Introduction Doty (1946) correlates intertidal zonation with critical exposure time and suggests that an organism's upper limit is determined primarily by its ability to with- stand the stresses which such exposure imposes; the more resistant an organism is to temperature and salinity varia- tion, dessication, and other factors resulting from exposure, the higher it can exist in the intertidal. Many species of barnacle inhabit the upper intertidal zones and consequent- ly might be expected to be resistant to these same stresses. Since barnacles are poikiotherms, it is reasonable to suspect that their body temperature would closely follow the temperature of their substrate. Indeed,Suzuki and Mori (1963) reported that the shell surface and the body tempera- ture of Tetraclita squamosa japonica remained the same, but this temperature was always slightly lower than that of the barnacle's substrate because of the cooling effect of evapo¬ ration of mantle cavity and body water. This evaporation would be expected to increase the salinity of the mantle cavity fluid. It appears then that under normal conditions of exposure high intertidal barnacles would be subjected to salinity extremes due to (1) fresh water from precipitation and consequent runoff and (2) high mantle cavity salinity due to evaporation resulting from high midday temperatures. It is, therefore, expected that these organisms might show adaptations to extreme salinities. This expectation has been in part confirmed by Borusk and Kreps (1929), who showed that B. balanoides (L) and B. crenatus Brug. can exist for three weeks in fresh water in a state of 'salt sleep', a state in which all cirral activity ceases, the opercular plates are closed, and res¬ piration is maintained at a low level. Furthermore, Barnes and Barnes (1958) reported that B. balanoides feeds normally in 60% to 90% sea water, opens but does not feed in 50% sea water, but closes in salinities of 25% or less. Barnes (1953) has shown that the nauplii of the same species remain healthy and active within a range of from 18'oto 300 (approx. 50% to 90% sea water). However, they cease movement in 6 %0 (approx. 20% sea water), and die in one hour in 3%0 (approx. 10% sea water). Barnes and Barnes observed that B. balanoides exhibited a beha- vioral response to exposure by extruding the mantle cavity water. However, they did not consider the question of tolerance to high salinity as important. The present study is an investigation of the effect of salinity on the adult and early-stage embryo of Balanus glan- dula Darwin (1854). This species occurs intertidally from about mean high high water (approx. + 10 feet) to the lowest high low water (approx. + 1 foot), thus spanning several intertidal zones. Since B. glandula is found up to + 10 feet, it would be expected to have a high tolerance to the temperature and salinity extremes which might occur during its normally long periods of exposure (up to two days). The results of this study indicate that Balanus glan- dula exhibits a remarkable tolerance to salinity extremes. The adult barnacles are able to withstand exposure to salinities of 0%6 to 300% sea water, while the developing embryos show somewhat less tolerance, dying when subjected to salinity below 50% sea water or above 200% sea water. Field observations suggested the normal occurence of increased mantle cavity salinities under natural conditions of exposure. No behavioral adaptations as noted by Barnes and Barnes were observed. Temperature To determine whether the temperature of the mantle cavity of B. glandula followed that of its substrate, a popula- tion attached to granite rock approximately eight feet above mean lower low water was monitored for temperature changes in the following manner. A hole was carefully made in the side of large specimens from the uppermost limits of the population and a Yellow Springs Tele-thermometer (Model 43 TD) small ani- mal microprobe inserted so that the probe entered the mantle cavity under the body. The hole was sealed with wax, effectively enclosing the probe inside the mantle cavity. At the same time, a small surface probe was taped to the shell adjacent to the microprobe and an air temperature probe attached so that it was at least a centimeter above the rock surface. The three probes were connected to a switch box which in turn was plugged into the Tele-thermometer. The temperature was monitored every five minutes for periods of 2 to 8 hours during both day and-night. The results, illustrated in figure 1, show that the mantle cavity temperature follows the shell surface temperature rather closely. The two temperatures remained within 5 degrees C. of each other and ranged between 8.9°0. and 38°0.; note that on May 3 and May 4 the barnacle temperature was somewhat higher than the surface temperature; however, since it was difficult to get the surface probe flush with the shell, the strong wind which occurred on these days could well have influenced these results by cooling the area adjacent to the shell so that the surface probe was actually recording the air temperature. In conditions of extreme exposure during midday in the summer or at night during the winter, the barnacles could well experience even harsher conditions. To check the field data, a similar test was run under laboratory conditions where ambient temperatures could be con¬ trolled and extended beyond those encountered in the field. In this as well as all following experiments involving adult barna- tilus cles only animals attached to the shells of the mussel My californianus were used so that the basal plates remained intact and the animals would not be subjected to injury. The same pro- cedure was used as in the field