Osmoregulation by six species of fiddler crabs (Uca) from the Mississippi delta area in the northern Gulf of Mexico

Journal of Experimental Marine Biology and Ecology 291 (2003) 233 – 253 www.elsevier.com/locate/jembe

Osmoregulation by six species of fiddler crabs (Uca) from the Mississippi delta area in the northern Gulf of Mexico Carl Thurman* Department of Biology, University of Northern Iowa, Cedar Falls, IA 50614-0421, USA Received 27 October 2002; received in revised form 6 December 2002; accepted 10 March 2003

Abstract Six species of fiddler crabs (Ocypodidae: Uca) were collected for osmoregulation studies from 25 locations near the delta of the Mississippi River in the northern Gulf of Mexico. Three of the species are classified as members of the Celuca subgenus, Uca spinicarpa, Uca panacea and Uca pugilator, while the remaining three are in the Minuca subgenus, Uca minax, Uca longisignalis and Uca rapax. In the field, U. minax, U. spinicarpa and, occasionally, U. longisignalis are found in freshwater habitats (FW; 0 – 299 mosM). Two Minuca species, U. longisignalis and U. rapax, are typically collected in brackish water habitats (BW; 300 – 629 mosM). On the other hand, U. panacea and U. pugilator are most abundant in eurysaline habitats (EH; >630 mosM). In the laboratory, populations of each species were challenged with media ranging from 30 to 3450 mosM (1 – 110x ). The FW species, U. spinicarpa and U. minax, did not tolerate osmotic concentrations >2100 mosM. The EH species, U. panacea and U. pugilator, however, tolerate concentrations >2800 mosM. The BW species, U. longisignalis and U. rapax, succumb to osmolalities between 2100 and 2800 mosM. Each species keeps its hemolymph concentration fairly constant in 30 – 1400 mosM solutions. The [ISO], isosmotic medium concentration (in mosM), is calculated for each taxon: U. minax, 659; U. spinicarpa, 682; U. longisignalis, 693; U. rapax, 769; U. pugilator, 816; and U. panacea, 822. In media with >1600 mosM, each species expresses different osmoregulating capabilities. The FW species, U. spinicarpa and U. minax, cannot control hemolymph osmolality above 1500 mosM while the BW-EH species, U. panacea, U. pugilator and U. rapax, regulate hemolymph values in media up to 2300 mosM. Within the FW/BW species U. longisignalis, the ability to osmoregulate corresponds with site of collection. Specimens from FW populations do not regulate as well as those from BW if challenged with hypertonic media. If adapted to a 1800 mosM in the laboratory, survivorship for U. longisignalis shifts to the right and the [ISO] increases to 832 mosM. This suggests that this species adapts to acute hypertonic conditions by tolerating elevated

* Tel.: +1-319-273-2276; fax: +1-319-273-7125. E-mail address: [email protected] (C. Thurman). 0022-0981/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0022-0981(03)00138-2

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internal osmolality. Generally, these observations extend our knowledge about the physiological capabilities of fiddler crabs from different salinity populations across the northern Gulf of Mexico. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Fiddler crabs; Uca spp.; Osmoregulation; Ecology; Ecophysiology; Biogeography

1. Introduction Fiddler crabs (Ocypodidae, genus Uca) are inhabitants of circumtropical lagoons, estuaries and intertidal regions where the salinity fluctuates from almost freshwater to suprasaline (Crane, 1975; Barnwell and Thurman, 1984). In a study of Pacific crabs, Jones (1941) found Uca crenulata from southern California to be excellent osmoregulators. In low salinity, the crabs kept their hemolymph osmolality well above that of the media (i.e. hyperegulation). On the other hand, under high salinity, hemolymph osmolality was maintained well below that of the media (i.e. hyporegulation). Species which were good hyporegulators could maintain a more constant internal osmotic pressure when exposed to air. Thus, the author felt that this ability has probably been of great importance in the development of a terrestrial life for some species. However, any role played by osmoregulation in the evolution of semiterrestrial fiddler crabs has been fairly obscure. Early studies on fiddler crab species from the east coast of North America, Uca pugilator, Uca pugnax and Uca minax, indicated that they were uniform in osmoregulation capability (Green et al., 1959; Baldwin and Kirschner, 1976a,b; Wright et al., 1984) despite their differences in salinity preferences and ecological distribution (Teal, 1958). More recent investigations, on the other hand, have found substantial differences in osmoregulating ability among species (Rabalais and Cameron, 1985; Graszynski and Bigalke, 1986; Lin et al., 2002). Consequently, it is now believed that physiological differences among species correlate with the habitat preference of each. Along this line, there is some evidence that regulatory capabilities may even vary between populations within a single species (Ferraris and Norenburg, 1997). Between the mouth of the Mississippi River in Louisiana and Cape San Blas on Florida’s western panhandle, six species of fiddler crabs are distributed across the northern Gulf of Mexico (Thurman, 1982; Barnwell and Thurman, 1984; Mangum, 1996). From a biogeographic perspective, this portion of the Gulf is a transition zone for temperate and tropical Uca. Interestingly, four of the six congeners from this area are close relatives. Since few studies have addressed salt and water balance by fiddler crabs in this area, the goal of this investigation was twofold. First, to document the ecological distribution of each species of fiddler crab with respect to habitat osmolality. Second, to examine the osmoregulatory capabilities of each species. To broaden our understanding of intraspecific variation, attention focused on comparing the physiological abilities of specimens of the same species collected in different ‘‘osmotic habitats’’. As a result of these observations, we now know the osmoregulatory capabilities of all six species from the region. It is clear that the pattern of tolerance and regulation expressed by each species correlates with the range of osmolality encountered in nature. Among the six species, only one, Uca

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longisignalis, expresses a substantial difference in osmoregulation between habitats. The findings presented here are similar to those reported previously for osmoregulation by Uca in other regions of eastern North America (Thurman, 2002, 2003).

