OSMOREGULATION IN TWO SPECIES OF PALAEMONETES (CRUSTACEA: DECAPODA) FROM FLORIDA1 SHELDON DOBKIN AND RAYMOND B. MANNINGz Institute of Marine Science, University of Miami ABSTRACT Individuals of the fresh-water shrimp Palaemonetes paludosus were held in the laboratory at a variety of salinities, after which blood samples were taken and the osmotic concentration determined by the freezing-point depression method of Gross (1954). Specimens of Palaemonetes inter- medius from high salinity were held at low salinity and vice-versa prior to blood sampling and determination of osmotic concentration. Results indicate that Palaemonetes intermedius is able to regulate over a wide range of salinities while regulation in P. paludosus breaks down at salinities in excess of 20%0' INTRODUCTION The caridean shrimp family Palaemonidae includes species which are found in a wide variety of habitats ranging from fresh-water to marine situations. Some species of the genus Palaemonetes are able to occupy such different habitats as fresh-water ponds and saline marsh pools. The ability to live in areas with a wide range of salinities imposes upon an animal the problem of regulation of internal environment. Panikkar (1941) studied osmoregulation in Palaemonetes varians, Palaemon serratus (as Leander serratus), and P. elegans (as Leander squilla), euryhaline species found in brackish and marine waters. His investigations showed that these animals could maintain hypotonicity in waters of high salinity while remaining hypertonic in brackish waters. The same ability has been observed in the caridean Crangon crangon by Broekema (1942), and in the penaeids Metapenaeus monoceros (Panikkar and Viswanathan, 1948), M. dobsoni, Penaeus indicus, and P. carinatus (Panikkar, 1951). Williams (1960) reported that Penaeus aztecus and P. duorarum were hypotonic to sea water at room temperature and hypertonic to dilutions of sea water below 30%0. Grapsoid and ocypodid crabs have also shown this pattern of osmoregulation (Jones, 1941). Parry (1957) investigated osmoregulation in the fresh and brackish water species Palaemonetes antennarius, and duplicated the findings of Panikkar (1941) on P. varians. Parry also determined the normal blood concen- tration of Palaemon longirostris, a brackish water form that breeds in the sea. IContribution No. 522 from The Marine Laboratory. Institute of Marine Science, University of Miami. This' work was supported by National Science Foundation grant G-16298, and the present paper constitutes a scientific report to that agency. ZPresent address: Division of Marine Invertebrates, U.S. National Museum, Washington, D.C. 150 Bulletin of Marine Science of the Gulf and Caribbean [14(1) Four species of Palaemonetes are found in south Florida. P. paludosus (Gibbes), a fresh-water species, has been observed in salinities of 0-10%0 in Everglades National Park (Tabb and Manning, ]962). P. intermedius Holthuis has been collected in salinities ranging from 6-38%0 in the same area. According to Holthuis (1952), P. pugio Holthuis inhabits brackish to almost fresh water, while P. vulgaris (Say) is found in brackish and marine areas. Nagabusham (1960) noted that P. vulgaris could tolerate salinities as low as 4~00 for up to one month. Palaemonetes paludosus and P. intermedius were chosen for intensive study because of their ease of capture and because their range of habitats were similar to those of P. antennarius and P. varians, respectively. The purpose of this study was to determine the normal blood concentration of P. paludosus and P. intermedius, and to investigate the internal reaction to osmotic stress in these species. ACKNOWLEDGMENTS The authors thank Dr. Charles E. Lane, Institute of Marine Science, University of Miami, for making available equipment and space in which to carry out this work. Dr. Lane and Drs. F. Bernard and C. Gifford pro- vided the authors with numerous suggestions on methods and interpreta- tion of results. METHODS AND MATERIALS Specimens of Palaemonetes paludosus were collected at Redskin Pond, an abandoned fresh-water rockpit just south of the Tamiami Trail about five miles west of Miami. Palaemonetes intermedius was collected in the Coral Gables Waterway at the Bird Road bridge and in Duck Pond on Virginia Key. Corrected hydrometer readings for the latter two areas showed the salinity of the water to be 10-11%0 and 39%0, respectively. This species was also taken from a Chara pond in the vicinity of Matheson Hammock; the corrected salinity at this locality was 5-6%0. In the case of Palaemonetes paludosus, two experiments were conducted. In May, 1962, shrimp were held in 16-oz. jars at salinities of 5, 10,20-21, and 30%0 for approximately 42 hours before