MARINE ECOLOGY PROGRESS SERIES Vol. 181: 125-129,1999 Published May 18 Mar Ecol Prog Ser I

Ontogeny of osmoregulation in the palaemonid Palaemonetes argentinus (Crustacea: )

'Laboratoire d'Ecophysiologie des Invertebres, Universite Montpellier 11, Place Eugene Bataillon, F-34095, Montpellier cedex 05, France Z~iologischeAnstalt Helgoland, Meeresstation, D-27498 Helgoland, Germany

ABSTRACT: Osmoregulation was studied in the zoeal stages I and VI, the first decapodid, the first juve- nile, and in adults of the palaemonid shrimp Palaernonetes argentinus. The larvae hatch in freshwater creeks or in adjacent brackish coastal lagoons of the warm temperate southwestern coast of the Atlantic Ocean; larval development is possible in low salinities. To cope with these demanding environments, the capacity for osmoregulation is well developed at hatching, increasing only slightly throughout development. All the postembryonic developmental stages hyper-regulated at low salinity (1 to 10Ym), hyper-osmoconformed at l?%, and osn~oconformed at higher salinities (26%0;up to 32%0 in adults). The type of osmoregulation did not change dunng developnlent from larval hatching through the adult phase. The ecological implications and the evolutionary sigruficance of osmoregulation in early llfe- history stages of P. argentinus and other aquat~ccrustaceans are discussed.

KEY WORDS: Osmoregulation . Ontogeny . Adaptation . Crustacea . . . Larva Palaemonetes

INTRODUCTION establishment of a species in a given habitat (review in Charmantier 1998). But the number of species in which Salinity is one of the main environmental factors the ontogeny of osmoregulation has been studied is exerting a selection pressure on aquatic organisms. still limited, and it was recently stated that future The ability of a species to adapt to environmental salin- investigations should be conducted in species sub- ity and its fluctuations is thus a major adaptive process mitted to harsh environments (extreme and/or highly achieved through different behavioral and/or physio- variable salinity) during their development (Char- logical mechanisms, among which osmoregulation is of mantier 1998, Charmantier et al. 1998). major importance for some groups, including Among the candidate species in a series of studies . In adult crustaceans, osmoregulation has in this field is the shrimp Palaemonetes argentinus been studied in numerous species (reviews in Mantel (Nobdi 1901) (Decapoda: Caridea: Palaemonidae). This & Farmer 1983, Pequeux 1995). More recently, several species is found in an area covering the north and studies have stressed the adaptive importance of center of Argentina, Uruguay and southern Brazil osmoregulation throughout the development of crus- (Spivak 1997). Although it is generally considered a taceans, underlining the fact that the ability of each freshwater species, it has also been found in brackish developmental stage to adapt to salinity and its varia- coastal lagoons extending along the coast of the warm tions is one of the factors permitting the successful temperate southwestern Atlantic Ocean (review in Spivak 1997), particularly in Mar Chiquita lagoon, Argentina, in which ample variations of salinity have been reported (Anger et al. 1994).

O Inter-Research 1999 Resale of fullartlcle not permitted 126 Mar Ecol Prog Ser

The larval development of Palaemonetes argentinus and premolt (D) near the end of the molt cycle. was described from laboratory-reared shrimp (Menu- Hemolymph samples were collected exclusively from Marque 1973). It comprises a variable number of zoeal stage C individuals. In zoeal, decapodid and first juve- and decapodid stages. Under natural conditions, the nile stages, this was defined as the middle of the instar biological cycle of P. argentinus can be accomplished period. The validity of this staging method was occa- in either freshwater or brackish water (review in sionally confirmed through rnicroscopical observations Spivak 1997). The life history of a population living in taking the telson or uropods as reference body parts the brackish lagoon of Mar Chiquita, Argentina, and (Drach 1939, Drach & Tchernigovtzeff 1967, Anger in adjacent freshwater creeks has recently been de- 1983). In adult , stage C individuals were scribed by Spivak (1997). All developmental stages of selected after microscopical observation of the uropods this species have been found together in this lagoon, (Drach & Tchernigovtzeff 1967). indicating larval development within the parental Preparation of media. Experimental media were pre- habitat (Anger et al. 1994). pared and stored in 5 1 plastic containers for the entire Since coastal lagoons like Mar Chiquita are charac- duration of the experiments. Dilute media were pre- terized by highly variable salinity regimes, fluctuating pared by adding desalinated freshwater to natural North between freshwater and seawater (Anger et al. 1994), Sea water. All experiments were conducted at a constant this species is exposed to a wide salinity range temperature of 24°C. Salinities were expressed as throughout its life cycle. The objectives of the present osmotic pressure (in mOsm kg-') and as salt content of