2. Methods 2.1. Collection, transport and care of Uca Between 18 and 22 June 2002, fiddler crabs and surface water samples were collected during low tide from 25 locations along the northern coast of the Gulf of Mexico (Fig. 1). In LaFourche Parish, LA, collections were made at (1) Golden Meadow (51 mosM) on State Highway 1 and (2) Leeville-State Highway 1 bridge (552 mosM) on Bayou LaFourche. In Jefferson Parish, collections were made at (3) Cheniere Caminada (449 mosM), (4) Caminada Pass (290 mosM) (29j12VN, 90j03VW), (5) Gulf Stream Marina (492 mosM) and (6) Grand Isle State Park (167 mosM). Along the Mississippi coast, crabs were collected from (7) upper Old Fort Bayou (106 mosM), (8) the Washington St. bridge (249 mosM), (9) the Gulf Coast Research Laboratory (GCRL) beach (538 mosM) and (10) Davis Bayou (463 mosM) in Ocean Springs, Jackson County (30j24VN, 88j51VW). In Mobile County, AL, collections were made from (11) Fowl River (30j23VN, 88j14VW)

Fig. 1. Collecting sites indicated by closed circles (.). A—Golden Meadow. B—Leeville. C—Cheniere Caminada. D—Pass Caminada. E—Gulf Stream Marina. F—Grand Isle State Park. G—Old Fort Bayou. H—North Washington St. bridge. I—Gulf Coast Research Laboratory beach. J—Gulf Islands National Seashore, Davis Bayou. K.—Upper Fowl River. L, M—Dauphine Island Causeway. N—West end Dauphin Island. O—East lagoon, Ft. Gaines. P—West bank Tensaw River. Q—Big Lagoon State Park. R—Millview Creek. S—Riverview. T—Tom King Bayou, East Bay. U—Jolly Bay. V—Co. 2321 Dam Rd. W—McKenzie St. dock. X—FL 77 bridge, Lynn Haven. Y—St. Andrew State Park. Bar = 100 km scale. North latitude and west longitudes indicated.

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near State Highway 188 (349 mosM) and from both the (12) Mississippi Sound (560 mosM) and (13) tidal channels (595 mosM) on the northern shoal of the Dauphin Island Causeway (State Highway 193). On Dauphin Island, crabs were taken from (14) west end tide pool near Bienville Blvd (101 mosM) and from the (15) east lagoon (800 mosM) near Ft. Gaines (30j15VN, 88j04VW). At the north end of Mobile Bay in Baldwin County, Uca were collected from (16) the west bank of the Tensaw River (47 mosM) near the US Highway 31 bridge (30j49VN, 87j55VW). In western Florida, fiddler crabs were captured in Escambia County at (17) Big Lagoon State Park (963 mosM), (18) Millview Creek (492 mosM) on the Perdido River and (19) Riverview (168 mosM) on the Escambia River (30j24VN, 87j13VW). In Santa Rosa County, crabs were collected from (20) Tom King Bayou (637 mosM) at East Bay (30j27VN, 86j55VW). In Walton County, a collection was made on the banks of (21) Jolly Bay (569 mosM) between La Grange Point and the Choctawhatchee River (30j24VN, 86j31VW). In Bay County, collections were made at (22) North Bay dam (153 mosM) on County Highway 2321, (23) McKenzie Commercial Dock (923 mosM) in Southport, (24) south end of State Highway 77 bridge (752 mosM), Lynn Haven (30j15VN, 85j39VW) and (25) St. Andrew State Park (947 mosM), Bitmore Beach, Panama City (30j07VN, 85j44VW). Crabs were transported by air to the University of Northern Iowa in plastic boxes containing water from the collection site. Within 24 h, they were submitted to laboratory conditioning at 23 jC. Only non-ovigerous, intermolt adult specimens with carapace width greater than 10 mm were used in experiments. The collections were divided into three groups. The first consisted of five crabs of each species held in habitat water. A second group of each species was submitted to the full regime of osmotic challenge within 7 days of collection. A third group (U. longisignalis) was slowly adapted to hyperosmotic solutions. First, they were placed in a 980 mosM solution for 7 days. Subsequently, these were transferred to 1450 mosM for 7 days then to 1800 mosM for an additional 10 days before receiving a regimen of osmotic challenge. In every case, crabs were able to leave the medium at will. For long-term maintenance, Uca were fed goldfish food (Tetra Werke, Melle, Germany) and frozen brine shrimp. 2.2. Osmotic solutions To obtain different concentrations of artificial seawater (ASW), Instant Oceank (Aquarium Systems, Mentor, OH) was mixed with distilled H2O to the desired density and the pH adjusted to 8.0 with HCl or NaOH. Groups of four to six crabs were placed in covered bowls (13  5 cm) containing 50 ml of ASW for 5 days. To eliminate waste and maintain a constant mosM, ASW solutions were replaced on day 2 and day 4 of each experiment. Uca were not fed 3 days prior to or during an experiment. Survivorship records were kept during the exposure period. A crab was considered deceased if it could no longer right itself. Only living Uca were used for osmotic measurements. 2.3. Osmolality measurements Following osmotic challenge, hemolymph was withdrawn from the ventral hemocoel via puncture of the arthrodial membrane between the basis and coxa of the fifth pereiopods

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using a chilled 1.0-ml tuberculin syringe (27-gauge needle). The osmolality (mmol kg 1) of each 10-Al hemolymph sample was determined immediately using a Wescork 5520 Vapor Pressure Osmometer (Logan, UT) by comparison to commercial standards. Water samples from finger bowls and habitats were also measured. Standard concentrations and mosM measurements were identical between 60 and 3200 mosM. However, between 1 and 55 mmol kg 1, the actual mosM was determined using a standard curve constructed from serial dilutions of a 100 mosM standard. The osmolality of double-glass distilled, deionized H2O was between 2 and 7 mosM. Fluid osmolality was taken as the average of all hemolymph samples from each crab or water sample. For each measurement, an average for all crabs is reported as mean F standard error. Statistical significance was assessed using Student’s t-test with p < 0.05 significant and p < 0.01 highly significant. In general, N refers to the number of crabs unless otherwise stated. Probit analysis (Finney, 1947) was used to estimate the 50% lethal osmotic concentration (LC50). The isosmotic medium concentration [ISO] for each species was calculated from the linear regression of hemolymph with medium osmolality in Figs. 3 –5. Only data points from the linear portion of the plot around the isosmotic line were used in computation.

