OF. VTEI TEMPERATURE A ALINIT GRIOPUS CALIFORNIC HOPKINS MARINE STATION ANFORD UNIVER June 1 UPON ON OF Introduction The high tidepool, with its tremendous variability in environmental conditions over very short periods of time, can provide a home for only the heartiest of animals. Tidepool dwellers must contend with large daily fluctuations in water temperature. Since such pools receive very little splash, evaporation causes severe extremes in salinity, as well. This paper attempts to examine light, temperature, salinity as high tidepool parameters and show their effect upon the behavior of one such inhabitant, Tigriopus cali fornicus. Tigriopus is an ideal test animal for work in behavioral studies. Approximately one millimeter in length, this copepod is a convenient size, large enough for easy counting with the naked eye, yet small enough so that experimental set-ups can contain large populations to decrease error without becoming cumbersome. The animal's abundancy provides large numbers of individuals from the easily accessible high tidepools. Correlations between activity and water column distribution will be explored. This behavior will then be related to the possible survival strategies of the animal. Although this paper only deals with one particular species, one may gain insight to population behavior in general. Materials and Methods (Lab Studies) Test animals at an approximate density of 1 per 2 ml. of sea water were placed in plexiglass test chambers, approximately 13 cm. high, 18 cm. long and 5 cm. wide. Activity was monitored using small infrared photorelays connected to an Estraline Angus event recorder (the circuitry by Dave Bracher). Figure 1 depicts the relay. Four relays mounted approximately 3 cm. apart on plexiglass strips were placed vertically in the chambers. In some experiments two such strips of relays were placed in one chamber as seen in Figure 2. An additional relay was placed in water devoid of animals to check for occasional spurious counts. In experiments run in constant darkness, photographic paper was placed immediately behind the chamber and an electronic strobe was flashed so that the animals cast a shadow on the paper. Once the paper was developed, these images could be easily resolved and the instantaneous distribution of the animals in the water column could be determined. Experiments were carried out in a controlled temper- ature room. In those runs which light and temperature were varied, the animals were placed in filtered sea water at a salinity of 34 ppt. Light sources consisted of incan- descent and fluorescent combinations. Glass trays filled with water were placed in front of the lights and acted as heat filters. In experiments which necessitated the con- struction of a saline gradient, "Instant Ocean" was used. The apparatus used to construct this gradient is shown in Figure 3. A beaker partly filled with an initial salt solution of 105 ppt. was continually mixed by means of a magnetic stirrer. Its concentration decreased in salinity at a fairly steady rate due to a constant inflow of distilled water. A siphon hose from the beaker fed the test chamber where the tubing was attached to a float. The continually changing saline solution was thus layered in the chamber With the higher concentrations near the bottom. Tigriopus were collected thirty minutes prior to each experimental run to minimize abnormal behavior brought on by extended periods in a lab environment. The animals were gathered during the months of April and May from a single high tidepool at Mussel Point, Pacific Grove, Cali- fornia. This pool received very little wave action. It was approximately 1 meter long and 1/2 meter wide, with its depth varying between five and twelve cm. during the two month interval. The salinity varied between 35 and 75 ppt. It is important to note that activity counts obtained show the number of counts per hour at a particular level in the water column. Counts alone, however, provide no feel for population distribution. A doubling of activity counts, for instance, could mean that: 1. the density of animals at that particular level increased by a factor of two, 2. the density remained constant and the animals doubled their activity, or 3. any combination of varying density and activity took place which would account for a two-fold increase in the number of counts. If the number of animals around a counter, therefore, stays constant for a given set of conditions, then the counter is an activity meter. Through the use of photographs, a population distribution can be observed. Horizontal lines were drawn through each detector in the photo and individuals were counted 1/8 inch to each side of the lines. This is shown in Figure 4. Each activity count for the hour prior to the