O Funkhouser - 2 STRAC An endogenous circatidal rhythm was described in both individuals and populations of the upper intertidal barnacles Balanus glandula and Chthamalus fissus. These rhythms persisted for at least 8 days under constant conditions with peaks of activity initially corresponding to times of high tide. A circatidal period of 27 hours was observed in activity of Balanus glandula, while the period of activity of Chthamalus fissus, 25 hours, was much closer to that of the tidal cycle. No circadian rhythms were observed in any individuals or populations studied under constant conditions or in the field. The hypothesis was made that the adaptive advantage of such an endogenous rhythm would be in the ability to anticipate periods of activity and inact¬ ivity and physiologically prepare for them. C ( Funkhouser — 3 TRODUCTION The upper intertidal barnacles Chthamalus fissus,,Darwin, 1854, and Balanus glandula, Darwin, 1854, are active only during periods of submersion at high tide. Since their activity is so greatly dependent on a predictable oscillating exogènous factor, it seems that anendogenous circatidal rhythm would be an adaptive character. An endogenous tidal clock would allow them to anticipate periods of activity and inactivity and physiologically prepare themselves. Persisting activity rhythms with approximate tidal periodicity have been described in many intertidal organisms. (Palmer, 1974) Several attempts have been made to establish tidal rhythmicity in barnacles to date, yet these have only resulted in observations of rhythms with periods of 25-35 days (Sommer, 1972) and 2-50 minutes. (Southward and Crisp, 1965) An unpublished student paper (Carlson and Roux, 1978) did describe a tidal rhythm in the activity of C. fissus and C. dalli with periodograms based on sampling every 3 hours. Besides these students' study, no work has yet been done docu¬ menting an endogenous tidal rhythm in barnacles. The objective of this study was to show conclusively whether the upper intertidal barnacles, C. Tissus and B. glandula exhibit endogenous tidal rhythms in cirral activity C Funkhouser -- 4 ERIALS AND METHODS Chthamalus fissus, all 3 mm. in width and of the morph with the slit aperature (Morris et al., 1980), were collected from both the RR tracks and breakwater collecting sites (Fig. 1), tidal heights of 1.2 m and 1.5 m respectively, and Balanus glandula, all 4 mm. in width, were collected from the RR tracks collecting site near Hopkins Marine Station in the spring of 1982. During low tide, barnacles, on small pieces of granite, were chiselled from rocks in the intertidal and then mounted in plasicene in rows on three shelves (Fig. 2). They were then submerged in a 2000 liter aquarium at the time they would normally have been submerged at the next high tide in the field. Many physical parameters have been shown to influence the activity of a barnacle, among them: oxygen concentration (Newell and Northcroft, 1965). pH (Southward and Crisp, 1965), water currents (Southward and Crisp, 1965). temperature (Cole, 1932; Southward, 1955) and food availability (Kuznetsova, 1978). To keep these physical factors constant and approximating ambient conditions, fresh sea water was circulated through the aquarium in a very slow, constant flow. An additional study was done in standing water with all other procedures identical, where the water running into the tank was shut off 12 hours before the barnacles were placed in the aquarium. In the circulating sea water, the temperature of the water was 13.7i 0.2 degrees Celsius, while in the standing water the temperature was 14.72 0.3 degrees Celsius. The barnacles were illuminated by a constant red light source reflected off a sheet of white plastic resulting in an intensity of 1.7 lux. All direct light was blocked. (Fig. 2) Activity of barnacles was monitored using a video camera and recorder. Each barnacle was viewed for presence or absense of cirral activity for 15 O Funkhouser -- 5 seconds each 10 minutes. The percentage of time barnacles were active was determined by the percentage of the 15 second periods in which cirral activity was observed each hour. C Funkhouser -- 6 RESU Activit Rhythms in Circulating Sea Water: Most individuals of both B. glandula and C. fissus showed persistant activity rhythms with circatidal periodicity upon visual analysis. (Fig 3, 4 and 5) Although the activity patterns are scattered with irregularity, due to the variability in activity of individuals, there is a definite correlation between the peaks of activity and the time of high tide. This