3. Results 3.1. Field studies Between Bayou LaFourche, LA, and St. Andrew Bay, FL, six species of Uca were taken from 25 habitats (Fig. 1; Table 1). From a systematic perspective, the six species are equally divided among two subgenera of Uca (Crane, 1975). Three species, U. minax, U. longisignalis and Uca rapax, are considered to be in the Minuca subgenus while three species, Uca spinicapra, Uca panacea and U. pugilator, belong in the Celuca subgenus. In general, Uca are abundant across the study area. However, in spite of the fact that collections sites ranged over only 1j17Vof latitude, species are not uniformly distributed from east to west. The three species common along the entire coast are U. minax, U. longisignalis and U. panacea. Two species, U. spinicarpa and U. rapax, are plentiful in the west but rare in eastern marshes. On the other hand, U. pugilator are found only in the most eastern marshes of St. Andrew Bay, Bay County, FL, and absent from western sites. Since 50% of the local Uca species exhibit range boundaries in this portion of the Gulf, the region between Mobile Bay, AL (87j55VW), and St. Andrew Bay, FL (85j39VW), represents a ‘‘longitude of transition’’ for the genus (Barnwell and Thurman, 1984). From an ecological viewpoint, each of the six species can also be classified into one of three salinity or osmotic habitats (Hedgpeth, 1957). An oligosaline or freshwater (FW) habitat is an environment with salinity between 1xand 10xor osmolality between 38 and 299 mosM. Habitats with salinity between 11xand 21xor osmolality between 300 and 630 mosM are mesosaline or brackish (BW). Eurysaline habitats (EH) have salinity greater than 21xor osmolality above 631 mosM. Two species, U. minax and U. spinicarpa are most often collected in FW or BW habitats (Table 1). U. longisignalis and U. rapax were typically collected from BW habitats. Also, U. longisignalis were frequently

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Table 1 Relation between habitat and hemolymph osmolality for Uca from the northern Gulf of Mexico Subgenus

Species

Habitata

Collection site preference

Habitat mosM

x

Hemolymphb (mosM)

Celuca

U. spinicarpa

FW/BW

U. pugilator

BW/EH

U. panacea

BW/EH

U. minax

FW

U. longisignalis

FW/BW

U. rapax

EH

Tensaw River Grand Isle SP Escambia River No Washington Br. Davis Bayou Millview Creek GCRL Beach Dauphin I Causeway E. Dauphin I North Bay So. End 77 Br. McKenzie Dock St. Andrew SP W. Dauphin I North Bay Grand Isle SP Gulf Stream Millview Creek East Bay So. End 77 Br. E. Dauphin I McKenzie Dock Big Lagoon Tensaw River Bayou Fourche Upper Old Fort Bayou North Bay Escambia River Millview Creek Tensaw River Bayou LaFourche W. Dauphin I. Upper Old Fort Bayou North Bay No. Washington Br. Upper Fowl River Cheniere Caminada Davis Bayou Millview GCRL Beach Leeville Br. Jolly Bay Dauphin I. Causeway E. Dauphin I Grand Isle SP Caminada Pass Cheniere Caminada

47 167 168 249 463 492 538 560 800 153 752 923 947 101 153 167 492 492 637 752 800 923 963 47 51 106 153 168 492 47 51 101 106 153 249 349 449 463 492 538 552 569 595 800 167 290 449

1.8 5.8 5.8 8.7 16.1 17.1 18.7 19.5 27.9 5.3 26.2 32.1 33.0 3.5 5.3 5.8 17.1 17.1 22.2 26.2 27.9 32.1 33.5 1.6 1.8 3.7 5.3 5.9 17.1 1.6 1.8 3.5 3.7 5.3 8.7 12.2 15.6 16.1 17.1 18.7 19.2 19.8 19.5 27.9 5.8 10.1 15.6

598 (1) 643 (1) 608 F 46 608 F 17 665 F 35 697 F 8 653 (1) 639 F 17 692 (1) 792 F 8 797 F 28 889 F 12 922 F 48 799 F 19 773 F 27 783 F 48 800 F 19 804 F 14 733 F 61 784 F 46 784 F 17 872 F 29 828 F 8 552 F 7 568 F 22 587 F 19 644 F 40 590 F 36 639 F 9 586 F 21 628 F 20 684 F 35 653 F 23 659 F 21 658 F 22 664 F 15 688 F 67 687 F 31 697 F 36 755 F 72 765 F 67 679 F 18 815 F 17 658 (1) 713 F 20 731 F 19 780 F 12

Minuca

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Table 1 (continued) Subgenus

Minuca

a b

Species

U. rapax

Habitata

EH

Collection site preference

Habitat mosM

x

Hemolymphb (mosM)

GCRL Beach Leeville Br. Dauphin I Causeway

538 552 560

18.7 19.2 19.5

758 F 36 780 F 15 778 F 35

FW: freshwater; BW: brackish water; EH: euryhaline. For each species, N = 5 specimens except where noted by ( ).

captured in FW. Both U. panacea and U. pugilator were common in EH habitats across the northern Gulf. Occasionally they were found in FW. In the western Gulf, other populations of these species occupy similar habitats (Thurman, 1984, 1998). The range of environmental conditions tolerated by different Uca species is illustrated in Table 1. For each species, the recorded habitat range (in mosM) is as follows: U. minax, 47 –492; U. spinicarpa, 47 – 560; U. longisignalis, 47– 595; U. rapax, 167– 560; U. pugilator, 153 –947; and U. panacea, 101 –963. For members of the Celuca subgenus (U. panacea, U. pugilator and U. spinicarpa), average hemolymph osmolality is different for each species across all habitats. For U. spinicarpa collected from nine localities, mean hemolymph osmolality is 644.8 F 33.9 mosM. For U. panacea from 10 sites, it is 796.0 F 34.4 mosM and U. pugilator from four sites is 850.0 F 56.7 mosM. Each is significantly different from its cohorts ( p < 0.05). For the Minuca subgenus (U. rapax, U. longisignalis and U. minax), the average hemolymph osmolality for U. minax from six sites is 596.7 F 34.1 mosM, U. longisignalis from 15 localities is 685.1 F 54.8 mosM, and U. rapax from six localities is 756.7 F 26.2 mosM. The hemolymph value for each species of Minuca is significantly different from its congeners ( p < 0.001). However, within each species, internal osmotic state appears to vary with habitat salinity. Among the Celuca, as habitat osmolality (X) changes around U. spinicarpa from 47 to 800 mosM, the hemolymph osmolality ( Y) increases from 598 to 692 in a linear fashion ( Y = 0.1193X + 598.6; r = 0.8042; p < 0.01). On the other hand, in U. panacea ( Y = 0.0458X + 770.9; r = 0.4064; p>0.05) and in U. pugilator (Y = 0.1344X + 756.7; r = 0.7608; p>0.05), there is no significant correlation between habitat and hemolymph osmolality. Among Minuca, there is no significant relationship between habitat and hemolymph in U. minax (Y = 0.1628X + 569.1; r = 0.719; p>0.05). As habitat osmolality changes from 47 to 800 mosM around U. longisignalis, there is a significant increase in hemolymph concentration (Y = 0.1454X + 631.6; r = 0.6036; p < 0.05). Likewise, the hemolymph concentration for U. rapax changes significantly with habitat (Y = 0.1607X + 688.2; r = 0.9120; p < 0.05). Since the hemolymph remains constant over a range of habitat osmolality in U. minax, U. pugilator and U. panacea, physiological regulation is implicated (Vernberg and Vernberg, 1972; Prosser, 1991). 3.2. Tolerance Uca from various habitats were exposed to ASW concentrations between 30 and 3450 mosM (1 – 120x ) for 5 days. Survivorship for species in the Minuca and Celuca subgenera are illustrated in Fig. 2 and summarized in Table 2. The hyposmotic and

