Tolerance of Tigriopus californicus, (Baker, 1912) to Slow Increases in Salinity Produced by Evaporation and Hypersaline Solutions. by David W. Stoller Biology 175H Hopkins Marine Station Pacific Grove, California 93950 Spring 1977 Abstract The susceptibility of Tigriopus californicus (Baker, 1912) from the coast of Central California to high salinities was studied using evaporation and hypersaline solutions. The EDgo for coma occurring during 2 days of evaporation was 144 %. with a 95% confi¬ dence interval of less than + .003. The EDgo., for three different rates of salinity increase produced by the addition of hypersaline solutions were 132.2, 145.2, and 154.5 % with fpp:g of 1.02, 1.01 and 1.01 with 100% coma occurring at 10, 40, and 70 hours respect¬ ively. Fifty percent of the animals became inactive but not comatose at a salinity of 93.3 %0 with a 95% confidence range of 97.97 to 88.86 %0 during a 2 day period of evaporation. Females with egg sacs appear to be more resistant than the general population. Tolerance of T. californicus to High Salinities Introduction The comatose condition as observed in Tigriopus californicus is one in which non-responsive body movement is observed following exposure to stress. The potential to recover to the active state separates coma from death. The ability of T. californicus to withstand in laboratory studies salinities in excess of 190%0 (Issel,1914) was confirmed by Egloff in his studies of a natural tide pool in which T. californicus were observed to be inactive subsequent to an increase in the pool's salinity over 44 days from 43 %o to 200 % (Egloff, 1966). After an additional 12 days in salinities in excess of 300 %the animals showed recovery within two weeks after being placed in normal seawater. Work on the copepod Tigriopus fulvus indicated that the time required for recovery in normal seawater was proportional to the hypertonicity of the affecting solution (Ranade, 1957). Studies on the effect of evaporation and the effect of increasing salinities prompted Patterson to suggest that T. californicus was capable of osmoregulation. However, this has not been confirmed. Patterson also described swarming and clumping behavior in T. californicu in salinities exceeding 200% seawater with loss of activity in 300% seawater (Patterson, 1967). Evaporation with resultant salinity increases is a common occurrence in high tide pools. Variation in salinities is an import ant variable in the environment of T. californicus. There have been few studies of the adaptation of T. californicus to gradual increases in salinities. Rates of salinity changes, specific salinities and time considerations relative to the comatose Tolerance of T. californicus to High Salinities and recovery states are key subjects which have yet to be examined. Differences in population density and sex ratio have been noted in the field (Nimkin, 1977). The effect of salinity changes with respect to population size and structure is an interesting subject for study as well. Past work has only superficially explored the physical and behavioral characteristics of T. californicus in coma and recovery. This study is an attempt to further elucidate the effect of hypersaline conditions on T. californicus and utilize both evapora¬ tion and solutions of high salinity as a means of producing gradually increasing salinities. Tolerance of T. californicus to High Salinities Materials and Methods T. californicus were collected from high tide pools with salinities of 35-45 %.at Mussel Point, Pacific Grove, California. Salinities were measured in the field using an American Optical Refractometer. Studies on the effect of evaporation were conducted in 8.9cm diameter petri dishes. Evaporation was studied at 20 C with or without the aid of a fan. Studies on the effect of elevated salinities utilized solutions prepared from Instant Ocean (Aquarium Systems, Inc., Eastlake, Ohio). These studies were carried out in a volume of 20 ml in 14½ x 1 3/4cm test tubes. Fifty percent effect doses for activity, coma, and death were obtained using an approximate probit analysis (Litchfield and Wilcoxon, 1949). 953 confidence limits for the EDgo are obtained as the product and quotient of the EDgo and its fyp.. Tolerance of T. californicus to High Salinities Results In the examination of salinities resultant to evaporation at which coma could be observed 20 petri dishes each containing 10 T. californicus of both sexes were placed in 10ml volumes of 35 %0 sea water. Figure 1 shows the result of this experiment. The 50% effect dose, EDg, was found to be 144 %owith a 95% confidence interval of less than +.003. Figure 2 shows percent of animals in coma and death resulting from exposure to increases in salinity produced by the addition of hypersaline solutions. The figure shows that not only do percent in coma and death increase with increasing salinity but percent death exceeds percent in coma after reaching a salinity of 175 % Some T. californicus can survive gradual increases in salinity to 175 %oand remain active