Environmental factors affecting the patterns of activity in the marine isopod Cirolana harfordi (Lockington, 1876). Chris Harrold Hopkins Marine Station of Stanford University Pacifio Grove, California. INTRODUCTION lirolana harfordi is a marine isopod which occurs most abundantly under rocks in the high intertidal zone from British Columbia to at least Ensenada (Ricketts and Calvin, 1968). The animal has also been observed at night actively swimming in the swirling water of breaking waves (author, personal obser- vation). Of the relatively small amount of information accumulated on C. harfordi very little has been directed toward the ecological relationships between the animal and its environment. Investigation of the aspects of feeding and energetics in C. harfordi has shown, among other things, that although it will actively seek bait in the form of pieces of fish during both day and night, activity is higher during the night than during the day (Johnson, 1973). Although generally considered to be a scavanger, Johnson feels they are primarily predators upon marine invertebrates such as small marine annelids. Brooding females are known to go for months at a time without eating anything. Ecological work on another cirolanid, Excirolana chiltoni, has demonstrated that activity patterns in this animal are closely tied to tidal rhythms (Enright, 1965). This species remains buried in the sand except during a 4- to 6-hour interval shortly after the tidal crest on the beach of collection, and the swirling action of the surf (as simulated in lab) initiates the pattern of activity. Isopod ecology studies performed at Hopkins Marine Station in 1973 suggested that within the period of high activity during nighttime hours there is a sharp peak in activity that has not been correlated to any obvious environmental factor. Finally, it has been shown that the benthic intertidal amphipod Synchelidium spp. shows bursts of swimming activity in response to small, short-term changes in hydrostatic pressure which may be experienced by the animals as waves peak just before breaking. This may be a mechanism by which the animals maintain themselves in this zone (Digby, 1961). A similar situation may exist in C. harfordi. The purpose of the present investigation was to establish the activity patterns of C. harfordi and to identify the environ- mental factors which were most influential upon these patterns. The factors which were felt to be the most important were light, depth of water, disturbance of substrate in which the animals occur, and water temperature. MATERIALS AND METHODS The investigation was divided into two parts. Activity patterns and the most probable influencing environmental factors were first measured in the field. The factors which most closely correlated to activity patterns were then simulated in the lab- oratory to establish the relative importance of each. Field study. The level of activity was ascertained by counting the number of animals caught in baited traps over a given length of time. 10 traps were spaced at 4-m intervals along a 45-m transect line running perpendicular to the shoreline. The traps were numbered from 1 to 10 going from +92 cm to -116 cm tidal level (see Figure 1). The cotton lines of the traps could be clipped on the transect line allowing easy removal and replace¬ ment of all traps in a short time period (see Figure 2). The traps could then be taken into the lab to remove and count the animals. A trap consisted of a plastic freezer jar 7.5 cm deep and 9 cm in diameter. 9 holes of 7 mm diameter were drilled in the bottom which allowed the animals to crawl in. The plastic screen cone guided them into the trap and prevented their exit. The cotton lines were used to attach a 1.5-pound diver's weight to the trap and to attach both the weight and the trap to the transect line. The transect line consisted of +inch polyethylene braid anchored at both ends by lag eye-bolts embedded in granite by expansion sleeves and epoxy. The bait consisted of weighed pieces of squid wrapped in fine nylon cloth to prevent the squid from being eaten. The bait was replaced every three hours. Since preliminary experiments showed essentially no activity during daylight hours traps were checked every hour from 1800 to 2400 hours on one night and from 2400 to 0600 hours the following night. As traps were collected depth, temperature, wave period, and wave height were recorded. Laboratory study. The behavior of C. harfordi in the laboratory made it a difficult subject in which to determine patterns of activity. If placed in a tub of seawater the animals ceaselessly swam about and showed continuous activity. If running seawater was placed in the tub the animals oriented toward the current and eventually crawled up the hose and invaded the seawater system. The experimental chambers therefore consisted of aquaria with one rock and 5 cm of sand on the bottom. Each rock was weighed to insure all aquaria contained a rock of similar size. These conditions closely matched the natural under-rock habitat of the animal. Once the animals were introduced into the aquaria they immediately burrowed into the sand or under the rock and remained there for indefinite periods of time. If, however, 10 ml of squid-water (one 40-g squid and 400 ml seawater put in a blender at high speed for 2 minutes, then strained) was added, within minutes the animals came out of the sand, swam about for a few minutes, and returned to their hiding place. In each experiment the response time (e.g., the time interval between the introduction of the food stimulus and the appearance of the first animal) was used as the criterion for level of activity: a lower response time indicated a higher level of activity. In some experiments 8 minutes was the time limit beyond which no response was considered to occur. 