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Fig. 2. Survivorship for Celuca and Minuca spp. after 5 days in 30 – 3400 mosM. U. spinicarpa, n = 151. U. panacea, n = 389. U pugilator, n = 70. U. minax, n = 83. U. longisignalis, n = 288. U. rapax, n = 92. LC50 in Table 2.

hyperosmotic portions of the graphs reflect the adaptive capabilities of each species. When Celuca are exposed to ASW < 100 mosM, U. spinicarpa and U. pugilator (each 100% survival) slightly more tolerant of hyposmotic concentrations than U. panacea (92% survival). Among the three Minuca, U. minax (92% survival) and U. longisignalis (75% survival) tolerate hyposmotic conditions better than U. rapax (66% survival). These laboratory experiments support the field observations that U. spinicarpa, and U. minax are Table 2 Summary of osmoregulation in Ucaa Subgenus

Species

Habitatb

[LC50]c

[Isosmotic]

[Maximum medium]

Celuca

U. U. U. U. U. U.

FW BW/EH BW/EH FW FW/BW BW/EH

2030 2975 3270 2085 2230 2450

682 822 816 659 693 769

2200 3100 3454 2200 2750 3100

Minuca

a

spinicarpa panacea pugilator minax longisignalis rapax

Values in mosM. [ ] indicates concentration. Habitats described in text. c LC50 is upper limit for survival under osmotic stress calculated by probit analysis. b

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more likely to be found in FW environments than the other four species. By the same measures, survival under hyperosmotic conditions in the laboratory correlates with habitat occupancy. In all cases, Uca are able to withstand osmotic pressures greatly exceeding that of seawater (1004 mosM or 35x). Among the Celuca, U. spinicarpa are more susceptible to osmotic pressures above 2100 mosM (LC50 = 2030 mosM) than either U. panacea or U. pugilator. The latter have osmotic LC50s of 2975 and 3270 mosM, respectively. For species in the Minuca subgenus, each is influenced differently by hypoand hyperosmotic conditions in the laboratory. U. minax withstands lower osmotic pressures than U. longisignalis or U. rapax. In hypertonic media, the osmotic LC50 for U. minax, U. longisignalis and U. rapax are 2085, 2230 and 2450 mosM, respectively. These findings support the observation that U. longisignalis and U. rapax are more common inhabitants of BW than U. minax. In summary, based on hypo- and hyperosmotic tolerance, U. minax and U. spinicarpa express physiological propensities for inhabiting FW environs while U. longisignalis, U. rapax, U. panacea and U. pugilator are more likely to be found in BW/EH niches. 3.3. Osmoregulation As indicated in Figs. 3 and 4 and Table 2, Uca spp. from the northern Gulf of Mexico have excellent osmoregulatory abilities. Their patterns are best described as type IV: hyper- and hyporegulators (Vernberg and Vernberg, 1972). In media < 600 mosM, each is able to maintain its hemolymph concentration above the solution. When challenged with osmolality >800 mosM, the crabs are able to keep hemolymph values below that of the medium. In general, all species are good regulators in media ranging between 100 and 1400 mosM (4xand 49x). However, significant variations are seen in the ability of each to successfully regulate under hyperosmotic stress. The regulatory patterns of the three species in the Celuca subgenus are shown in Fig. 3 whereas patterns for the three species in the Minuca subgenus are show in Fig. 4. The hemolymph responses of the three species in the Celuca subgrouping to the osmotic regime are also summarized in Table 2. In media < 200 mosM, U. spinicarpa appear to maintain a more constant hemolymph concentration than U. panacea or U. pugilator. In U. spinicarpa, average hemolymph concentration between 200 and 1800 mosM (7xand 62.5x) is 702 F 60 mosM. However, there is a significant increase in hemolymph ( Y) concentration with medium (X) osmolality ( Y = 0.1278X + 595 mosM; r = 0.9093; p < 0.01). In osmolality ranging from 200 to 2200 mosM (7– 76.5x), the hemolymph osmolality in U. pugilator increases from 746 F 18 to 980 F 74 mosM (average = 854 F 89 mosM), while in U. panacea, hemolymph increases from 757 F 31 to 954 F 78 mosM (average = 843 F 69 mosM). Both possess a hemolymph which is significantly more concentrated than in U. spinicarpa ( p < 0.05). In both U. panacea and U. pugilator, hemolymph concentration is a linear function of the medium over the same osmotic range. For U. pugilator, the linear relation between bath and hemolymph is Y = 0.1487X + 695 mosM (r = 0.9521; p < 0.01), whereas in U. panacea, the relation is Y = 0.2350X + 629 mosM (r = 0.8869; p < 0.01). When the medium osmolality equals that of the hemolymph, the bath is said to have an isosmotic concentration [ISO]. Using the linear algorithms, the [ISO] for U. spinicarpa, U. panacea and U. pugilator are 682, 822

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Fig. 3. Celuca spp. Relation between medium and hemolymph solute concentration (mosM) after 5 days. Isosmotic line indicated (iso). Open circle (o) is mean for several populations shown as closed circle (.). U. spinicarpa, n = 102. U. pugilator, n = 62. U. panacea, n = 320. Curves fit by eye.