study were thus (1) to determine the ability of selected the medium (in %o);a value of 3.4%0is equivalent to postembryonic stages of development to osmoregu- 100 mOsm kg-' (29.41mOsm kg-' per l %o).The osmotic late, and (2) to relate their osmoregulatory capabilities pressure of the media was measured with a micro- with available data on the developmental ecology of osmometer Model 3 MO (Advanced Instruments, Need- this species. ham Heights, MA, USA) requiring 20 pl sample-'. Media with the following osmolalities (mOsm kg-') and cor-

responding salinities (%o) were prepared and used MATERIALS AND METHODS for all stages: 31 mOsm kg-' (l.l%o),77 (2.6), 156 (5.3), 302 (10.3), 500 (17.0),and 755 (25.7).Adult shrimp were Specimens: origin and culture. Juvenile shrimp also exposed to seawater, 947 mOsm kg-' (32.2%). Palaemonetes argentinus were collected near the mouth Osmoregulation. Zoeal and decapodid stages were of a freshwater creek, Arroyo Sotelo, flowing into the exposed to the experimental media in covered petn Mar Chiquita lagoon, Argentina (for more details of the dishes. In decapodids, a particular escape behavior habitat see Spivak 1997), and transported live in water (jumping) had to be prevented by placing additionally from the capture site (1%0 salinity) to the marine bio- a sheet of nylon gauze (200 pm mesh size) on the water logical station Helgoland, Germany. They were reared surface. Later stages (juvenile I, adults) were placed in to adulthood at 1%0,24°C and a 12 h light:12h dark glass vials (250 m1 and 1 1 capacity, respectively). cycle, with Artemia sp. nauplii or isopods (Idotea spp.; These were covered with a convex glass lid, the bot- in late juvenile stages only) given as food. tom of which touched the surface of the media to pre- Since preliminary experiments (Anger unpubl.) had vent jumping; at the margins of the vials, a ring of air shown that larval development requires brackish allowed for gas exchange. water, larvae were reared from hatching through Hemolyrnph osmolality was determined for each stage metamorphosis at 5%0,using aerated beakers (1 1;other after a period of osmotic stabilization in each medium. conditions as in juvenile rearing). Water and food Based upon results from previous studies on different (freshly hatched Artemia sp, nauplii) were changed species (Charmantier 1998), larvae and first juveniles daily. The developmental stages used in experiments were kept for 24 h in each medium before sampling; the were the first and the last zoeal stage (zoea I, VI), the acclirnation time in adults was 48 h. Larvae and juveniles first decapodid (postembryonic instar VII), the first were superficially dried on filter paper, then quickly juvenile stage (reached after a total number of 6 zoeal immersed in mineral oil to avoid evaporation and desic- and 2 to 5 decapodid stages; for terminology of larval cation. The remaining adherent water was aspirated stages see Williamson 1982) and adults (males and through a glass micropipette. Another micropipette was females, total body length ranging from 2.4 to 3.6 cm). then inserted in the heart to sample hemolymph. In adult The duration of successive larval stages was 2 to 3 d. shrimp, hemolymph was collected from a cut fourth or Stages within each molting cycle (Drach 1939) were fifth pereiopod, after carefully drylng it with filter paper. determined according to the time elapsed since the last For all developmental stages, hemolymph osmolality preceding ecdysis, distinguishing between the initial was measured with reference to the medium osmo- postmolt stages (A and B), intermolt (C) in the middle, lality on a Kalber-Clifton nanoliter osmometer (Clifton Charmantier & Anger: Ontogeny of osmoregulation in Palaemonetes argentinus

Technical Physics, Hartford, CT, USA) requiring about Medium (%o) 30 nl. The results were expressed either as hemolymph 1.1 2.6 5.3 10.3 17.0 25.7 32.2 I.I.I.I.,., osmolality or as osmoregulatory capacity (OC), defined 1 I I ddd C Palaemonetes argentinus as the difference between the osmolalities of hemo- d bd lymph and the medium. Analysis of variance (ANOVA) D zoea l and Student's t-tests were used for multiple and pair- El Zoea V1 wise statistical comparisons of mean values, respec- Decapodid tively, after appropriate checks for normal distribution B JuvenileI Adult and equality of variance (Sokal & Rohlf 1995).