in 140 %. The difference between death and coma was determined by returning the animals to a salinity of 35 %0. Death became the dominant effect when a salinity of 175 %owas reached. When the salinity was increased more slowly over a two day period even greater adjustment by the animals is indicated as shown in Figure 3. An even greater percent of the animals showed an ability to recover at 175 %0. Figure 4 shows the effect of the rate of salinity increase produced by evaporation. Animals were placed in 5, 15, and 25ml's of 35 %sea water and salinity increased at different rates due to the differences in the effect of evaporation. A faster rate of salinity increase from the smallest volume resulted in a higher percent coma at lower salinities. This affect, however, seems to be Tolerance of T. californicus to High Salinities less pronounced when 15ml and 25ml volumes are compared. The EDgoeg for the 3 different rates of salinity increase from starting volumes of 5, 15, and 25mls were 132.2, 145.2 and 154.5%respectively and the fepgoes for the 3 rates were 1.02,1.01 and 1.01 respectively. In Figure 5 ability to recover as a function of time after 100% of the population was in coma is presented. Ability to recover was determined by returning thesalinity to approximately the starting condition of 35 %0 by the addition of distilled water. The results indicate that the slower the rate of salinity increase due to evaporation the greater the percent recovery. However, the longer the animals remained in coma the smaller the percent recovery, Populations of T. californicus can be divided into three segments based on their response to increasing salinity due to evaporation. See Figure 6. As salinity increases the percent of free swimming animals declines. Many of the inactive animals can, however, be prompted into activity through physical stimulation. There exists a range of salinities between 125 % and 135 % where all the animals are quiescent but can be prompted into movement. Beyond this range coma becomes conspicuous. The EDgo for inactivity was 93.3 % with a 95% confidence range of 97.97 to 88.86 %0. Physical stimulation by stirring leads to only very temporary activity. As salinity is increased through evaporation T. californicus tends to group together in lose aggregations. The salinity at which this curious phenomena can be observed is dependent upon the population density. Figure 7 shows that high population density leads to aggregation at a lower salinity than is observed with intermediate or low popula¬ tion densities. A greater percent of the animals at high and medium Tolerance of T. californicus to High Salinities densities seem to participate in the grouping behavior. Figure 8 again using three different population densities illustrates that at low density coma is conspicuous at lower salinities than is observed with medium or high population densities. These differences diminish as the salinity is increased. A comparison of the susceptibility to coma of females with egg sacs and a mixed population is presented in Figure 9. The EDgo for coma in females with egg sacs is 148.25 %o salinity with an frp. 1.004. The EDgo for the mixed population is 144.05 % with a fyp-a of 1.04. Similarly, in the mixed population the females of copula¬ ting pairs appeared to be somewhat more resistant. Although ability to recover upon return to normal salinity was used to differentiate between coma and death, the occurrence of a 90° dorsal retroflexion of the tail before or after return to normal salinity appeared to be related to an inability to completely recover. Tolerance of T. californicus to High Salinities Discussion This study has explored the ability of T. californicus to withstand high salinities produced by evaporation or the addition of hypersaline solutions. The observed changes from active to inactive, to coma and death have improved the characterization of these animals' response to this stress. The relationship between high tolerance to high salinities and the rate of salinity increase is compatible with Patterson's hypothesis of osmoregulation (Patterson, 1968), however, a real demonstration of an osmoregulating ability requires an analysis of the body fluids of the animal. High tide pools in the field undergo slow changes in salinity from evaporation, therefore, populations of T. californicus would be faced with gradual salinity changes over a period of time and appear to be quite tolerant of such slow increases in salinity. Egloff's study with long term adaptation of T. californicus to rising salinity in a natural tide pool suggests that some animals showed activity in salinities near 200 % (Egloff, 1966). In the present study 150 % was found as an extreme upper limit for activity in short time acclimatization. The rate of increase of salinity is crucial in determining the salinities at which coma occurs and from which recovery is possible. Percent recovery can be elevated through slower rates of salinity increases. Most natural tide pools of significance to T. californicus contain greater volumes of sea¬ water than this study used in evaporation experiments and even longer time would be available for acclimitization under natural conditions, The mechanisms involved in adaptation to slow salinity increases remains to be explored. Tolerance of T. californicus to High Salinities The increased resistance to high salinities of females with egg sacs perhaps represents an additional insurance for the main¬ tenance of T. californicus in its changing environment. Acknowlegement: I wish to express my appreciation to Dr. John Phillips for his valuable contribution in thought and efforts in making this paper possible. 