OSMOREGULATION IN TIGRI IS CALIFORNICUS by Thomas C. Merchant Charles Baxter - Advisor Hopkins Marine Station Biology 175H INTRODUCTION Tigriopus californicus is a harpactecoid copepod inhabiting high intertidal splash pools, which are subject to rapid and large changes in salinity. Since they are found active between 5 and 120 0/00, their osmoregulatory behavior is of considerable interest. This is especially true because the two studies on the subject appear to be contradictory. Patterson (1968), based on analysis of gut fluid, claims that Trigriopus is a conformer while Fisher and Bassett (1976), using fluid from whole body squashes, found a unique mode of osmotic regulation. Both conformation and regulation have been demonstrated for euryhaline crustacea. This may involve hyperosmotic regulation, hyposmotic regulation and, in some cases, both (Prosser 1973). The unique behavior of Trigriopus involves conformation at intermediate salinities with hyposmotic regulation at high salinities and hyperosmotic regulation at low salinities. MATERIALS and METHODS A. Body Fluid Osmolarity Tigriopus were collected for each trial from a high tidepool (40 0/00 salinity) on Mussel Pt., Pacific Grove, California during May 1977. 100 animals were acclimated to each of the salinities: 5, 10, 15, 20, 25, 35, 40, 45, 50, 55, 60, 68, 75, 85, and 120 0/00 for 38 - 44 hours (trial 1) and 47 - 53 hours (trial 2). Females carrying eggsacs were not used. Acclimation was begun on the same day the collections were made. A sample of total body fluid was obtained using the technique of Fisher and Bassett (1976) as follows. The acclimated animals were pipetted onto a millipore suction filter and washed with 2 - 4 mls. distilled water to remove any residual salt from their bodies. The animals and filter were centrifuged for 2 minutes at 1500 r.p.m. to dry the animals. The filter was placed on dry ice and the frozen animals scraped off into a watch glass where they were ground with dry ice into a powder. This powder was scraped into a capillary tube (1.1 - 1.2 mm internal diameter, 25 mm long and fused at one end) and centrifuged for at least 3 hours at 1300 r.p.m. to separate body fluid from body parts. A melting point determination was done using the comparitive melting point method of Gross (1954). Time points were taken as the last crystal to melt. Controls to correct for errors such as condensation from the air onto the groud sample were done. A potato starch and water (salinities 40, 50, 60 0/00 were tested) suspension was made in a watch glass and frozen with dry ice. This was ground with dry ice scraped into a capillary tube and centrifuged for 2 hours at 1300 r.p.m. The melting point was found for the controls and the samples could be corrected by the average error. B. Gut Volume vs. salinity About 50 animals were acclimated for four hours in 25 mls. of salinities 5, 20, 35, 55, 85, .5, and 120 0/00 (the latter two sets of animals were in torpor - the state of apparent death). Several animals at a time were placed under a compound microscope using pleces of broken coverslip under the coverslip to prevent crushing of the animals. Measurements of the width of the posterior and anterior portions of the gut and of the length of the animal were made. Animals with fecal pellets were not used because the pellet caused a substancial increase in gut width. The gut is nearly cylindrical and its length is entirely dependent on the length of the animal, therefore width of anterior portion plus width of posterior portion of the gut divided by length of the animal is used to give an index of gut volume corrected for animal size. C. Metabolism study Several hundred animals were acclimated for 24 hours in 30 ml. baths of salinity 5, 10, 20, 35, 45, 55, 60, and 85 0/00. A Gillson constant pressure respirometer was used and three replicates for each salinity were tested. Readings were taken every 15 minutes for an hour to make sure the O2 consumption rate was constant. After the run, the animals were dried in an oven (70 degrees F.) to constant weight, then weighed. RESULTS A. Body Fluid Osmolarity The data given in figure one shows a pattern of regulation in low salinity, conformation in intermediate salinity and regulation in high salinity. The data points are corrected by 10 0/00 which represents