RESULTS

Osmoregulation by the developmental stages was evaluated over a wide range of salinities. The results are given as variations in the hemolymph osmolality and as OC in relation to the osmolality and salinity of Medium(mosmlkg) the medium (Figs. 1 & 2). Fig. 2. Palaemonetes argentinus. Variations of the osmoregu- The pattern of osmoregulation did not change during latory capacity (OC) in different stages of postembryonic de- development. All tested stages hyper-regulated in the velopment in relation to the osmolality and salinity of the medium at 24°C; error bars: X*SD; n = 4 to 10 (zoeae I and VI) dilute media (< 17 %o), hyper-osmoconformed at 17.0 Oh and 7 to 12 (later stages) individuals; different letters above and isoconformed at 25.?%0; the adult shrimp isocon- error bars indicate significant differences between stages formed also in seawater (32.2%0).Thus, the pattern of (p < 0.05) hyper-iso-osmoregulation was present already in the zoea I, and it persisted in the decapodid, juvenile and adult stages of the Me cycle (Fig. 1). podid stage. No important variation in OC was noted in The ability to hyper-osmoregulate at low salinity later developmental stages, i.e. in the juvenile I and tended to increase slightly during early development, adults. At moderately low salinities (5.3and 10.3 %o), the as demonstrated by variations in hemolymph osmolality hyper-OC increased significantly from zoea I to zoea V1 (Fig. 1) or in OC (Fig. 2). At very low salinities (1.1and and the decapodid stage. In subsequent stages, it 2.6%0),the hyper-OC did not change between zoeal remained almost unchanged, with a slight tendency to stages I and VI, then it increased slightly in the deca- decrease in adults at 5.3%0.At 17.0 and 25.7%0,there was little ontogenetic variation in OC. At 25.7%0, all Medium(%o) stages osmoconformed, as did the adults at 32.2%0. Since 32.2Y00 is an unusually high salinity for Palae- monetes argentinus under natural conditions, a group Paluemonetesargentinus of 13 adult shrimps was exposed for several days to this medium. The water was aerated and changed regu- larly, and the shrimps were fed. The median lethal time (LT 50) for 7 of them was 11d. No mortality was noted in a control group of shrimp maintained for the same time at l%o.

0 0 --+-- Decapodid 4 N Juvenile I DISCUSSION R , +- Adult Among the decapod species which have been stud- ied for their osmoregulation, most marine and brack- Medium(mosmlkg) ish-water caridean shrimps hyper-hypo-osmoregulate as adults. In particular, they display an efficient hypo- Fig. 1. Palaernonetes argentinus. Variations of the hemo- regulation in seawater (925 to 1000 mOsm kg-', ca 31.5 lymph osmolality in different stages of postembryonic devel- to 34.0%0;review in Mantel & Farmer 1983). In the opment in relation to the osmolality and salinity of the medium at 24°C; error bars: E+ SD; n = 4 to 10 (zoeae I and VI) genus Palaemonetes, reported hypo-osmoregulatory and 7 to 12 (later stages) individuals; dashed line: isocon- capacities in seawater were -320 mOsm kg-' in P. var- centration ians (Potts & Parry 1964), -380 mOsm kg-' in P. inter- 128 Mar Ecol Prog Ser