experiments except that tempera- ture changes were induced by immersing the animals in 9°0. sea water, 14"C. sea water or by exposure to room temperature or the heat from a 60 watt bulb. Figure 2 shows the results of this experiment. Again, the barnacle's temperature followed the shell surface temperature, although the internal temperatures tended to lag slightly behind those at the shell surface. Nevertheless, the barnacle's tempera¬ ture changed quickly, in one case falling 14.2'0. in five minutes. Exposure to high temperature might be expected to raise the salinity of the mantle cavity fluid, as suggested by Suzuki and Mori (1963). To test this possibility the mantle cavity fluid was removed by inserting a syringe through a hole made in the shell and the salinity determined with a refractometer (A0, 0-30 C.) before and after 16 hours exposure to the heat generated by a 60 watt bulb. Initial mantle salinities were 47Zwhile the final reading was 51.7%. This increase suggests that higher salini- ties would be produced under harsher conditions. Thus the bar- nacles might be subjected to high mantle cavity salinities during periods of midday exposure, suggesting wide salinity tolerance limits for both adults and embryos. Adult Tolerance Limits Specimens of Balanus glandula which were attached to mussels were cleaned, other species of barnacles removed, and the excess mussel shell broken away. The specimens were placed in a pan of sea water to induce cirral activity; the shells were numbered and the number of active and inactive barnacles noted. Barnacles not showing activity were considered to be dead and were not included in the subsequent experiments. At least fifty "active" barnacles were utilized for each experiment. These were placed in a refrigerator pan containing well-aerated water of the desired salinity. Salinities were determined with an AO refractometer and a Gemware hydrometer. A standard curve was made for the refractometer by taking readings of 50%, 100%, and 200% sea water and adjusting these to the reading of a sample of "Normal water". The hydrometer readings were converted by means of standard tables. The pans were covered to prevent evapo- ration and aerators inserted through holes in the cover to pro- vide continuous aeration. These pans were floated in refrigera- ted sea water, keeping the solutions at a constant temperature of 9° to 10°0. At intervals of 1, 4, 24, 48, and 72 hours, ten 5. barnacles were removed from each of the test solutions and placed in aerated sea water (13°to 16°0.) for two days. The barnacles were then removed from the shells and placed under a dissecting scope. They were considered to be alive if they reacted to the light or to a prod with a dissecting needle. Figure 3 indicates the results of these experiments and shows that the adults can withstand exposure to salinities ranging from 0% to 300% sea water. The one animal which died in the 125% sea water solution was not considered to be signi- ficant since there were no observed mortalities in solutions of 90% or 150% sea water. Embryo Tolerance Limits The two ovigerous lamellae were removed from each mature adult, one was cut in half and the other left intact. The length of the clear outside covering of the embryo and the length of the embryo itself were measured with an ocular micrometer under a compound microscope. The lamellae were then placed in aerated beakers containing water of various salinities. The beakers were floated in refrigerated sea water at a temperature of 9 to 10 degrees C. The lamellae were removed after 1, 4, 24, 48, and 72 hours and measured as before and the percent of lysed or crenated eggs noted. Unlike the adults, the embryos appear to be sensitive to salinities below 50% and above 200%, as is shown in figure 4. Since the results for the intact and the cut lamellae are essentially the same, it is evident that the elastic sac surroun¬ the lamella provides no protection to the eggs. Also the eggs never behaved as osmometers; essentially they either resisted the osmotic stress with no apparant volume change or they lysed. Salinity of the Mantle Cavity Fluid Since the adult can withstand distilled water, it seems probable that the animal may be excluding water by closing the opercular plates. The opercular plates remain open in fifty percent sea water, but there is no feeding; in 25% sea water the opercular plates close tightly. To investigate the efficacy of this behavior in excluding the ambient salinity, the mantle cavity fluid of B. glandula was sampled in the following manner. A hole was made in the shell and the mantle cavity fluid removed with a small syringe. The mantle cavity fluid from seve- ral barnacles was pooled so that there was enough fluid to use in the refractometer. The animals were placed in aerated beakers containing water of various salinities and these were floated in refrigerated sea water maintained at 8°to 10°0. After 1, A. 24, 48, and 72 hours, the salinities of the mantle cavity and of the test solution were determined. In 10% to 50% sea water, where the opercular plates were closed, the mantle cavity fluid remained significantly more saline than the test solution. In 125% to 200% sea water, where the barnacles were actively feeding, the mantle cavity salinity rose to that of the test solution. The method utilized is not precise since it is impossible to avoid getting blood and small amounts of eggs and tissue in the sample; likewise, the presence of excretory products might increase the refractive index of the fluid. This could account for the variability in the salinity readings. Notwithstanding, the mantle cavity salinity and the external salinity were different enough in the lower salt solu- tions to