the decrease in concentration during the processing of controls. The two trials are stat- istically different with respect to hyposmotic regulation (T test: p£.02) and hyperosmotic regulation (T test:p (.003). On all figures, the vertical dashed line is the approximate EDgo for torpor. B. Gut Volume vs. Salinity As figure 2 shows, the gut volume changes significantly with the onset of the torpor state. A 54% decrease in volume is observed between 5 0/00 and low salinity torpor while an increase of 92% is observed between 85 0/00 and high salinity torpor at 120 0/00. In order to determine if the gut volume at 5 0/00 was significantly larger than at the other non-torpor salinities, a predicted value for 5 0/00 was obtained using the method of least squared linear reg¬ ression to extrapolate the non-torpor salinity data to 5 0/00. The standard error for this value was found using the method of Sokal and Rohlf, Box 14.3. Using a student T test, the actual and predicted values were found to be different (p).025). C. Metabolism Study Figure 3 shows that Tigriopus' O, consumption decreases with increasing salinities. There appears to be no significant change in O, consumption between 35 and 60 0/00. Each O consumption value is the mean of 3 data points for a given salinity. DISCUSSION Euryhaline animals exhibit a wide variety of osmotic behavior. Some conform, though they in general, buffer the intracellular environment against ionic change by adjusting the amino acid concentrations. Regulating animals do so over a range of salinities, and show conformance on either side. The arthropod Artemia, like Tigriopus, is exposed to large variations in osmotic pressure. Their response is to regulate their body fluid at a relatively constart level, markedly hypotonic to all except the lowest salinities (Croghan 1958). Tigriopus' osmotic behavior is unusual but highly adaptive to its habitat - high tidepools of constantly changing salinities, and hence changing osmotic pressures, as a result of the interaction between weather, tides and high waves. Most salinities are between 30 and 60 0/00 but pools are commonly found substantially beyond these values on both sides. Tigriopus were found to conform between salinities 25 and 60 0/00 which corresponds to the usual tide pool salinities. Since regulation requires energy, it is adaptive for Tigriopus to conform to the common fluctuations in salinity. Outside of the conforming range, Tigriopus regulate their body fluid until a state of torpor is induced. This greatly inhances Tigriopus' ability to survive in their tidepool habitat, which often exceeds the salinity levels over which they conform. The state of torpor, induced by extreme osmotic stress (2 0/00 and 120 0/00), seems to be a shutting down of most body functions. In this state Tigriopus can survive the extreme salinity levels for extended periods of time (David Stoller 1977). Patterson (1968) reported Tigriopus to be conformers based on osmotic concentration of gut fluid which was assumed to be isosmotic with body fluid. This assumption is probably incorrect since Tigriopus drink while active (Patterson 1968). Further, the studies reported here and by Fisher and Bassett (1976) indicate that the fluid from whole animal squashes is regulated. In addition, gut volume changes occurring during torpor can be best explained by assuming the gut to be nearly isosmotic with the environment while body fluids are regulated. Upon entry into low salinity torpor, the gut volume decreases drastically which is what would happen if the gut were isosmotic with the environment and the animal stopped regulating in torpor. For the same reasons, the gut is expected to, and does, expand with the onset of high salinity torpor. Hence the gut must be allowing water passage in torpor which is not compensated by some active process. An arguement may be made that the gut epithelium is the site of regulation. The gut volume was larger than normal in a 5 0/00 environment. This may imply that the gut is pumping water out of the body to maintain hypertonicity of the body fluids. Another line of evidence is the gut volume changes observed when individuals enter torpor. Thus when regulation ceases or is diminished in the gut lining the osmotic flux begins. The gut also presents a major surface area with