Circadian rhythms are endogenously controlled physiological or behavioral activities with periods of about a day and have been demonstrated in nearly all major groups of living organisms. Several species of crustaceans, including crabs (Rebach, 1985), amphipods (Kennedy, et al., 2000), crayfish (Page and Larimer, 1972), and the New Zealand lobster (Williams and Dean, 1989), exhibit circadian rhythms of locomotor activity. Comparatively fewer studies have focused on physiological changes in crustaceans, but several parameters have been measured, including color changes (Brown and Webb, 1948), oxygen consumption (Brown, et al., 1954), and heart rate (Pollard and Larimer, 1977).
Heart rate has been shown to be strongly affected by time of day in some crustacean species. A nocturnal increase in heart rate has been demonstrated in shore crabs (Aagaard et al., 1995) while circadian control of heart rate has been demonstrated in crayfish (Pollard and Larimer, 1976). In other crustacean species daily or circadian control of heart rate is unknown. In lobsters heart rate does increase during forced exercise (Rose, et al., 1995) and in response to temperature or salinity changes (Jury, et al., 2000) but its temporal modulation is unclear. The goal of our experiments was to determine if locomotion and heart rate was modulated by time of day and/or the circadian system of the American lobster, Homarus americanus .
Animals and Environmental Conditions :
Lobsters Homarus americanus used in Experiment 1 (2 male/4 female, 450-670 g, carapace length of 82-94 mm) was obtained from a local commercial supplier in January, 2001. Lobsters used in Experiment 2 (1 male/5 female, 440-620 g, carapace length of 83-93 mm) were obtained from the same supplier in September, 2001. They were promptly placed in a 55-gallon marine tank (Oceanic 55", Jewel Industries Inc., Chicago, IL). Seawater was obtained from UNH marine coastal laboratory in Newcastle, NH or the Jackson Estuarine lab in Durham, NH. The water temperature was recorded continuously (HOBO, Onset Computer Corporation, Bourne, MA) and maintained at 17 o C + 1.5 o C. The salinity of the water was determined three times per week and maintained between 28 and 36 ppt. To maintain a relatively constant volume in the tank we added distilled water approximately once weekly. The pH level of the water was maintained at 8.0 + 0.5 by adding Sea Buffer (Aquarium Systems, Mentor, OH) approximately weekly. Two bags of Green-X phosphate/nitrate remover (Hagen, Inc., Mansfield, MA) were added to the filtration system in order to stabilize the NO 3 levels between 25 and 100 mg/1. Light was provided using two broad-spectrum, 20-watt fluorescent lights (Simkar Corp., Pittsburgh, PA). The light level at the surface of the water was 25 lux.
We constructed the locomotor activity recorders (running wheels) used in our experiment from five-gallon high-density polyethylene pails. They were perforated extensively in order to allow water circulation and also to provide a surface that the lobsters could grip. The inner diameter of each wheel was 30 cm and the inner width was 10 cm. A hollow, 1 cm diameter axle was placed in the center of the wheels and then fixed to a PVC frame. Two magnets were affixed 180 degrees from each other on one side of the outer surface of the wheel. A magnetic reed switch to detect wheel movement was located on one leg of the stand. Activity data from this system were continuously recorded using a Drosophila Activity Monitoring System (Trikinetics, Bourne, MA) and a Macintosh computer.
Experimental Design :
Experiment #1:
Lobsters were placed into the running wheels and subjected to a 14:10 LD cycle for five to six weeks before we began the experiment. Several days passed before the lobsters showed obvious signs of walking rhythmically. After this time period, wires were inserted through the dorsal carapace dorsal to the heart and the lobsters kept in LD for an additional ten to fourteen days. Subsequently, a 13-day period of constant darkness (DD) ensued. Data that are presented here were recorded from 2/13-3/14/01.