time of the photograph was then divided by its corresponding visual count and a true activity, or activity per individual was determined. By combining all of the counts one can assay for overall activity. Results and Discussion (Lab Studies) Activity and position was monitored in constant darkness, at 15 degrees C. and at a salinity of 34 ppt. over a 52 hour period; the results for the top and bottom counters are shown in Figure 5. At the top there occurred a very high level of initial activity during the first four hours. The diagram in Figure 6 therefore reflects data after this initial time period. This histogram shows the mean of the overall activity in which each bar represents a six hour interval. A decreasing trend is observed for the first 12 hours which then seems to remain relatively flat for the following thirty. At this point the activity begins to drop off slightly, perhaps due to a depleted source of food. All further data were, therefore, taken during this thirty hour period to assure a steady state condition. Figure 7 describes the mean activity distribution during the steady state interval. The bulk of the activity consistently takes place in the top and bottom regions. It is important to note that the relative flatness shown during the steady state interval in figures 5, 6 and 7 indicates that any endogenous rhythm in activity, if present, is sufficiently slight, and it can be neglected while interpreting further results. After 52 hours, a piece of plankton netting was used to remove the animals from the surface to a depth of approximately one inch. The run was then resumed; Figure 8 shows the results. The first set of bar graphs shows the mean counts per hour at each level in the water column of the four hours prior to the removal of the top layer of animals. The next two depict mean values after two and four hours respectively. At four hours the distribution appears quite similar to that before removal. It appears, therefore, that there are few, if any, individuals entirely of top or bottom dwelling behavior. The vast majority seem to be moving up and down in the water column. It is this majority causing the distribution with which the behavioral study deals. Figures 9 and 10 show the activity per individual at different levels for three different times during the run. One observes that an animal linearly decreases its activity as that animal nears the surface. Near the top it may reduce its activity to a level required to overcome the negative buoyancy of the animal. One can possibly describe the distribution as one of orthokinesis. Since an animal near the bottom is much more active than another near the top, those lower in the water column are more apt to spend less time in that region. Thus there is a higher probability of finding a particular animal in the upper areas. After a baseline was established, various stimuli were added and their effects compared. Figures 11 and 12 show the response of Tigriopus to an artificial light cycle over a 24 hour period. These runs were made at 15 degrees C. and 34 ppt. The top histogram shows counts per hour on top as a function of light intensity and time. As the intensity is increased from darkness to 110 LUX, there appears a slight increase in activity. A dramatic increase in activity on the top then occurs upon the transition to 1600 LUX. As the intensity is further increased to 3500 LUX, however, a drop in surface activity is apparent. When the intensity is again set to 1600 LUX, the high activity resumes. The decreasing levels of light intensities are then characterized by the corresponding decrease in surface activity. The bottom histogram, showing a plot of activity per individual, takes on the same relative shape as the upper graph. Light, therefore, greatly affects activity. At some intensity around 1600 LUX there is a marked increase. If activity in this case is directly related to actual numbers of animals, then at this level more animals will be in the water column, and in particular on top. This may be a selective advantage. Perhaps the animal can readily detect food, predators, waves, etc. at this level. This would make the early daylight hours and the hours slightly before dusk conducive to feeding activities. During the bright midday, a decrease in activity would certainly help protect the animal from predation as well as harsh conditions brought about by exposure to direct sunlight. The effect of temperature upon the animals' behavior was tested and the results are shown in Figure 13. The salinity and constant darkness conditions were maintained as in previous baseline studies. The temperature was kept at 5 degrees C. for four hours, then linearly increased to 23 degrees C. spanning a 24 hour period. Both activity on the top and