ELSEVIER Journal of Experimental Marine Biology and Ecology,

206 (1996) 149-164

JOURNAL OF EXPERIMENTAL MARINE BIOLOGY AND ECOLOGY

Respiratory, acid-base and ion status during voluntary immersion of the air-breathing crab Curdisoma carnifex

assessed in situ

Stephen Morris*, Agnieszka M. Adamczewska

School of Biological Sciences (AOS), University of Sydney, Sydney, NSW 2006, Australia

Received 9 October 1995; revised 13 May 1996; accepted 30 May 1996

Abstract

The respiratory haemolymph gas and acid-base state of the crab Curdisoma carnifex during submersion were investigated in situ on the Cocos (Keeling) Islands. Crab burrows less than 90 cm deep contained no water. Burrow water was brackish, between 60 and 370 mOsm osmotic pressure but C. carnifex was observed to voluntarily immerse in 50% seawater. Post-pulmonary haemolymph 0, partial pressure and content were reduced after 30 min submergence. The internal hypoxia was accompanied by a severe hypercapnic acidosis exacerbated by a lactacidosis after 30 min in water. This haemolymph acidosis caused a decrease in Hc-0, affinity such that the in vivo P,, increased from 6 to 18 torr, which further decreased 0, transport. During submersion C. cumifex released minimal amounts of air from the branchial chambers. A trapped air-bubble represents a finite 0, source and CO, sink and could thus account for the hypercapnic hypoxia. The haemolymph osmotic pressure of C. curnifex taken from burrows among the trees and sand flat margins ranged from 699 to 803 mOsm whereas those sampled on the sand flats on their way to immerse in saltwater had lower osmotic pressure, between 678 to 687 mOsm. While there was no correlation between burrow water osmotic pressure and that of the haemolymph it is possible that the crabs make periodic forays to salt water to replenish haemolymph ions. C. curnifex

occasionally immerses itself but it is an air-breather and not a truly amphibious crabs. C. curnifex

do not require water for respiratory and acid-base homeostasis and thus, further studies must examine in more detail ionic and water balance in field situations.

Keywords: Cardisomaa; Air-breathing; 0,; CO,; Acid-base; Immersion

*Corresponding author.

0022.0981/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved

PII SOO22-098 1(96)02658-5

150 S. Morris, A.M. Adumczewskcr I .I. Exp. Mar. Biol. Ecol. 206 (1996) 149-164

1. Introduction

The ecophysiology of land crabs and their ability to survive in air is increasingly well understood as are the problems associated with the evolution of air-breathing in this group (Burggren and McMahon, 1988; Greenaway, 199 1; Morris, 199 1; Wolcott, 199la,b; Wolcott T.G., 1992; Burggren, 1992; Morris and Bridges, 1994 for reviews). Air and water are two very different respiratory media. The acute problem for aquatic animals adopting air-breathing, especially amphibious species, is the maintenance of O2 uptake and the excretion of CO,. The study of amphibious crabs can provide

information as to mechanisms important in the evolution of air-breathing and in deriving the likely ecophysiology of species ancestral to extant air-breathing crabs.

Land crabs of the genus Cardisoma have been portrayed as intermediate or amphibious species, requiring regular access to water for immersion (Gifford, 1962; Hartnoll, 1988). The burrows of Cardisoma often reach down to free water (Pinder and Smits, 1993; Adamczewska and Morris, 1996) but these crabs may prefer breathing air. Burggren et al., 1985 as well as Shah and Herreid, 1978 described C. guanhumi as “striving” to prevent the entry of water into the

branchial chamber and C. hirtipes avoids breathing water when submerged under field conditions (Adamczewska and Morris, 1996). C. curn{fex and C. guanhumi trap air in their branchial chambers when submerged and hypocapnic alkalosis could be induced only in crabs in which water had been artificially introduced into the branchial chamber (Cameron, 198 1; O’Mahoney and Full, 1984). Submersion without access to air can be lethal for both C. carnifex (Cameron, 1981) and C. hirtipes (Adamczewska and Morris, 1996). However, C. guunhumi

and C. carnifex are also reported to sustain aquatic respiration for many hours (Gifford, 1962; Cameron, 198 1, respectively). Respiration in submerged C. guanhumi appears to be at the expense of increased ventilatory work (O’Mahoney and Full, 1984), partially supported by anaerobiosis (Shah and Herreid, 1978). The different Cardisoma species might thus represent different degrees of terrestrialism.