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Fig. 4. Minuca spp. Relation between medium and hemolymph solute concentration (mosM) after 5 days. Isosmotic line indicated (iso). U. minax, n = 65. U. longisignalis, n = 218. U. rapax, n = 62. ( ) indicates osmolality of habitat water. BW-EH indicates average for crabs from habitats with osmolality >450 mosM in Table 1. Curves fit by eye.

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and 816 mosM, respectively (Table 2). Above 1800 mosM, U. spinicarpa lose control of hemolymph osmolality. On the other hand, U. pugilator and U. panacea appear to lose control of hemolymph osmolality in media with osmotic pressure greater than 2200 mosM. Specimens from all four populations of U. pugilator seem to respond to hyperosmotic stress in a similar fashion. In U. panacea, there appears to be considerable variation in response among populations to hypertonic solutions. However, no cohesive explanation for this could be determined. Thus, hemolymph averages (open circles) for all population (closed circles) under similar hypertonic stress between 2200 and 3185 mosM are shown in Fig. 3. Consequently, the osmoregulatory capacities of U. panacea and U. pugilator were similar. However, both differ significantly in ability from U. spinicarpa. Hemolymph responses for the three species in the Minuca subgroup to the osmotic regime are shown in Fig. 4 and Table 2. In hypotonic media, U. minax appear to maintain a more constant hemolymph concentration than U. longisignalis or U. rapax. In media < 200 mosM, U. minax maintain a hemolymph osmolality of 579 F 2.6 mosM while U. longisignalis and U. rapax are 634 F 18 and 713 F 5.0 mosM, respectively ( p < 0.001). The hemolymph response ( Y) of U. minax to increasing medium osmolality (X) between 105 and 1250 mosM is linear ( Y = 0.2143X + 518; r = 0.8707; p < 0.01). However, the slope of the algorithm is not significant ( p>0.05) and average hemolymph mosM is 641 F 64 across the range. In U. longisignalis, the hemolymph response ( Y) to changing bath osmotic pressure (X) is linear from 200 to 1300 mosM ( Y = 0.1644X + 579; r = 0.6040; p < 0.05) with an average measure of 710 F 48 mosM. Since the slope of the regression algorithm is highly significant ( p < 0.001), hemolymph is not as independent of the external medium as seen in other species. In medium >1400 mosM, two distinct hemolymph patterns are seen. U. longisignalis from hypotonic habitats (FW < 349 mosM or 12x) cannot control hemolymph osmolality as well as those from populations in BW or EH locations. In Fig. 4, data for U. longisignalis taken from habitats with osmolality >450 mosM are indicated as BW/EH. Specimens from FW habitats do not regulate hemolymph mosM in media >1800 mosM whereas those from BW/EH habitats are capable of regulating hemolymph up to 2400 mosM. On the other hand, U. rapax are capable of regulating hemolymph osmolality in media of 200– 2200 mosM (hemolymph average = 801 F 52 mosM). Over this range, the linear relationship ( Y = 0.0947X + 697; r = 0.8848; p < 0.01) has a slight but significant slope ( p < 0.05). In media >2400 mosM, U. rapax lose control of hemolymph osmolality. However, it appears that some individuals can tolerate an acute internal osmolality as high as 2200 mosM. Using the linear algorithms, the [ISO] for U. minax, U. longisignalis and U. rapax are 659, 693 and 769 mosM, respectively (Table 2). In general, the physiological properties of U. minax and U. longisignalis are similar but distinct from those of U. rapax. Like crabs in the Celuca group, the osmoregulatory capability of each Minuca species reflects its niche preference. Consequently, U. minax and U. longisignalis should be encountered most often in FW or BW while U. rapax would be in BW or EH habitats. 3.4. Osmotic adaptation From the data in Fig. 4, obviously, U. longisignalis are flexible in osmoregulation. The physiological capacity of this species appears to depend upon habitat condition. This is not

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Fig. 5. U. longisignalis (n = 64) adapted to 1800 mosM then exposed to osmotic regimen for 5 days. Hyposmotic LC50 = 62 mosM; hyperosmotic LC50 = 2633 mosM. [ISO] = 832 mosM. Curves fit by eye.

indicated by data for the other five species. To examine this mechanism in detail, 75 specimens of U. longisignalis collected at Dauphin Island Causeway (Fig. 1; site L) were placed first in 980 mosM medium, then sequentially transferred to 1450 and 1800 mosM over a 2-week period. After 10 days in the last hypertonic media, specimens were exposed to the 30– 3400 mosM regimen shown in Fig. 5. Since the hypertonic-adapted crabs are more susceptible to hypotonic media (LC50 = 62 mosM) and survive better in hyperosmotic media (LC50 = 2633 mosM), survivorship (Fig. 4) is shifted to the right. The relationship between medium (X = 100– 2250 mosM) and hemolymph ( Y) osmolality is linear ( Y = 0.2920X + 589; r = 0.9622; p < 0.05). However, in lieu of the fact that the maximum tolerated medium remains between 2700 and 2800 mosM, the slope of the algorithm has increased by 178%. In the hypertonic-adapted state, the [ISO] is shifted from 693 to 832 mosM. Consequently, it appears that U. longisignalis adapt to acute hypertonic regimens by allowing interstitial fluid osmolality to rise and tolerating the new internal state (Prosser, 1991).