medius (Dobkin & Manning 1964), and -275 mOsm low salinities increases, but the pattern of hyper- kg-' in P. pugio (Roesijadi et al. 1976). This is in con- isoregulation remains the same. This also applies, trast to adult P. argentinus, which hyper-regulate at without important changes in regulation efficiency, to low salinities and hyper-osmoconform or isoregulate at the first juvenile and the adult stages. higher salinites up to 32.2Ym. This ontogenetic pattern in the capability for osmo- Many freshwater caridean shrimps are actually regulation can be assigned to 1 of 3 principal types euryhaline and can thus adapt to relatively high salin- described by Charmantier (1998). Palaemonetesargen- ities in the range of 25 to 33%0.Some of these have tinus belongs to the second category, in which the retained a capacity to hypo-regulate in seawater, e.g. adult type of efficient osmoregulation is established as Macrobrachium rosenbergii(Sandifer et al. 1975) and early as in the first postembryonic stage. Among the M. petersi (Read 1984), while others osmoconform at other crustaceans belonging to this category there are 700 mOsm kg-' (ca 23.8%) and above, e.g. - several branchiopod cladocerans (Aladin & Potts 1995), etes paludosus (Dobkin & Manning 1964). P. argenti- Artemia salina (Conte 1984), the amphipod Gammarus nus clearly belongs to this latter group. It is a strict duebeni (Morritt & Spicer 1995), the isopods Cyathura osmoconformer in salinities of about 20 to 32.2%0.Sur- polita (Kelley & Burbanck 1972) and Sphaeromaserra- vival under these conditions is limited to a few days, tum (Charmantier & Charmantier-Daures 1994), and, and, in brackish-water habitats such as Mar Chiquita among the decapods, the palaemonid shrimp Macro- lagoon, the shrimps tend to avoid high-salinity areas brachiumpetersi (Read 1984) and the crayfish Astacus (Anger et al. 1994). leptodactylus (Charmantier-Daures & Charmantier At the other end of the salinity spectrum, Palaemon- 1997). A feature common to these species is that they etes argentinusis well adapted to low and very low live in osmotically harsh habitats. Since they are salinities through efficient hyper-osmoregulation. This exposed to high osmotic stress upon hatching, these supports the general view of this shrimp as a freshtva- species have adapted through the appearance of effi- ter species (Spivak 1997). At 31 mOsm kg-' (l.l?&;i.e. cient osmoregulatory processes in the first (and subse- at conditions close to freshwater), adult individuals quent) postembryonic stages. In freshwater species maintain hemolymph osmolality at about 410 mOsm (including P. argentinus)this translates into high val- kg-'. This average value is within the 350 to 550 (most ues of hemolymph osmolality at very low salinity. As frequently 400 to 500) mOsm kg-' range reported for the ability to osmoregulate depends on specialized tis- hemolymph osmolality in freshwater decapods, includ- sues and organs (review in Charmantier 1998) their ing caridean shrimps (e.g. P. antennarius:400 mOsm structural and functional ontogeny will be an interest- kg-', Parry 1957; P. paludosus:470 mOsm kg-', Dobkin ing field for future research throughout the develop- & Manning 1964), crayfishes, and some potamid crabs ment of P, argentinus.Since, in this species, the pattern (review in Mantel & Farmer 1983). The strong ability of of osmoregulation is basically the same from early P, argentinusto hyper-regulate in media close to fresh- zoeae to adults, additionally the question arises water most probably depends, at least in part, on active whether the same or different osmoregulatory tissues ion pumping by specialized osmoregulatory tissues in and organs are involved throughout ontogeny. the branchial chamber. Their location would be worth There is mounting evidence that the ability of young studying. In addition, it has been demonstrated that developmental stages to osmoregulate is an important some freshwater candean shrimps can, as do cray- adaptive process. The possession of high osmoregula- fishes, produce a hypo-osmotic urine to prevent ion tory capabilities at hatching is one of the adaptive fac- loss (reviews in Mantel & Farmer 1983, Pequeux 1995). tors permitting the development of Palaemonetes Very few palaemonid shrimps have been studied with argentinus in freshwater or in coastal areas such as respect to urine concentration, and since several of Mar Chiquita lagoon. This shallow lagoon covering them, including P. argentinus,are known to penetrate 46 km2 is constantly fed with freshwater (salinity ca into freshwater, the contribution of the excretory l%), and seawater (ca 33.5~%~)can intermittently enter organs to hyper-osmot~c regulation in this species and leave according to different factors associated with would be another interesting field of research. the topography of the mouth of the lagoon, the lunar The pattern of osmoregulation does not change dur- cycle, tidal amplitude, and the direction and strength ing the postembryonic development of Palaemonetes of winds. The resulting salinity in the habitat of P. argentinus.The zoea I and V1 and, most probably, all argentinuscan be low (1 to 5%) for extended periods other zoeal stages are hyper-isoregulators, with a of several days, or it can vary between 1 and 30%0 slight ontogenetic increase in the ability to hyper- within a few hours (Anger et al. 1994). Given the im- regulate at 5 and 10n4~.In decapodids, which are mor- portant osmoregulatory abilities of this species at all phologically and physiologically transitional between postembryonic stages in media ranging from fresh- zoeae and juveniles, the ability to hyper-regulate at water to seawater, the palaemonid shrimp P, argenti- Charmantier & Anger: Ontogeny of osmoregulation in Palaemonetes argentinus 129