Previous respiratory gas measurements have shown arterial haemolymph not to be O2 saturated in air-breathing C. curnifex (Wood and Randall, 198lb; Burggren and McMahon, 1981) but these studies did not sample the efferent pulmonary haemolymph which mixes with the post branchial flow. Field measurements of haemolymph gas in submerged C. hirtipes showed an internal hypoxia (Adamczewska and Morris, 1996) and ventilatory drive in C. guanhumi is CO, rather than 0, driven, similar to air-breathing animals (Cameron, 1975; McMahon and Burggren, 1988). However, Wood and Randall ( 198la) suggest that air-breathing C. curnifex retain pools of water in the branchial chamber for a CO, sink. The burrow water of Cardisoma is typically both hypoxic and hypercapnic (Wood and Boutilier, 1985; Pinder and Smits, 1993; Adam- czewska and Morris, 1996) and presents an unattractive medium for respiratory gas exchange. Thus, while there is some doubt as to whether Cardisoma are amphibious or obligate air-breathing crabs they are adept air-breathers. Comprehensive field studies are necessary to determine the extent to which Cardisoma breathe water in their natural environments. Comparisons between species will aid in identifying important adapta-

S. Morris, A.M. Adamczewska / J. Exp. Mar. Biol. Ecol. 206 (1996) 149-164 151

tions in the evolution of a terrestrial life style. The requirement of Curdisoma for immersion might be primarily for either ion or water balance, which in aquatic species normally involves the gills. If so, it is likely that various Curdisoma species exhibit differing dependency on water in this regard.

Curdisomu retain gills, as well as having lungs, (Farrelly and Greenaway, 1992, 1993) and these might be used in ionic regulation (Greenaway, 1989; Wolcott, 1991b). C. hirtipes has access to freshwater only (Greenaway, 1989; Adamczewska and Morris, 1996), while C. guunhumi and C. curnifex can inhabit burrows containing water with a range of salinities up to full strength seawater (e.g. Wood and Boutilier, 1985; Pinder and Smits, 1993). However, C. hirtipes and C. curnifex can maintain ion balance in water with very low ionic concentration (Greenaway, 1989; Wood and Boutilier, 1985). Adamczewska and Morris, 1996 suggested that preventing flooding of the branchial chambers might reduce diffusive Na loss to freshwater and thus the cost of ion regulation. However, this would seem to be of less importance to species such as C. curnifex which can immerse in seawater. These uncertainties as to the need for immersion by the various Curdisoma species prompted the current re-examina- tion.

Preliminary observations of C. curnifex on the Cocos Islands revealed that some burrows contained water but that this species had access to seawater as well as fresh ground water. The possibility that colonization of areas around freshwater by C. hirtipes discouraged immersion could be tested by examining C. curnifex for similar aspects of respiratory and ionic regulation. Is the branchial air trapping behaviour of C. hirtipes a consequence of increased terrestrialness or is it common to Curdisomu generally? Does C. curnifex in the field show a greater propensity and ability to breathe water than C. hirtipes?

C. curnifex has an Indo-West Pacific Ocean distribution (Hartnoll, 1988). The islands of the southern Cocos atoll are low (max 9 m), comprised of coralline material and the distance to the ocean seldom exceeds 300 m. Rainwater collects as a subterranean freshwater lens which reaches to the shore environments (Falkland, 1994). C. curnifex are plentiful, especially on West Island (Fig. 1). They inhabit palm plantations and native forest but with a clear preference for the lagoon side of the islands where some crabs roam the sand flats to feed (Morgan, 1994; pers obs.) and to immerse in lagoon water (pers obs.). While some burrows and puddles provide freshwater for immersion (Morgan, 1994), the lagoon and burrows on sand flats provide up to full strength seawater; presenting a variety of ionic environments. A field census of haemolymph and burrow water osmotic pressure and ion composition was carried out to establish whether decreased ionic strength of the burrow water promoted ion loss and reduced the concentration of the haemolymph.

The present study will provide new ecophysiological data for C. curnifex by determining haemolymph respiratory gas, acid-base and ionic state during submersion in situ. It will also determine if trapping air in the branchial chambers is a shared feature of Curdisomu by comparison with C. hirtipes under similar circumstances. The data will be valuable in assessing the crucial transition from amphibious to obligate air-breathing and the adoption of a fully terrestrial habit.

152 S. Morris, A.M. Adamczewska I J. Exp. Mar. Biol. Ecol. 206 (1996) 149-164

Emphemeral 1 Freshwater m

Fig. 1. Location of Cocos (Keeling) Islands with position and description of the experimental site

2. Materials and methods

2. I. Field location and sampling

Cardisoma carnifex (mean mass - 3 10 g) were sampled on West Island of the Cocos Islands atoll during February 1995 (Fig. 1). Since the Islands are comprised mostly of coralline material the rainfall drains through the porous soil and thus there are few surface water sources. The experimental site was located on extensive sand flats on the lagoon side of West Island. Here C. carnifex both fed on sea-grass debris and immersed themselves in a natural drainage channel (creek; Fig. 1) fed by water percolating from the flats at low tide. Freshwater might also have leached into this drainage, therefore, the ionic status of the water was assessed (below). The “creek” was - 30 m removed from the mature palm growth on both sides of the flat and was visited by C. carnifex in the 2-3 h immediately after dawn before the sun discouraged their forays from the forest. Only a small percentage of the population engaged in this behaviour at any time. Other crabs were observed early in the morning apparently “stripping” dew from grass leaves but most crabs remained in the forest. There were few burrows on the sand flats but a number of relatively shallow burrows occurred on the margins. The sampling site was 11 min removed from the measurement station where blood gas parameters were measured. All blood samples were taken in 1 ml plastic syringes that were chilled and maintained in an ice bucket for the duration of blood gas analysis.