activity per individual, or total activity are plotted. Again the shpaes are relatively the same. The interesting feature is the flat region in which activity does not vary as a function of temperature. These findings seem to be in accord with Newell's work (Newell, 1967 in Prosser, 1973) dealing with the resting metabolism of four marine invertebrates. His graphs showed a similar region where standard metabolism was independent of temperature. The active metabolism of Tigriopus (Fahey, 1977) appears to have a 0.10 of about 1.8 in this temperature region. The animals monitored by the activity counter, however, were undisturbed, having a minimal amount of activity. This would more accurately be a measure of resting metabolism. It is interesting to notice that the flat region in the curves do correspond to approximate temperatures for the tidepool during the months of April and May. This may be another selective advantage in that the activity of a naturally occurring population of animals may be temperature compensated. Another experimental run compared activity to various saline concentrations. A salinity gradient was set up in a container, varying from a maximum of 61 ppt. at the bottom to a minimum of 6 ppt. at the top. All other parameters were identical to the baseline conditions. The results in Figure 16 clearly show an activity peak around 35 ppt. one hour after the population was introduced into the gradient. Activity rapidly falls off to each side of this peak. A similar shape can be seen from data at 14 hours. The curve, however, is shifted to a much lower salinity, with the peak centered at 18 ppt. At eighteen hours, the shape remains the same, again peaking at about 18 ppt. with a substantial decrease in activity. At the three time points under observation, photo- graphs were taken. Note the bands of high animal concen- tration in the photo of Figure 17. Points for activity per individual were calculated and plotted as a function of salinity in Figure 18. (Photographic counts less than five were not used as data points since the reliability of the value was poor). The three-line plot clearly shows a decrease in true activity as time increases. It is important to note that the slope of the 1 hour and 14 hour lines are equal. If the animals were using orthokinesis to cluster at a given salinity, one would expect that, in that region, the animals would be least active and that activity would increase as the animals deviated from that salinity. The Figure 16 data, however, contradicts this by the low activity readings at the higher salinities. Materials and Methods (Field Studies) Field studies were carried out in the same high tidepool from which test animals for the lab studies were gathered. A sampler, depicted in Figure 17, was construc¬ ted to examine water column distribution. It consisted of two pieces of plexiglass, approximately 1/2 meter long. 13 cm. wide and 2 cm. thick. Holes 2 cm. in diameter were drilled in one piece with centers approximately 3cm. apart. A sheet of plankton netting was glued to one face of this piece. Both plexiglass sheets were vertically placed in the center of the tidepool. The side with holes faced the solid sheet at a distance of about 20 cm. The drilled piece was laterally moved toward the other until both were flush, trapping the animals in various compartments. The number of individuals in each was counted and an overall water column distribution was determined. Samples were taken under various conditions. Results and Discussion (Field Studies) The findings of four field samples are shown in Figure 18. It is interesting to note the distribution in darkness. The histogram for actual distribution is quite similar to that for mean activity shown in Figure 7. Large differences, however, do appear in comparison to that distribution shown in Figure 4. Discrepancies may result in that animals on the bottom may be swept up as the sampler is moved. If one examines the distribution on the top, similarities to the light experiments can be noticed. There are the greatest number of individuals at the top for intermediate intensity. For instance more animals are congregated near the surface at 500 LUX than either at darkness or 3600 LUX. Combining this with the lab findings, one can hypothesize that at some intensit perhaps around 1600 LUX, the increase in surface activity correlates to an actual increase in surface density, Summai 1. A population of Tigriopus californicus in constant darkness, 15 degrees C. and 34 ppt. salt water will distribute themselves such that the majority of the animals are at the top and the bottom. Those nearest the bottom, with the exception of those resting on the bottom, have a higher activity than those animals higher in the water column. 