nus has been able to invade freshwater habitats, while (Woods Hole) 175.102-110 retaining the ability of adapting to brackish habitats Charmantier G, Charmantier-Daures M, Anger K (1998) with variable salinity. Ontogeny of osmoregulation in the grapsid crab Armases miersii (Crustacea, Decapoda). Mar Ecol Prog Ser 164: Similar ecophysiological correlations have been es- 285-292 tablished in species living in habitats with demanding Charmantier-Daures M, Charmantier G (1997) Osmoregula- salinity features, for instance under variable conditions tory ability of early juvenile crayfish Astacus leptodacty- in shallow coastal waters or lagoons (Sphaeroma serra- lus. Am Zool 3?:140 (abstract) Conte FP (1984) Structure and function of the tum: Charmantier & Charmantier-Daures 1994; Gam- larval salt qland. Int Rev Cvtol91:45-106 marus duebeni: Morntt & Spicer 1995), temporary rain- Dobkin S, ~anningRS (1964) bsmoregulation in two species fall puddles (Uca subcylindrica: Rabalais & Cameron of Palaemonetes (Crustacea: Decavoda). . from Florida. Bull 1985) and supratidal rockpools (Armases miersii: Char- Mar Sci Gulf ~arjbb14:149-157 mantier et al. 1998), in extremely hgh salinities (Artemia Drach P (1939) Mue et cycle d'interinue chez les Crustaces Decapodes. Ann Inst Oceanogr Monaco 19:103-391 salina: Conte 1984), in freshwater (Astacus leptodacty- Drach P, Tchernigovtzeff C (1967) Sur la methode de determi- lus: Charmantier-Daures & Charmantier 1997), and in nation des stades d'intermue et son application generale species that accomplish ontogenetic migrations between aux Crustaces. Vie Mheu 18A:595-609 habitats with different salinity regimes (Macrobrachum Kelley BJJr, Burbanck WD (1972) Osmoregulation in juvenile and adult Cyathura poljta (Stimpson) subjected to salin- petersi: Read 1984; Penaeus japonicus: Charmantier et ~tychanges and ionizing gamma irradiation (Isopoda, al. 1988). An improved understanding of the ecological Anthuridea). Chesapeake Sci 13:201-205 implications and of the adaptive and evolutionary sig- Mantel LH, Farmer LL (1983) Osmotic and ionic regulation. nificance of the ontogeny of osmoregulation should In: Mantel LH (ed) The biology of Crustacea, Vol 5. come from further studies conducted on other species Internal anatomy and physiological regulation. Academic Press, New York, p 53-161 living in diverse habitats, and from investigations on the Menu-Marque SA (1973) Desarrollo larval de Palaemonetes physiological basis of the ontogeny of osmoregulation. argentinus (Nobili, 1901) en el laboratorio (Crustacea, Caridea, Palaemonidae). Physis (B Aires) 32:149%169 Morritt D, Spicer JI (1995) Changes in the pattern of osmo- Acknorvledgements. G.C. thanks Prof. Dr F. Buchholz, Head regulation in the brackish water amphipod Gammarus of the Helgoland Section of the Stiftung Alfred-Wegener- duebeni Lilljeborg (Crustacea) during embryonic devel- Institut, Biologische Anstalt Helgoland, for receiving him at opment. J Exp Zool 273:271-281 the marine biological station. The authors thank Dr M Char- Nobili G (1901) Decapodi racolti dal Dr Filippo Silvestri nell' mantier-Daures for help and critical reading of the manu- America meridionale. Boll Mus Zool Univ Torino 16:l-15 script. K. Riesebeck, K. Paschke, K. Pfaff and G. and L. Parry G (1957) Osmoregulation in some fresh water prawns. Gimenez for occasional technical help, U. Alexander and J Exp Biol 34:417-423 M. A. Garcia for secretarial assistance. This is a contribution Pequeux A (1995) Osmotic regulation in crustaceans. J Crus- to a cooperative research programme, MAR-8, between the tac Biol 15:l-60 Universidad Nac~onal de Mar del Plata (Argentina) and the Potts WTW, Parry G (1964) Sodium and chloride balance in Biologische Anstalt Helgoland (Germany), funded by the the prawn, Palaemonetes varians. J Exp Biol 41:591-601 Federal Ministry of Science and Technology (Bonn) and Rabalais NN, Cameron JN (1985) The effects of factors impor- SECYT (Buenos Aires). A grant from the French Ministitre tant in semi-arid environments on the early development des Affaires Etrangeres was awarded to G.C. of Uca subcylindrica. Biol BuU (Woods Hole) 168:147-160 Read GHL (1984) Intraspecific variation in the osmoregula- tory capacity of larval, post-larval, juvenile and adult Mac- LITERATURE CITED robrachiurn petersi (Hilgendorf). Comp Biochem Physiol 78A:501-506 Aladin NV, Potts \VTW (1995) Osmoregulatory capacity of the Roesijadi G, Anderson J, Petrocelli S, Giam C (1976) Osmo- Cladocera. 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Editorial responsibility: Otto Kinne (Editor), Submitted: October 27,1998; Accepted: January 26, 1999 Oldendorf/Luhe, Germany Proofs received from authorjs): May 4, 1999