4. Discussion In order to fully appreciate this study, three divergent topics need to be addressed: biogeography, ecology and physiology. Since it represents a longitude rather than latitude of faunal transition, a brief review of the area’s geology in relation to Uca

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biogeography is warranted. Within this region, each species occupies a particular niche configured from a spectrum of environmental characteristics. Osmolality is but one challenging facet confronting congeners. The following will compare the patterns of osmoregulation seen in the six Gulf species to those reported for Uca in other studies. Since osmoregulation exhibits a relationship to environment, the ability to invade a new habitat is, in part, related to the physiology of adaptation. Most likely, the patterns of adaptation observed in the laboratory correlate with the capability to adjust to a new osmotic regimens in nature. 4.1. Origins of coastal habitats and Uca spp. in the northern gulf The region for this study was chosen due to its unique geological history and role in Uca biogeography (Barnwell and Thurman, 1984). The history of the coast between the Mississippi River in Louisiana and Cape San Blas in Florida (Fig. 1) is closely related to the distribution of several species. During the Miocene (13 – 25 million years before present: MYBP), the Okeefenokee Trough formed a shallow sea separating Georgia, Alabama and Mississippi from the north end of insular Florida (Riggs, 1984; Bert, 1986). This passageway provided a dispersal route for Gulf species into Carolinian bays on the east coast of the United States. Closure of the connection around 1.75 MYBP isolated temperate populations in the Gulf from their Atlantic counterparts. During the late Pleistocene and the Holocene, the physiography of the northern Gulf was continuously molded by sea-level transgressions and delta lobe formation (Otvos, 1997). At its maximum, glaciation during the Wisconsin period (19,000 YBP) extended the eastern Atlantic and Gulf coastal plains to the edge of the continental shelf. Since average annual temperature along the Gulf coast was probably 5 – 10 jC cooler than today, temperate species were forced to seek warm refuges in south Florida and Mexico. Over the past 6000 years, the current alluvial fan of the Mississippi River in Louisiana was formed from 16 different deltas. These ‘‘lobes’’ are classified into five general complexes. Individual deltas within each complex are the result of successive distributary networks along the main river course (Frazier, 1967). The impact of prehistoric freshwater inflow is clearly evident in the topology of present-day submarine canyons and the stratification in marine sediments (Van Andel and Poole, 1960). The St. Bernard delta complex (4000 – 1000 YBP) has been a significant influence on the coastline between Louisiana and Apalachicola, FL. The original Miocene phosphogenic carbonates (neogene) sediments of the ancient Ocala platform extend from Cape San Blas westward to Mobile Bay along the Gulfport– Ingleside barrier ridge (Otvos, 1997). From the St. Bernard delta, siliclastic sediments from the Mississippi River were distributed eastward to Mobile Bay. As the sea level transgressed, barrier islands were overlaid by river silts closing Pontchartrain Bay (2600 YBP) and producing extensive marshes in eastern Louisiana, Mississippi and Alabama. As the Mississippi River turned south forming the LaFourche and Plaquemines – Modern delta complexes about 1000 YBP, sediments along the northern Gulf coast stabilized (Frazier, 1967). Thus, successive geological events over the last million years appear sufficient to create a biogeographic barrier in the northern Gulf of Mexico. Consequently, it is not surprising to see a transition of benthic fauna in this area.

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The origins and present-day distribution of the six fiddler crab species in this region correlates with geology. One species, U. minax, due to its discontinuous distribution along shores of the southeast United States appears to be the oldest (Thurman, 1982). It (or its direct ancestors) probably ranged across the shores from Texas to the Carolinas during the Miocene. With closure of the Okeefenokee Trough, populations were isolated along the Atlantic and Gulf. On the other hand, the sibling pair, U. panacea and U. pugilator, have evolved more recently. During glaciation, the predecessor of U. pugilator was isolated in south Florida and on some Caribbean Islands while the progenitor for U. panacea was forced into Mexico but could not survive on the Yucatan peninsula. Most likely by a similar mechanism, U. spinicarpa evolved in the western Gulf from an ancestral Celuca stock. On the other hand, U. rapax is an older, widely distributed, neotropical form with the sibling species U. herradurensis and U. galapagensis along the Pacific coast. The sixth species, U. longisignalis, endemic to the temperate Gulf, is related to U. minax and the Atlantic species, U. pugnax (Barnwell and Thurman, 1984). During the recent transgression of sea level and the recession of the glacial climate, U. longisignalis and U. pugnax evolved independently in sympatry with the older U. minax in either the Gulf or Atlantic, respectively. The transition from siliclastic sediments to neogene carbonates is associated with a disruption in distribution of three species, U. rapax, U. spinicarpa and U. pugilator. The distributions of the latter two species are associated with the substrate transition. However, for the neotropical species, U. rapax, it appears to be a simple coincidence. The low winter temperatures between Ocean Springs and Apalachicola appear to minimize its occurrence in the area. 4.2. Ecophysiology and species distribution Among Crustacea, patterns of ionic and osmotic regulation are well established for a number of species. Excellent reviews on the subject have been written by Mantel and Farmer (1983) and Greenaway (1988). More recent works by Wolcott (1991) and Ahearn et al. (1999) focus on the cellular aspects of ion transport and water balance. Altogether, these reveal a vast knowledge of salt and water balance for crustaceans. In crustaceans, the fundamental mechanisms for fluid and ion balance are consumption, elimination and transport across epithelial barriers. Historically, Uca spp. are known for their ability to regulate their internal milieu using these mechanisms (Jones, 1941; Gross, 1964). The concentration of water and ions in hemolymph remains somewhat constant over a wide range of osmotic pressure. As the intensity of the challenge increases beyond the regulated range, internal osmotic concentration begins to waiver, and the relationship becomes one of conformity and tolerance. At the extremes of the range, the organism may not tolerate the pressure and, eventually, dies. From a perspective of comparative ecophysiology, most osmoregulation studies document similarities and differences in species which are phylogenetically and ecologically unrelated. Few studies deal with closely related species living in sympatry. In fact, most studies with fiddler crabs have been limited to a single species (Table 3). In cases where more than one is studied, the data are presented in such a way that few, if any, distinctions between congeners can be made (Green et al., 1959; Baldwin and Kirschner, 1976a,b; Wright et al., 1984). The present study examines osmoregulation by six sympatric species

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Table 3 Habitat preference and osmoregulatory capabilities of Uca Subgenus

Species

Reference

Habitata

Isosmotic concentration

Minimum hemo (mosM)

Maximum tol. med. (mosM)

Amphiuca Deltuca Thalassuca

U. U. U. U. U. U.

Spaargaren (1977) Lin et al. (2002) Lin et al. (2002)