2. The bulk of the population is somewhat homogeneous in behavior in that there appears to be few, if any, having distinct bottom-loving or top-loving tendencies. The majority seems to periodically move up and down. There seems to be some intermediate light intensity, around 1600 LUX, causing density increases at the top of the water column. 4. The resting metabolism of a Tigi riopus population over the temperatures of a naturally occurring tidepool seems to be temperature independent. 5. An actual preference to a salinity lower than sea water, about 18 ppt., seems to be apparent in an animal population. te ure ile at Hopkins Mari hed paper on ahe; 977. Unpublis. M., . Comparati sser C. J. 3 Animal. Physiolog y, W. Saunders Co., Philadelpl Aknowledgemer thank Robin Burnett for all of his wish ingeniu the one about the salinity ons, es cial. gradier Thanks also to Dave I 3ahe for the i itry and to all the members of the Burnett I a fun quar fo 1- Fig Figur e Fig adient, Enlarged tector w of i lexiglas test chamber and mounted Set-up for cons ructing salinit 2 FIGURE 1 PLASTIC TUBING FLOAT PHOTORELAYS FIGURE 2 INITIALLY 3X SEA WATER. MAGNETIC STIRRER DISTILLED 1,O FIGURE 3 opus gure Photograph Tig chamber at constant darkne 34 ppt. butior C. and FIGURE 4 Fig Baselin data showi r hour period of counts in constant d C. and 34 ppt. hou arknes: — 4 — —) — + + + —X— — — — X 8 +X —+ 1 10 X + * + 1 â 4 X 11 - + —*— — 1 + 2 —1— 4 0 —— — Li — — -+ 9 — — +--- — 1 1 .. .. —— + gure rap show baselil ivity. data for overall a ach bar sents a 6 hour period., raph Figure showing the mean act: of the steady state region in the baseline d +. . t o 10 8 10 1 1 u 1 0 —— S —+ ) n 1 — — — — B — OVERLL ACTIVITY n- + — o 5 10C + 0 0 +r0. 0. 6 1) 1 0 910 0 1 90 56 + u u — u 1 — n 1 11 n 14 0 I 0 IX 1 9 + ME (EACHA2S — IGURE + TV MEAN ACTIVI —— X n230 2 — 10— 10— 6 + 0 ++ 1 L 08 — FGURE + distributior Figure The top histogram shows bef ore the tor of animals was remove The layer middleg 2 hours aftert es the di tribution removal and the bottom gr aph shows the populati at 4 hour: PRIORTO REMOVAL (N=4) + + X 1— O — — 0 11 — 0 0 1 — 10 A 10 4 —11— e — — 1 + BOTTOAE + + HouR5 (N=2) 10 O + +0 in 0 1 J2o 1 1 — 1— +4 11 + 0 4 11 1 — — 1 1 — TOP 60770 — — + A ROUR c 0 1 0 x 0 9 1 1 + + — — — — 11— 1 0 — — . + GURE + LLttt + Figure plot activity per individual as l location funtion o fverti in constant darkness 5 C. ind 34 ppt. Tigure 10 showing data fr A table om which ph in was obtaine — DE GUre9 .. 1.0 o + . — â . — —0.! — StHus 4 ou E 1 E Bor IDA 1o ACTNTT ACT. PER 19 — —.. PNDTO COUNTS INDIVID. 43 1.30 12 HRS BOTTON 14 1.17 3.80 2 6.9 11 10 — 22 46 0.48 BoOA 3RR 28 —.18 3. 9 0.82 11 12 0.58 7 2 10P O.24 17 — — —1— 48 135 SOTTOA 28 1.07 30 9 11 O.82 2.5 6 041 177 23 77 —— FIGURE10 MhE: — - he top re 1 graph the pro dure shows carrying out light response experiments Nine different steps were used beginning with darkness over up to 3500 LUX and back to darknes: a 2 hour per he middle pic ture bal of the mean activity on tor for lig t interval The bottom shows overall activit 75 u — + 584 — — 5 3500 —0 1.00f 100O — — + tie 0 +0 455E + 4.5 T 0 —— — — + — + E (S 8 — EON ACEVH op (CounTs 9 9 0 20 8 9 9 -0 0 + 05 o u — 8 — H oy S A1 - + (PERHOUR — 0 0 0 0 9 1 0 — 0 c 10 9 0 0 — + — + — FIGURE + — igure rap ba shownf interval expressing activity in counts a function of vertical distribution. each light hour 3 + 88 0 111 O n — ++ t — ODARKMESS t + — — 0 a ato + 0 o 9 1U 010 NN N N1 ke IV + OLuX + — — —N O — ++ O 1n + N- n1 X 1 4 0 10 0 1 0 10 O O 0— 0 11 V V 1 - 0 1X 90 1 FE N n 1 o O N 1 „ n — — + â +0 4.5 Lu 10 ++ + + — o + —+- 8 0 NO 0 u 0 J0 1 0 u b IXIX MO I Ix 11 S — + 3500 1UX Ftoottug — + — o 10 S ONO 1 0 E X 1 1 e 10 IVIXE N U N — 10 DARENE 9 4.5 Ux ( FguRe — igure top as ature l ast Counts per hou for both act al activity as functions oft . — + ++ + 10 + 5 p E + .: St + — — — HOTA 8 er X + ANACEV I cous 3 —+ TOP S S S + — + —— + — 20 10 15 C. EMPERATURE C H FIGURE Vs. hour Counts per salinit s after being int. roducedt 14 hrs, and hour, the salinity gradient I. 44 0 6 50 40 8 S o + +2 —3 0 Jo + + 0 + 1+ + ++ X HOUR 14 HRS — —18Hes *4 50 10 60 30 40 20 SALINIT (eer) I + FIGURE Photograph of gure alinity gradient at 14 hou distribution 12 FIGURE 15 -70 salini Plot of individual act ivt E HOUR M=-0.0370 Ré=0.94 S 0 L 1 + + — 4 HOURS 2 A- S -O.05 —R0.85 1 — 1 — + — a S +e 18 HOURS — — — — A--0.04 — -0.48 S Ra 4 + + + — — + — 40 50 10 20 — 30 SALINT (PPT) — FIGURE 16 ++ 2 —— — E from disrt igu Apparatus used to gather animals their vertica! idepools ande turalt bution in the water column. Ooo D ge O O O D00 —13cm- J FIGURE 17 plexiglas Tigur Histograms showing of data taken in ield. fferent * 11 o E R — S os L o 0 W o R C T do1 lg W O 9 S 8 sndob 4 L 4