strongly suggest that the barnacles were able to exclude surrounding water by closing their opercular plates. Discussion Both field and laboratory experiments tend to substan- tiate the close correlation of ambient temperature and barnacle mantle cavity temperature. Since water remains in the mantle cavity, even after two days of dessication, a hot day could con- cievably raise the salinity of this fluid significantly. B. glan- dula seems to have the capacity to withstand very high salini- ties: the adults can survive in 300% sea water, while the embryos can withstand up to 200% sea water. In the embryos, the sac surrounding the ovigerous lamellae does not appear to provide protection since there is no volume change or difference in the rate of lysing between intact and cut lamellae. It is suggested that the presence of the clear outside covering of the embryo is the most likely means of protection. The mechanism by which adults endure these high salinities is not known. However, the properties of the animal's exoskeleton might be involved in main- taining this high tolerance. The embryos are less tolerant to low salinities; how- ever, it seems that they would never be subjected to salinities of less than 50% sea water, for the adult barnacles close their opercular plates at salinities lower than this, completely sealing themselves from the environment by this means. High salinities, on the other hand, may be unavoida- ble when the organisms are subjected to high temperatures. Eva¬ poration of the shell water and the consequent transfer of man- tle cavity water to the shell as well as direct evaporation from the mantle cavity might occur even though the opercular plates were closed. It is not surprising, therefore, that B. glandula has adapted to high salinities by some mechanism other than oper- cular closure. The adult and brooded embryos of Balanus glandula can withstand the salinities normally imposed by exposure in the in- tertidal. Their distribution would not be limited by salinity unless the extreme conditions were so persistant as to cause starvation, an excessive accumulation of waste products, or in- hibition of larval settlement and development. The high tolerance of B. glandula to salinity is one factor enabling it to inhabit the highest intertidal zone. Summary 1. The mantle cavity temperature of Balanus glandula closely follows its shell surface temperature. Adults can withstand from 0% to 300% sea water; they exclude the lower salinities by closing the opercular plates, effectively sealing themselves off from the surrounding water. Embryos can tolerate from 50% to 200% sea water. Salinity does not appear to be a limiting factor in the dis- tribution of Balanus glandula. C 8. Acknowledgements I would like to thank Dr. Welton Lee for advice given on the experimental procedure and editorial assistance given on the paper. Literature Cited The effect of lowered salinity on some barna¬ BARNES, H., 1953. cle nauplii. J. Animal Ecol. 22: 328-330. BARNES, H. & M., 1957. Resistance to dessication in intertidal barnacles. Science 126: 358. BARNES, H. & M., 1957. Note on the opening response of Balanus balanoides (L') in relation to salinity to certain inorganic ions. Veroff. Inst. Meeresforsch Bremerhaven 5: 160-164. BORUSK, V. & E. KREPS, 1929. Untersuchungen über den respira¬ torischen Gaswechsel bei Balanus crenatus bei verschiede- nem Salzgehalt des Aussenmediums. Part III. Pflug. Arch. ges Physiol. 2 371-380. DOTY, MAXWELL S., 1946. Critical fide factors that are correla¬ ted with the vertical distribution of marine algae and other organisms along the Pacific coast. Ecology 27: 315-328. SUZUKI, N. & S. MORI, 1963. Adaptation phenomena concerning the water content of the barnacles. Japanese Journal of Ecolo- gy 13 (1): 1-9. Q S 40 - 20 r 40 20 940. 30 — 20 — 0200 te l ++ 0500 — May 15,196 May91967 May6,1967 7 + ke Nay 31967 3 " 1200 Time 1600 S S —— ltaataa- — 90 Jnodwe O — T E h ————— T 7 AIV 1u9 H HE — — —— — 0 S O 8 0 — S 8 S — — % AJJUIIDS 8 9 C CAPTIONS FOR FIGURES Figure 1. Temperature changes under field conditions. The straight line indicates barnacle temperature, the dashed line shell surface temperature and the cross, air tempera- ture; the length of the cross indicates the tempera- tured limits. Figure 2. Temperature changes under laboratory conditions. The straight line indicates barnacle temperature, the dashed line shell surface temperature and the cross, air tem- perature; the length of the cross indicates the tempe- rature limits. Percent of adults alive after 72 hours in test solu- Figure 3. tion (top); percent of embryos unlysed after 1 hour in test solution (bottom); the cross-hatched bar in the bottom graph is the control. Figure 4. Mantle cavity over 72 hours. The straight line repre- sents mantle cavity fluid salinity; the dashed line indicates the test solution salinity. CAPTIONS FOR FIGURES Figure 1. Temperature changes under field conditions. The straight line indicates barnacle temperature, the dashed line shell surface temperature and the cross, air tempera- ture; the length of the cross indicates the tempera- tured limits. Temperature changes under laboratory conditions. The Figure 2. straight line indicates barnacle temperature, the dashed line shell surface temperature and the cross, air tem- perature; the length of the cross indicates the tempe- rature limits. Percent of adults alive after 72 hours in test solu- Figure 3. tion (top); percent of embryos unlysed after i hour in test solution (bottom); the cross-hatched bar in the bottom graph is the control. Figure 4. Mantle cavity over 72 hours. The straight line repre- sents mantle cavity fluid salinity; the dashed line indicates the test solution salinity.