BW/EH FW HS BW/EH BW EH

1016 756 786 860 860 800

790 500 400 487 750 700

1430 996 1274 1679 1775 1040

EH EH

750 990

600 850

1500 1900

EH EH BW/EH

960 840 850

700 500 525

1660 1679 2600

FW BW/EH BW/EH FW BW FW/BW

682 816 822 790 1000 750

567 614 553 550 700 500

2200 3454 3100 1600 1800 3400

EH

900

784

2000

EH FW FW/BW BW/EH

750 659 693 769

670 524 563 505

4000 2200 2750 3100

Celuca

inversa arcuata vocans formosensis crenulata pugilator

U. speciosa U. lactea U. subcylindrica

Minuca

U. U. U. U. U. U.

spinicarpa pugilator panacea minax pugnax longisignalis

U. burgersi U. U. U. U.

rapax minax longisignalis rapax

Jones (1941) Baldwin and Kirschner (1976a) Wright et al. (1984) D’Orazio and Holliday (1985) Spaargaren (1975) Lin et al. (2002) Rabalais and Cameron (1985) Current data

Wright et al. (1984) Holliday (1985) Rabalais and Cameron (1985) Schmidt-Nielsen et al. (1968) Zanders and Rojas (1996) Current data

Hemo = hemolymph; tol. med. = tolerated medium. a See text for abbreviations.

in the same genus take from 25 different locations in the northern Gulf of Mexico. Using this approach, it is clear that most species are physiologically distinct and that an osmoregulating capability is related to habitat. Without question, Uca spp. from the northern Gulf of Mexico are excellent osmoregulators. As reported for many other species (Table 3), they are able to maintain hemolymph fairly constant over a wide range in media (100 – 1800 mosM; 3– 65x). Since most inhabit salinity ranging from 5% to 95% SW (Table 1), this capability seems adequate for survival along the shores of the northern Gulf. Few are likely to encounter concentrations equal to 185% SW for any length of time. On the other hand, survival in hypotonic environmental may represent a more realistic challenge. Estuaries here are often shallow with an underlying ‘‘salt tongue’’ penetrating several miles up the river (Hedgpeth, 1953). Since they are excellent hyperosmotic regulators in hypotonic media, species such as U. minax and U. spinicarpa (and frequently U. longisignalis) are common along riverbanks and creeks. Due to their patterns of osmoregulation over intermediate

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salinity ranges, U. spinicarpa, U. longisignalis and U. rapax are more likely to be encountered in BW habitats. On the other hand, U. rapax, U. panacea and U. pugilator may be uncommon in the upper estuary due to either an inability to tolerate the low osmolality for extended periods or an inability to forage on silty, anaerobic muds (Thurman, 1984, 1998). The true capability of organism is revealed only when osmotic pressure is near the regulatory limits. In the majority of the early osmoregulation studies, Uca were exposed to a limited range of osmotic stress (Table 3, maximum tolerated medium osmolality). For example, in a study comparing U. pugilator and U. tangeri, a species from the eastern Atlantic, Graszynski and Bigalke (1986) found U. tangeri to be a more effective osmoregulator over a range of 330 –1500 mosM. Spaargaren (1975, 1977) found U. inversa from the western Indian Ocean and U. speciosa from the Caribbean to tolerate up to 1430 and 1660 mosM (50xand 58x ), respectively. However, studies by SchmidtNielsen et al. (1968), Rabalais and Cameron (1985), and Zanders and Rojas (1996) challenged their specimens with much stronger hypertonic solutions to reveal their regulatory capacities. After 3 –4 days in 2000 mosM (70x ), U. burgersi from tide pools near Kingston, Jamaica, had hemolymph of 1000 mosM (Schmidt-Nielsen et al., 1968; Barnwell, 1986). To compare the capabilites of two Gulf endemic species, Rabalais and Cameron (1985) exposed U. longisignalis and Uca subcylindrica to a regimen of 2.2 – 2490 mosM (0.08 – 120x ) for 5 days. They found significant differences between the two with U. longisignalis having the best osmoregulating abilities. As Zanders and Rojas (1996) acclimated U. rapax from Venezuela to salinity between 48 and 3000 mosM (1.7xand 105x ), the hemolymph increased from 700 to 1000 mosM. In a study of Pacific fiddler crabs, Lin et al. (2002) exposed four sympatric congeners, U. arcuata, U. formosensis, U. vocans and U. lactea, from Taiwan to solutions ranging from 0 to 1679 mosM for 7 days. Both U. formosensis and U. lactea survive in 1679 mosM (60x ) while U. arcuata and U. vocans succumb to 1300 (45x ) and 1679 (60x ), respectively. All but U. formosensis failed to survive in 0 mosM. Although the crabs were not tested above 1680 mosM, they do not appear to be as good osmoregulators as their relatives in the Western Hemisphere. In the present studies, among the Celuca, U. spinicarpa can tolerate media of 2200 mosM while U. pugilator and U. panacea survive in 3454 and 3100 mosM, respectively. From the Minuca subgenus, U. minax is able to survive in 2200 mosM while U. longisignalis and U. rapax are able to withstand media up to 2750 and 3100 mosM, respectively. For most cases, the present study documents greater hypertonic tolerance in Uca than previously observed. An interesting exception is U. longisignalis where none of the populations in the northern Gulf were able to withstand 2800 – 3400 mosM solutions as reported by Rabalais and Cameron (1985). From this perspective, it is most interesting that U. longisignalis illustrate an ability to adapt physiologically to local conditions (Figs. 4 and 5). 4.3. Patterns of adaptation in osmoregulation If it is important for a species to maintain physiological flexibility for survival in various habitats, capacity adaptation allows the organism to modify its ability to hyperand hyporegulate economically in different media (Prosser, 1991). As the organism