permiability, access to internal and external fluid environments, circulation of both by peristaltic contractions and literature support for gut epithelia in¬ volvement in regulation in other Crustacea (Artemia regulate via the gut (Croghan 1958)). On the other hand the body surface is covered with an exoskeleton of presumed low permeability (Fisher and Bassett 1976) and shows no obvious areas of morphological specialization for exchange. If the gut fluid is isosmotic with the environment and it makes up a significant portion of the total body fluid measured, the hemolymph and other internal body fluid must be at a different concentration than the values obtained during regulation. A corrected value for real body fluid may be obtainable. Gut volume and total water content (from dessication experiments) used to estimate real body fluid volume would be a good place to start. The concentration of the body fluid could then be calculated using the gut volume change in torpor. In trial one the onset of regulation occurys sooner than it does in trial two. Trial two was acclimated for nine hours longer which suggests that Tigriopus when given a longer acclimation period may tolerate a broader range of salinities without regulation. This must be tested with longer and shorter acclimation periods. Measurement of O, consumption at various salinities shows a respiratory pattern similar to that of most euryhaline species (Prosser 1973). Metabolism is high at low salinities and decreases as salinity increases. The line appears to be fairly level between 35 and 60 0/00 which coincides with the conforming zone - no metabolic change in response to salinity change. There was no monitoring of activity or other metabolic changes that may occur with salinity variation (Lockwood from Newell 1976), therefore O, consumption changes related to osmoregulation may be at least pertially masked. SUMMARY Tigriopus californicus exhibit a unique osmoregulatory behavior which is highly adaptive in the high splash pool habitat. They conform osmotically in intermediate salinities and regulate hypo and hyper osmotically in high and low salinities respectively. Gut fluid appears to remain isosmotic with the environment. Evidence is presented to suggest the gut may be a regulatory surface in Tigriopus. The range of osmoconformance depends on the length of acclimation to a given salinity. O consumption in Tigriopus is high in low salinities decreasing as salinity rises. Metabolism appears not to change significantly over the conforming range 35 to 60 0/00. LITERATURE CITED Bassett, J.B. Jr. and G.A. Fisher Jr., 1976, Aspects of Osmoregulation in Tigriopus californicus, Unpublished M.S. on file at Hopkins Marine Station library. Croghan, P.C., 1958, Osmotic and Ionic Regulation in Artemia, J. Exp. Biol., 35: 219 -233, 243 - 249. Gross, W.J., 1954, Osmotic Responses in the Spunculid Den¬ drostomum zostericolum, J. Exp. Biol. 31:402 - 423. Lockwood, A.P.M., from Newell, 1976, Adaptations to Environment, Butterworths and Co., London. Patterson, R.E., 1968, Physiological Ecology of Tigriopus californicus, a High Tidepool Copepod, M.A. thesis U. C. Berkeley. Prosser, C.L., 1973, Comparitive Animal Physiology Vol. 1, W.B. Saunders Co., Philadelphia. Sokal, R.R. and F. James Rohlf, 1969, Biometry, Box 14.3, W.H. Freeman Co. San Francisco. Stoller, D.W., 1977, Tolerance of Tigriopus californicus to Slow Increases in Salinity Produced by Evaporation and Hypersaline Solutions, Unpublished M.S. on file at Hopkins Marine Station Library. ACKNOWLEDGEMENT. Many thanks to staff and students for a valuable experience, Robin Burnett for his interest, help and nice sets, and especially to Chuck Baxter for his patience. ideas and help with my project and this paper - and for kicking my mind into gear once in a while. Figure 1 - Body Fluid Concentration (0/00) vs. Environmental Salinity (0/00) Trial 1 - O Trial 2 - X Diagonal line is the isosmotic line D H BODY FLUID9 0 + t ++++ t 4 0 C Figure 2 - Mean Gut volume vs Environmental Salinity (0/00). Error bars are one standard error on either side. D Z GUT VOLUME + 4 H H ++ + H c Figure 3 - Mean O, consumption vs. Environmental Salinity (0/00).o consumption units are h ot. Error bars are one standard error on either side of the data point. — 0, CONSUMPTION e + H H H 0)