0 Figure 1. Plot of the percent of animals in coma, i.e. no activity subsequent to physical stimulation vs. increasing salinity in parts per thousand produced by evaporation over a two day period. The bars indicate the standard deviations obtained from 14 to 18 replicates. Figure 2. Plot of mean percent coma and mean percent death vs. number of hours exposed to a one day regime of increasing salin¬ ity. Salinity changes were made at 6 hour intervals. Closed circles indicate percent in coma. Open circles indicate percent dead. Bars indicate one standard deviation obtained from five replicate experiments. C Figure 3. Mean percent coma and mean percent death vs. number of hours exposed to a 2 days regime of increasing salinity. Salinity changes were made at 6 hour intervals. Closed circles indicate percent in coma. Open circles indicate percent dead. Bars indicate one standard deviation obtained from five replicate experiments. 0 Figure 4. Mean percent of animals in coma vs. salinity in parts per thousand resulting from evaporations over a three day period. Closed circles are results from evaporation of an initial volume of 5ml of 35 % sea water; open circles are results from evaporation of an initial volume of 15ml and open triangles are the results from evaporation of an initial volume of 25ml of 35 %. Times for 1003 coma are indicated for each of the three types of preparations. 0 Figure 5. Histogram of the percent of animals recovering from coma vs. time after 100% of the animals became comatose. Ability to recover was tested by returning the salinity to approximately 35 %. Open bars represent results from evaporation of an initial volume of 5ml, and linedbars represent preparations from evaporation of an initial volume of 15ml and solid bars represent preparations from evaporation of an initial volume of 25ml of 35 % sea water. Figure 6. Plot of mean percent active, inactive and coma or death vs. increasing salinity produced by evaporation over a 2 day period. Open circles indicate percent inactive. Open triangles indicate percent active and closed circles indicate percent of animals in coma. Bars indicate+ one standard deviation obtained from five replicate experiments. Figure 7. Histogram of percent of animals aggregating ys. increasing salinities in parts per thousand during 2 days of evaporation of 10ml of 35 % sea water. Solid bars represent pre¬ parations with more than 150 animals, lined bars represent prepara tions with 25 to 50 animals and the open bars represent preparations with less than 10 animals. Figure 8. Histogram of percent animals in coma)i.e. not showing movement in response to physical stimulation,vs. increasing salinity in parts per thousand during a 2 day evaporation of 10ml of 35 %. sea water. Solid bars represent preparations with more than 150 animals, lined bars represent preparations with 25 to 50 animals and open bars represent preparations with less than 10 animals. c Figure 9. A comparison of a mixed population containing males and females with and without egg sacs, and females with egg sacs,in coma,vs. salinity. Closed circles indicate females with egg sacs and open circles indicate the mixed population. PERCENT IN COMA 100 90 80 70 60 50 10 20 140 141 SALINITY %. 142 143 144 145 146 147 PERCENT COMA OR DEATH 80 SALINITY CHANGES 70 60 105 50 40 70 30 20 10 A 0 18 12 HOURS 140 - 24 175%. 30 S 6 S 8 5 a He U 8 U 8 S D — 8 88 8 a e kata- Q 3 S t X — â + 3 —— —— 1 t + 3 III E IL 4 25 8 10 Literature Cited Egloff, D.A. 1966. Ecological Aspects of Sex Ratio and Reproduction in Experimental and Field Populations of Marine Copepod Tigriopus californicus. Stanford University PhD. Dissertation, Hopkins Marine Station 1967. Issel, R. 1914. Vita latente per concentrazione dell' acqua (anbiosi osmotira) e biologia delle pozze di scoglura. Mill Zool. Sta. Neapil. 22: 191-254. Litchfield, J.T. and Wilcoxon, F. 1949. Graphic Method for Evaluating EDgo. J. Pharm. and Exptl. Therap. 96: 96-113. Nimkin, K. 1977. Environmental Effects on Sex Ratios in Tigriopus californicus. Biology 175H Research Paper, Hopkins Marine Station, Spring 1977. Patterson, R.E. 1961. Physiological Ecology of Tigriopus californicus a High Intertidal Copepod. Master of Arts Thesis, U.C. Berkeley. Ranade, M.R. 1957. Observations on the resistance of Tigriopus fulvus (Fisher) to changes in temperature and salinity. J. Mar. Biol. Assoc. U.K., 36: 115-119.