circatidal rhythmicity in some individuals (430, #20 and #13) seems to have persisted for at least 8 days. By summing the activity of the individuals in each of these three pop- ulations, the individual variability was averaged out and the rhythms in activity became much more apparent. Each of the two C. fissus and the B. glandula populations demonstrated clearly visible circatidal rhythms, (Fig 6 and 2) which persisted in constant conditions for at least 8 days. When the two populations of C. fissus, from the two similar collecting sites, were analyzed as a combined population, the rhythm became even more obvious. (Fig. 8) The general increase in activity of C. fissus populations and decrease in activity of the B. glandula population were due to an increase or decrease in the number of indivuals active,rather than a change in the act¬ ivity level of the already active individuals. The period of the rhythm, as determined by visual analysis with special attention directed to the peaks of activity after the 5 day gap in data, was different in B. glandula than in Q fissus, even from colecting sites very close to each other. The rhythm of activity in B, glandula was deter- nined to be approximately 27 hours, while is was approximately 25 hours in C. fissus. No circadian periodicity was observed in any of the data. Funkhouser - 7 Activity Rhythms in Standing Sea Water: Most C. fissus individuals monitored in still sea water expressed tidal activity rhythms similar to those in circulating sea water, (Fig. 9) persisting for at least 60 hours. However, many of the individuals' rhythms were not consistantly in phase with each other or the tidal cycle, so pop¬ ulation analysis gave arhythmic results. The average period of the activity rhythms, as determined by the same method used in the circulating sea water study, was determined to be approximately 25 hours, very near that of the tidal cycle. As in the study with circulating sea water, no circadian rhythm was observed. Funkhouser — 8 IISCUS TON AND CONCL Populations as well asindividuals of both B. glandula and C. fissus, from sheltered locations, showed persistant rhythms in cirral activity when submerged in constant conditions in the lab. The fact that not all indiv¬ iduals', or even populations', activity rhythms had the same period and were not always in phase with each other or the tidal cycle supports the conclu¬ sion that the rhythmic nature of activity was endogenous rather than cued by an exogenous factor. This is corroborated by the result that the C. fissus tested in standing, rather than circulating, sea water also exhibitted rhythmic activity with circatidal periodicity. During a qualitative field study conducted by the author over a two month period, cirral activity in both B. glandula and C. fissus was observed during any period of submersion, regardless of the day-night condition. Considering this, it is not at all surprising that no circadian rhythms were observed in constant conditions. Perhaps, being submerged for such short periods each day, 5-20% of each tide cycle for barnacles at a tide height of 1.6 m.(Glynn, 1965), and sometimes not for 23 days in a row (Glynn, 1965), it is important for these upper intertidal barnacles to be active whenever possible, even at the risk of predation during daylight hours. On the other hand, the utilization of an endogenous circatidal clock would be of tremendous adaptive advantage. Since these barnacles are sub¬ merged for such short periods of time, it is probably very important for them to maximize their feeding time within these periods. An endogenous circatidal clock would enable them to prepare themselves physiologically just before potential periods of inundation — enabling immediate activity response upon submersion. Without such a clock, they would either have to unneccessarily remain continuously prepared for activity, even at times of low tide, or possibly Funkhouser - 9 miss periods of potential feeding at the start of each inundation while physiologically changing from an inactive state. By using an endogenous clock, they would be able to predict times of activity and utilize times of in¬ activity accordingly. This hypothesis is supported by a study in which Balanus balanoides were observed to show a differential response to wetting with respect to time before the next high tide, becoming most responsive just before inundation. (Arnold, 1970) This suggests that some interesting physiological and biochemical correlates of this cirotidal rhythm may prove fruitful for further investigation. Funkhouser -- 10 ERATURE CITED Arnold, D.E. A tidal rhythm in the response of the barnacle Balanus Balanoides to water of diminished salinity. J. Mar. Biol. Assoc. U.K. 50, 1045-1055 (1970). Endogenous tidal feeding rhythms in the intertidal barnacles Carlson, S.J. Chthamalus dalli and Chthamalus fissus. Unpublished paper and Roux, R.D. on file at Hopkins Marine Station Library (1978). The relation between temperature and the pedal rhythm of Cole, W.H. Balanus. J. gen. Physiol. 