Experiment #2:
The goal of this experiment was to begin to determine if daily heart rate modulation was simply a reflection of running wheel activity or perhaps modulated by time of day directly. Lobsters were placed in running wheels and activity and heart rate recorded within two days of being purchased. They were exposed to a 14:10 LD cycle throughout the experiment. After 10 days, a clear plexiglass partition was placed in the running wheel that prevented the lobster from moving forward or backward more than a few cm or turning the wheel. Four of the six lobsters were also further restrained with cable ties to prevent them from rolling laterally. This experiment was performed from 10/14-11/7/01.
Wire Placement : 
Two holes were drilled 0.5 cm apart on the midline of the dorsal carapace of each lobster, superficial to the heart, using a 28 guage needle. Lengths of wires that had had 1mm of outer insulation stripped were inserted through these holes approximately 1mm. These wires were fastened to the carapace by cyanoacrylate and a small 1" by 2" rectangle of duct tape. Two additional layers of duct tape and cyanoacrylate provided strain relief. A 2" piece of aquarium tubing was placed between the exit points of the wires from the tape and the axle of the running wheel. This helped to prevent the lobsters from chewing or detaching the wires. The wires were connected to an impedance converter (Model 2991, UFI, Morro Bay, CA). Output from the impedance converter was sent via a coaxial cable to a Powerlab 200 A/D converter (iWorx, Dover, NH). The converter was connected to a Macintosh computer, which continuously saved the heart rate data.
Average heart rates were calculated for five-minute intervals for each lobster using a macro program in Chart for Powerlab (version 3.6.1, iWorx, Dover, NH). Results were spot checked to ensure accuracy. Occasionally heart rate data were not recorded due to signal drift or damaged wires. In the case of missing data for an entire five-minute period, negative values were inserted in order to create missing data for the statistical program. If, within a five-minute period, a fragment of intelligible data remained, then those data were averaged and the result was used in place of a full five-minute entry.
Our results demonstrate for the first time circadian rhythms of locomotion in Homarus americanus . Our findings are similar to several other species of crustaceans including crabs (Rebach, 1985), amphipods (Kennedy, et al., 2000), crayfish (Page and Larimer, 1972), and the New Zealand Rock Lobster (Williams and Dean, 1989). Overall, our results demonstrate the existence of an endogenous circadian clock controlling locomotor activity in Homarus .
Our results also demonstrate for the first time that heart rate of Homarus americanus is modulated by time of day and by the circadian system. The 50-100% nocturnal increase in heart rate is similar to the nocturnal increase seen in LD cycles in crayfish (Page and Larimer, 1972), and crabs (Aagaard, et al., 1995). In addition, endogenous circadian control of heart rate has been previously shown in crayfish (Pollard and Larimer, 1977), and crabs (Rebach, 1995). Our experiment demonstrated that the circadian clock also controls heart rate in Homarus .
An important issue to be addressed is whether the increase in heart rate is simply a reflection of the increase in locomotor activity in LD and DD. Rose et al. (1998), using a forced walking technique, previously demonstrated a tight coupling between exercise and heart rate. When exercise began, heart rate quickly increased to a certain level regardless of walking speed. Not surprisingly, we also found evidence for a tight correlation between heart rate and locomotor activity in both LD and DD. The activity patterns in LD were virtually identical and the free running periods in DD were statistically indistinguishable. Our actograph data shows apparently identical temporal pattern of activity in heart rate and locomotion (Figure 1).
We have two lines of evidence however which suggest that there may be some differential control of wheel-running activity and heart rate. First, in LD there is evidence for anticipatory increase in running wheel activity several hours before D, but no anticipatory increase in heart rate activity (Figure 2). This is quite different than the nearly instantaneous increase in heart rate when shore crabs begin to move (Aagaard, 1995). As a second line of evidence, when lobsters are substantially prevented from moving, heart rate rhythms are present in 3/6 individuals. While our experimental design does not rule out the possibility of isometric activity (which could drive heart rate rhythms directly), the results do suggest that the daily (and perhaps circadian) modulation of heart rate is at least in part independent of the daily (and circadian) modulation of wheel-running activity.