DD with an average Tau of 24.14 ±0.26 h. Alpha, the length of the main bout of activity, was significantly longer (paired t-test, p<0.01) in DD than in LD (Fig 5).
Free running rhythms were also observed in 13 out of 15 animals exposed to LL. Figure 4 provides representative examples of one animal that exhibited significant rhythmicity in LL (Tau=29.5 h) and another where significant circadian periodicity was not detected. Overall, the average free-running period in LL was 23.38 ±0.50h. The Taus in LD, DD, and LL were not statistically different (F test (2,22)=1.17, p>0.3). There was no effect of sex, LL intensity on Tau, activity amounts, alphas or phase angles (STATS, t tests).
The overall amount of activity was lower in LD (mean ±SEM M/d) than in DD (mean ±SEM m/d) but statistically indistinguishable from LL (mean±SEM m/d)(Figure 5; F(2,22)=4.10,p<0.05).