12, 599-608 (1929). Community composition, structure... Endocladia-Balanus Glynn, P.W. assn. in Monterey Bay, Amsterdam,,Zoological Museum of the University of Amsterdam, 12(148) (1965). Kuznetsova, I.A. Feeding habits of cirripedia, HydroBiol. Journ. 14(3) 29-33 (1978) Cirripedia: the barnacles, in Morris R.H., Abbott, D.P. and Newman, W.A. Haderlie,E.C., Intertidal invertebrates of California, and Abbott, D.P. Stanford University press, 690 pp (1980) Relation between cirral activity and 0 uptake in Balanus Newell, R.C. balanoides. J. Mar. Biol. Assoc. U.K. 45,387-403 (1965). and Northeroft, H.R Biological clocks in marine organisms. New York, John Wiley Palmer, J.D. and sons, 198 pp (1974). Endogene und exogene periodik in der aktividat eines nierderen Sommer, H.H. krebses (Balanus balanus). Zeitschrift vergl. Physiol. 177-192 (1972). 76(2 On the behaviour of barnacles I. The relation of cirral and Southward,A.J. other activities to temperature. J. Mar. Biol. Assoc. U.K. 34, 403-422 (1955). Activity rhythms of barnacles in relation to respiration Southward A.J. and Crisp, D.J. and feeding. J. Mar. Biol. Assoc. U.K. 45, 161-184 (1965). C Funkhouser - 11 JOWLEDGEMENTS I'd like to thank Chuck Baxter for his fruitful words of wisdom and all the people that gave me enthusiastic encouragement throughout the study. C Funkhouser — 12 FIGURE LEGE Fig. 1 —- Diagram showing location of collection sites along the south shore of Monterey Bay in California. Fig. 2 —- Diagram showing front and side views of set-up used. Fig. 3 —- Plot of activity of 3 individual C. fissus, taken from the break- water collecting site and observed in circulating water, plotted ys. time. The tidal and diurnal cycles are included below for visual comparison. The curved zig-zag between 5/21 and 5/26 represents a 5 day gap in the data. Fig. 4 —- Plot of activity of 3 individual C.fissus,,taken from the RR tracks collecting site and observed in circulating water, vs. time. The tidal and diurnal cycles are included for visual comparison. The curved zig-zag between 5/21 and 5/26 represents a 5 day gap in the data. Fig. 5 -- Plot of activity of 3 individual B. glandula, taken from the RR tracks collecting site and observed in circulating water, ys. time. The tidal and diurnal cycles are included for visual comparison. The curved zig-zag between 5/21 and 5/26 represents a 5 day gap in the data. Fig. 6 — Plot of activity of populations of C. fissus, taken from each collecting site and maintained in circulating water, vs. time. The tidal and diurnal cycles are included for visual comparison. The curved zig-zag between 5/21 and 5/26 represents a 5 day gap in the data. Fig. 7 -- Plot of activity of population of B. glandula, taken from RR tracks collecting site and observed in circulating water, vs. time. Tidal and diurnal cycles are included for visual comp¬ arison. The curved zig-zag between 5/21 and 5/26 represents a 5 day gap in the data. O C Funkhouser - 13 Fig. 8 — Plot of activity of C. fissus population, taken from both collecting sites combined and maintained in circulating water, vs. time. The tidal and diurnal cycles are included for visual comparison. The curved zig-zag between 5/21 and 5/26 represents a 5 day gap in the data. Fig. 9 — Plot of activity of 3 individual C. fissus, taken from the RR tracks collecting site and observed in standing water, vs. time. The tidal and diurnal cycles are included for visual comparison. The curved zig-zag between 5/21 and 5/26 represents a 5 day gap in the data. . 99 52 9 0 —— - -- EEE 25 C O 1 O 5o TIDE HEICH 90 NO TIME DATA ACTIVE TAKEN - — — — — — e SN O Go 0 S S SN 8 0 — 0 3 0 0 0 — - — TIME ACTIVE NO DATA TAKEN O — — - - TIDE HEIGH o % - - — DS S DO DC SN 0 20 0 00 0 5 60 65 S % TIME ACTIVE — - — J--- - - - NO DATA TAKEN — -— — — —————— — - HEIGH 00 HEIGHT 9 - - — — - — — - - - NC TIME — - DATA ACTIVE ------- - TAKEN C0 + 0 Ge SN O — O 0 % O 0 O O O — O N O O 00 — — 00 — 0 0 0 % TIME ACTIVE —————— — — ——— TAKEN DATA NO ö C. HEIC Io O 0 0 O - 0 - 60 TIDE HEIGHT o 2 — — NO — — TIME DATA ACTI TAKEN — — — Q N O Q SN C0 — 0 60 — N o 2- 0 ON N 0 0 — TIDE HEICHT o 2 TIME ACTIVE 8