10 minutes was the time limit used in later experiments. While many environmental factors interact to influence the activity patterns of C. harfordi, laboratory experiments were restricted to light, depth, and water turbulence. Experiments to test the affects of light upon activity patterns were conducted in three aquaria illuminated by three General Electric F72712 cool-white fluorescent tubes placed 40 cm above the water surface. Each aquarium containing 20 animals was placed under constant light, constant dark, and light-dark conditions (lights off from 2000 to 0600 hours in the light-dark cycle). Response time and water temperature were recorded every two hours for 24 hours. Experiments to test the affects of tidal rhythm upon activity wore conducted in three aquaria, each containing 20 animals, and placed under the three light conditions described above. In the constant light and the light-dark aquaria the water level was lowered from the normal 25 cm depth to 3 cm above the sand from 0400 to 1000 hours during a 48-hour period. In the constant light aquarium the water level was lowered from 2100 to 0300 hours for a 24-hour period. This duration of low water simulated the duration of low tide at the time and location from which the animals were collected. Response time and temperature were recorded every two hours except for a 4-hour break in the 48-hour experiments. Experiments to test the affects of water disturbance upon activity were conducted in three aquaria, each containing 20 animals and placed under the three light regimes previously de- scribed. Water disturbance was created in the aquaria by a 120- volt, AC electrio stirrer connected to a timing device allowing the motor to run for 4 seconds, stop for 9 seconds, and run for 4 seconds repeatedly for six hours, after which six hours of quiescence followed. This cycle was repeated for 24 hours. The response time and temperature were recorded every two hours. In all experiments, the flow rate in the aquaria was ad- justed to between 1240 ml/min and 1320 ml/min in order to keep the dilution rate of squid water the same. During times of low water in the tidal rhythm experiment the squid-water was diluted by the appropriate amount to account for the reduced volume of seawater. Laboratory animals were trapped as previously described. Males and non-brooding females will satiate themselves if given a large enough piece of food and will not eat for a week or two afterward. Presumably they will not respond to a food stimulus during this time so the traps selected for hungry males and non- brooding females. This eliminated a potentially large source of error in response time in the laboratory. Animals were used no longer than 8 hours after collection. They were allowed approximately 6 hours to acclimatize to the experimental conditons. SULTS Field study. Original data showed that variations in temperature. wave period and wave height were insufficient to account for changes in activity, and the patterns in the fluctuations of these faotors did not correlate with fluotuations in activity levels, as shown in Figure 3. The activity pattern of the field population is presented in Figures 4-6 which present both the number of animals found as a function of time and the water depth at trap sites 6. 7. 8, and 9, respectively. Due to an unexpectedly low tide on May 28 traps 1-5 were above water throughout the entire 6-hour period. Consequently data from these traps were not collected. No more than one animal appeared in trap 10 at any time so the results were not presented. Figure 4-a shows that peak activity occured at 0100 hours. This the is the time at which the water level reached 25 cm. Figure 5-a shows peak activity again occured at 0100 hours. Figure 5-b shows the close correlation between time of peak activity and the time at which the water level reached 25 cm. Figuro 6-a shows that the activity peak occured at 0200 hours, an hour later than in any of the othor previous graphs. Figure 6-b shows that water depth reached its shallowest point between 2400 and 0200 hours. Peak activity oocured within this time in- terval. Figure 7-a shows that a much lower peak occured an hour later, corresponding to the ebbing tide which was occuring at this time. The water depth never even approached the 25 cm depth found in Figures 4, 5, and 6. The corresponding activity level did not approach the height that it did in Figures 4, 5. and 6. Figure 8 shows that a definite peak in activity occured at the 20-40 cm depth interval, corresponding to the peaks in activity found at the time the water level was 25 cm at traps 6 and 7, and corresponding to the lower peaks of activity which occured at the time minimum depth was reached at traps 8 and 9. Laboratory study. The results from the experiments testing the effect of light on activity patterns are represented in Figures 9-a through 9-c. The response times shown here are aver- aged response times from three different experiments. A response time above 9.5 minutes was considered no response since after 8 minutes the response-time determination was ended in one ex- periment and after 10 minutes it was ended in two other exper- iments. Activity level was much lower (higher response time) in constant light than in constant dark. No significance was ascribed to the fluctuations during the 24-hour test period in constant conditions. The activity level increased markedly during the dark period of the light-dark cycle. Some antici- pation of the change in light conditions was shown. The results from the experiments testing the affect of tidal rhythm upon activity level in the three light regimes are shown in Figures 10 and 11. Response times shown are those ob¬ tained from one experiment. 8 minutes was the time limit within which a response had to occur in Figure 10, and 10 minutes was the time limit in Figure 11. In constant dark the activity level was very much affected by tidal cycle; the level of activity was lower during time of low water and increased when the level of water was raised. In the light-dark cycle activity was cued on the light only, tidal levels notwithstanding. The results of the experiments testing the affect of water water disturbance upon activity level in the three light regimes re shown in Figures 12-a through 12-c. Response times shown are those obtained from a single experiment. 