sampling. Others were held at 25 and 36.7%0 for about 18 hours. In December ]962, shrimp were held in 64-oz. jars at salinities of 10, 15, 20-21, 25 and 30%0 for seven days prior to determining blood concentrations. Control specimens in both experiments were held in water from Redskin Pond in a ]5-gallon aquarium. At most salinities, animals in three categories, ovigerous females, ripe females, and juveniles, were distinguished. In the course of the analyses, only one male P. paludosus was found. This may have been due to occurrence of males in areas other than the shallow-water grass in which the females were collected, or it may be a result of a high 1964] Dobkin & Manning: Osmoregulation in Palaemonetes 151 proportion of females in the shrimp population. Monthly collections of P. paludosus at Redskin Pond and the Krome Avenue Canal indicate that the high proportion of females is a normal occurrence. Specimens of P. intermedius collected at the Coral Gables Waterway in salinities of 1'0-11%0 were kept in running water from Biscayne Bay (35-37%0) for a period of about two weeks prior to blood analysis in May 1962. Some of those collected in Duck Pond (39%0) were held at 10%0 for a period of about 42 hours, while the others were held in their habitat water. Water temperatures varied from 19°-25°C during the December experi- ment, and from 26°-29°C in May. Blood was collected from the shrimp by inserting a capillary tube of approximately 0.4 mm inside diameter under the postero-dorsal border of the carapace and into the heart. Before this was done, the shrimp were wrapped in absorbent tissue and all extraneous water was carefully removed from the surface of the body, gill chambers, and particularly the point of insertion of the capillary. Approximately 1-2 ftl of blood were collected from each shrimp, depending upon the size of the animal. Two- microliter samples were easily obtained from larger specimens of P. .nalu- dosus, but in the case of P. intermedius and juvenile P. paludosus it was often difficult to obtain one microliter. The blood of P. intermedius was difficult to sample due to rapid coagulation within the capillary. The osmotic concentration of the blood samples was determined by the freezing-point method of Gross (1954). Samples of standard solu- tions of 0.8, '0.6, 0.5, 0.4, and 0.2 molar NaCI were used in the determi- nations (three or four standards per rack depending upon the expected range of osmotic concentration of blood samples) as well as samples of distilIed water. Possible impurity of some of the distilled water samples was indicated, in that on several occasions the distilled water had a lower freezing point than samples of pond water analyzed simultaneously. The salt concentration was converted into terms of freezin~ point depression by the relationship ~oC=285 mM NaCI (Parry, 1957). RESULTS In contrast with the general pattern of blood hypotonicity in a con- centrated medium and hypertonicity in a dilute medium, P. paludosus was found to be hypertonic to its medium in a variety of concentrations. The freezing point of its blood increases with increasing salinity of the medium, remaining hypertonic in salinities as high as 30%0' P. paludosus did not survive a salinity of 36.7%0. The results of holding experiments in different salinities are summarized in Table 1 and Figure 1. The blood approaches isotonicity with the medium at a salinity of approximately 25%0, near the point at which the blood of P. intermedius is isotonic. 152 Bulletin of Marine Science of the Gulf and Caribbean [14(1) 1.8 o l:>. CI 1.4 .2 3 _________________~4 1.0 2 0.6 0.2 o 0.2 0.6 1.0 1.4 I.B r:. C E FIGURE1. Freezing-point depression of the blood of: a, Palaemonetes paludosus held at salinities of 0, 5, 10, 15, 20-21, 25 and 30%0.-b, Palaemonetes inter- medius held at salinities of 5-6, 10,36.7, and 39%0.Line c represents isotonicity. Numerals indicate the number of blood samples analyzed. A wide range of internal concentrations was found in P. paludosus from fresh water. u DC of blood ranged from 0.48 to 1.17 with a mean of 0.87. Ovigerous females were slightly less concentrated than non-ovigerous ones. Ripe females were within the range of variation of ovigerous females in two specimens examined. All observations on P. paludosus from fresh water are summarized in Table 2. The blood of P. intermedius is hypotonic in a concentrated medium and hypertonic in a dilute medium. The blood is isotonic with the medium at a salinity of about 25%0, Specimens collected in a concentrated medium (sea water, 39%0) and held in a dilute medium do not differ markedly from those collected in a dilute medium. Over a salinity range of 5-39%0 the A °c of the blood changes from 1.01 to 1.21.