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gradually adapts to a new osmotic environment, the curve of regulation is modified depending upon external conditions. The central portion of the curve, representing a physiological optimum changes slightly, while the terminal portions are shifted either to the right or the left. Since there is little genetic diversity among the Uca populations in the northern Gulf (Felder and Staton, 1994; Mangum, 1996), physiological change is most likely due to alteration in gene expression. The expression of enzymes associated with ion transport varies during the adaptation of U. minax to extreme osmotic concentrations (Wanson et al., 1984). As crabs adapt to media of increasing osmolality, there is a concomitant decrease in Na/K-ATPase activity in the posterior gills (Wanson et al., 1984; Holliday, 1985) as well as a Na/H exchanger (Towle and Wiehrauch, 2001). On the other hand, when assaulted with hypotonic medium, Na/K-ATPase and carbonic anhydrases will increase whereas water permeability will decrease. These changes begin within 24 – 72 h. Consequently, adaptation producing new osmoregulatory capacity takes only a few days (Rabalais and Cameron, 1985; Zanders and Rojas, 1996; Lin et al., 2002). From an intraspecific perspective, descriptions of differences in volume regulation, ionoregulation and osmoregulation among populations of grapsid species are uncommon. Ferraris and Norenburg (1997) reported that U. rapax populations in Florida, Belize and Panama differ substantially in their fluid- and electrolyte-regulating abilities. Staton and Felder (1992) have reported variation in osmoregulation among populations of Sesarma reticulum from Georgia and Louisiana. Capacity adaptation in osmoregulation has been illustrated in populations of U. subcylindrica and U. minax (Thurman, 2002, 2003). From the current data, populations of U. longisignalis from different habitats express different physiological capacities. Crabs from FW habitats are limited in survival above 1800 mosM while those from BW and EH can withstand osmotic pressure up to 2700 mosM. Similar patterns of capacity adaptation were not obvious in other fiddler crab species from the Mississippi delta region. The only other study of osmoregulation in U. longisignalis focuses on a single population at the species’ western boundary in south Texas. After adapting to 30xSW, Rabalais and Cameron (1985) found it to be a better osmoregulator than U. subcylindrica (n = 120). In media between 300 and 1800 mosM, the [ISO] for U. longisignalis (n = 70) is approximately 750 mosM. As the medium increases from 2100 to 3400 mosM, the hemolymph increases from 800 to 1600 mosM. In the present study, 288 specimens from 25 locations in the northern Gulf were examined while previous studies of osmoregulation in U. longisignalis by the author examined 110 specimens from three locations in Texas and another 47 taken near Apalachicola, FL. (Thurman, 2002, 2003). Essentially, osmoregulation has been examined in populations throughout the geographic distribution of this species. From eastern to western Gulf, the [ISO] for U. longisignalis increases from 653 to 732 mosM. However, the upper limit for medium regulation does not exceed 2700 mosM across Gulf populations. Populations from FW habitats succumb to solutions >1800 mosM. Even when slowly adapted to a hypersaline condition of 1800 mosM (Fig. 5; n = 62), U. longisignalis do not survive above 2800 mosM although the [ISO] rises to 832 mosM. Based on these studies, the species identified by Rabalais and Cameron (1985) is suspect. When compared to other Gulf Uca, it appears that the physiology of U. rapax rather than U.

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longisignalis was reported. In U. rapax populations across the Gulf, the [ISO] varies from 769 mosM in the east to 932 mosM in the west. Regardless of a population’s average [ISO], U. rapax withstand media up to 3100 mosM. Zanders and Rojas (1996) reported a similar physiology for populations of this species in Venezuela where the [ISO] is 750 mosM. Consequently, with no museum voucher specimens, the findings reported for U. longisignalis by Rabalais and Cameron (1985; Fig. 2) are more likely for U. rapax. In summary, this study reports on the osmoregulatory abilities by six species of fiddler crabs from populations across the northern Gulf of Mexico between St. Andrew Bay, FL, and Bayou LaFourche, LA. All six are excellent osmoregulators in media between 200 and 1400 mosM. FW species have [ISO] < 700 mosM while BW and EH species are >700 and >800 mosM, respectively. The BW and EH species also regulate hemolymph osmolality over a greater medium range than FW species. Among the six species, only one, U. longisignalis, exhibits significant physiological variation between populations. This species adjusts its physiological capability to local conditions. Although many factors determine the success of benthic invertebrates occupying coastal niches along the northern Gulf of Mexico, osmoregulation appears to be an important physiological property influencing their distribution. References Ahearn, G.A., Duerr, J.M., Zhuang, Z., Brown, R.J., Aslamkhan, A., Killebrew, D.A., 1999. Ion transport processes of crustacean epithelial cells. Physiol. Biochem. Zool. 72, 1 – 18. Baldwin, G.F., Kirschner, L.B., 1976a. Sodium and chloride regulation in Uca adapted to 175% sea water. Physiol. Zool. 49, 158 – 171. Baldwin, G.F., Kirschner, L.B., 1976b. Sodium and chloride regulation in Uca adapted to 10% sea water. Physiol. Zool. 49, 172 – 180. Barnwell, F.H., 1986. Fiddler crabs of Jamaica. Crustaceana 50, 146 – 165. Barnwell, F.H., Thurman, C.L., 1984. Taxomony and biogeography of fiddler crabs of the Atlantic and Gulf coasts of eastern North America. Zool. J. Linn. Soc. 81, 23 – 87. Bert, T.M., 1986. Speciation in western Atlantic stone crabs: the role of geological processes and climatic events in the formation and distribution of species. Mar. Biol. 93, 157 – 170. Crane, J., 1975. Fiddler Crabs of the World. Princeton Univ. Press, New Jersey. D’Orazio, S.E., Holliday, C.W., 1985. Gill Na,K-ATPase and osmoregulation in the sand fiddler crab, Uca pugilator. Physiol. Zool. 58, 364 – 373. Felder, D.L., Staton, J.L., 1994. Genetic differentiation in trans-Floridian species complexes of Sesarma and Uca. J. Crustac. Biol. 14, 191 – 209. Ferraris, J.D., Norenburg, J.L., 1997. Volume and ion regulation during repeated exposure to temperature change: physiological divergence in trans-isthmian cognate pairs and latitudinally distance populations of decapod Crustacea. Mar. Ecol. 18, 193 – 209. Finney, D.J., 1947. Probit Analysis. Cambridge Univ. Press, London. Frazier, D.E., 1967. Recent deltaic deposits of the Mississippi River: their development and chronology. Trans. Gulf Coast Assoc. Geol. Soc. 17, 287 – 315. Graszynski, K., Bigalke, T., 1986. Osmoregulation and ion transport in the extremely euryhaline fiddler crabs Uca pugilator and Uca tangeri. Zool. Beitr. 30, 339 – 358. Green, J.W., Harsch, M., Barr, L., Prosser, C.L., 1959. The regulation of water and salt by the fiddler crabs, Uca pugnax and Uca pugilator. Biol. Bull. 116, 70 – 87. Greenaway, P., 1988. Ion and water balance. In: Burggren, W.H., McMahon, B.R. (Eds.), Biology of the Land Crabs. Cambridge University, New York, pp. 211 – 248.

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