10 minutes was the time in which a response had to occur. Figures 12-a and 12-5 show no detectable pattern in activity level. Figure 12-c shows the typical pattern of response time in the presence of the light- dark cycle seen in Figures 9-c and Figure 10. DISCUSSION It is not surprising that C. harfordi did not appear to cue its activity pattern to temperature. During cold, foggy 10 days the water in the high intertidal probably does not fluctuate any more than the ambient sea temperature and would not signal to the animals that conditions are favorable for high levels of activity. The confirmation of the nocturnal habits of C. harfordi is not unexpected. Birds are known to be heavy predators on terrestrial isopods. Marine birds, which are likely to show diurnal activity patterns, may have been an important predator upon C. harfordi and through evolution have applied heavy selective pressure for the isopods to adopt a nocturnal lifestyle. Day- time would also provide heavy physiological stress upon an ani¬ mal venturing out at a low tide under a high noon sun. The data strongly suggest that the animals have an opt- imum depth of 20 to 40 cm which raises many interesting questions. Do they remain in their homes until the water level directly over their heads reaches 20 to 40 cm, or do they all come out at sunset and merely follow the tidal cycle up and down main- taining a position at 20 to 40 cm? It is impossible to tell from the present data. What are the advantages in maintaining this optimum depth? Perhaps under selection pressure from deeper vater predatory fish a behavioral pattern has evolved keeping isopods from venturing into deep water. At the same time ex¬ cursions into shallower water presents the possibility of being caught in a high tide pool as the tide was ebbing away. 11 Bofore any dofinite conclusions can be drawn, however, much more field data must be collected to present a statistically valid sample size during all phases of the tidal cycle. The possibilities are nonetheless intriguing. MIARY C. harfordi shows definite nocturnal activity patterns. Of the four environmental factors examined, light and water depth affect the animals' level of activity. Light is the most important of the two; only in the absence of this factor does water depth appear to influence the activity of the animal. 12 References: Burbanch, W.D., 1962, An ecological study of the distribution of the isopod Cyathura polita from brackish waters of Cape Code, Mass. Amer. Midland Na ralist, 67(2): 449-476. Digby, P.B.S., 1961, Mechanism of sensitivity to hydrostatic pressure in the prawn, Palaemonetes varions, Nature, 191: 366-367. Enright, J.T., 1962, Response of an amphipod to pressure changes, Comparative Biochemist try and Physiology, 7: 131-146. - 1965, Entrainment of a tidal rhythm, Science, 147: 864- 867. Hayes, W., 1969, Ecological studies on the high-beach isopod Tylas, PhD thesis, University of California, San Diego. Johnson, W., 1973, Personal communication. Jannson, B.O., 1968, The diurnal activity of some littoral pera- carid crustaceans in the Baltic Sea, Journal of Experimental arine Biology and Ecology, 2: 24-36. Lockwood, A.P.M., Aspects of the Physiology of Crustacea, W.H. Freeman & Co., San Francisco, 1967. Ricketts, E.F., and J. Calvin (revised by J. Hedgpeth), Between Pacific Tides, Stanford, California, Stanford University Press, 1968. The Marine Isopod Cr taceans, Dubuque, lowa, Schultz, S.A., Wm. C. Brown Co., 1969. CAPTION Figure 1. Profile of the study area defined by the transect. Trap site are marked by solid triangles. Figure 2. Trap used to determine levels of activity of C. arfordi in the field. Figure 3-a through 3-d. Water temperature as a funotion of time for traps 6 through 9, respectively. Figure 4-a. Activity of the field population at trap 6 as a function of time on May 26 and 28, 1973. Arrows indicate sunset and sunrise. Figure 4-b. Water depth as a function of time at trap 6 on the same two days. Figure 5-a. Activity of the field population at trap 7 as a function of time on May 26 and 28, 1973. Arrows indicate sunset and sunrise. Figure 5-b. Water depth as a function of time at trap 7 on the same two days. Figure6-a. Aotivity of the field population at trap 8 as a function of time on May 26 and 28, 1973. Arrows indicate sunset and sunrise. Figure 6-b. Water depth as a function of time at trap 8 on the same two days. Figure 7-a. Activity of the field population at trap 9 as a function of time on May 26 and 28, 1973. Arrows indicate sunset and sunrise. Figure 7-b. Water depth as a function of time at trap 8 on the same two days. Figure 8. The total number of animals in the field population found at a given 20-cm depth interval throughout the 12-hour eriod. Figure 9-a. The activity pattern of laboratory animals under constant light conditions. Figure 9-b. Activity pattern of laboratory animals under constant dark conditions. gure 9-c. The aotivity patterns of laboratory animals under light-dark cycle. The double line on the horizontal axis indicates the period of darkness. Figure 10. The affect of tidal rhythm upon activity level in the absence and in the presence of the light-dark cycle. Verticle dotted lines indicate duration of high and low water. Double line on horizontal axis of light-dark graph indicate periods of darkness. Figure 11. The affect of tidal rhythm upon activity level in constant light. Verticle dotted lines indicate periods of high and low water. Figure 12-a. The affect of water disturbance upon activity level under constant light conditions. Figure 12-b. The affect of water disturbance upon activity level under constant dark conditions. Figure 12-0. The affect of water disturbance upon activity under the light-dark cycle. Verticle dotted lines indicate periods of water disturbance created by stirring. 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