“Air-breathing” crabs were completely dry and therefore had been breathing air for

S. Morris, A.M. Adamczewska I J. Exp. Mar. Biol. Ecol. 206 (1996) 149-164 153

some time. Air-breathing crabs were captured as they emerged onto the sand flats (Fig. 1) and the carapace was drilled (OMNI 1000 battery drill fitted with dental bits) to provide access to the pericardial cavity and the pulmonary vessel. The sampling

apertures were completed within 20 s and immediately thereafter a haemolymph sample (300 l.rl) was taken from the pulmonary vein (pulmonary haemolymph, 6 s). A second (800 l.~l) sample was taken from the pericardial cavity (arterial haemolymph, 6 s) and a third directly from the infrabranchial venous sinus (venous haemolymph, 10 s) at the base of the last walking leg by puncturing the arthrodial membrane (total handling time was less than 1 min).

Samples from “water breathing” crabs were obtained from animals that had been completely immersed in water for 30 min. On reaching the water the crabs submersed in the creek and remained quiescent under the bed of sea grass (Thalussiu hemprichii), occasionally small isolated bubbles were released by the crabs. Each crab voluntarily entering the creek was observed from a distance to ensure it remained immersed for the 30 min period. After 30 min of immersion the animal was restrained in situ, that is, held submerged and rapidly drilled and sampled as described above. Samples of pulmonary, arterial and venous haemolymph were taken from seven crabs in each treatment.

2.2. Haemolymph analysis

Haemolymph samples were immediately analysed for blood gas and pH. The PO, was determined with a micro oxygen probe at 28°C (Microelectrodes, Ml 16-730) connected to a PHM73 pH/blood gas monitor (Radiometer, Copenhagen, Denmark). Haemolymph oxygen content (C,J was measured at 32°C with a modified Tucker chamber method

(Tucker, 1967; Bridges et al., 1979) using a Strathkelvin meter (model 781b). Haemolymph CO, content (CcoJ was measured with a Pc02 electrode (model E5037/

SI) connected to a PHM73 pH/Blood gas monitor using a Cameron chamber also at 32°C (Cameron, 1971) and calibrated with fresh 15 mmol*ll’ NaHCO, standards. The changes in PC, and PO, were timed until a linear rate was achieved and interpolated to

injection time. kaemolymph pH was measured with a micro pH-probe (Microelectrodes,

MI16-705) at 28”C, connected to the PHM73. The in vivo haemolymph PC,, was

calculated according to the Henderson-Hasselbalch relationship using a CO, solubility coefficient of 0.037 1 mmol.1~ ’ - to6 ’ for air-breathing crabs (28°C) and 0. 0441 mmol.ll’~torr-’ for submerged crabs (24°C) from the tables of Cameron (1986) together with pK=6.03. These two temperatures were the mean measured air and water temperatures between 6 and 9 am. Part of the remaining haemolymph sample was mixed with 0.6 mol.l-’ HClO, (ratio of 1: 1) to denature proteins and neutralized with K,CO,. The sample with denatured proteins and a sample of whole haemolymph were frozen (< - 10°C) until further analysis in the laboratory in Sydney.

The haemolymph samples were analysed for lactate (Boehringer Mannheim, test kit # 138084), glucose (Sigma test kit #510-A) and urate (Sigma test kit #685). Haemolymph osmotic pressure was measured using a Wescor 5100C vapour pressure osmometer. For ion analysis haemolymph was mixed with 0.1 molell’ HNO, (ratio 1: 1) to denature proteins. Haemolymph Cl was measured with a CMTlO chloride titrator

154 S. Morris, A.M. Adamczewsku / J. Exp. Mar. Biol. Ecol. 206 (1996) 149-164

(Radiometer, Copenhagen) and Na, K, Mg and Ca with an atomic absorption spec- trophotometer (GBC 906, Melbourne). To suppress interference, samples for measure- ment of Na and K were diluted with 5.9 mmol.l-’ CsCl, while for Mg and Ca the dilution was with 7.2 mmol.l-’ LaCl,. Maximum Hc-0, carrying capacity was determined in the field on fresh haemolymph by equilibrating a small sample (40 ~1) with air in a tube. The 0, content of the blood was then measured using the Tucker chamber. Relative Hc saturation was then calculated for each individual crab sampled.

Statistical analyses on differences between air or water breathing animals and between pulmonary, arterial and venous haemolymph samples were by ANOVA, using the Systat 5.03 statistical package. Bartletts x2 test was used to test for homogeneity of variances. Post hoc testing was by CONTRAST analysis of pairs of means. Significance level was taken as P =0.05. All data are presented as mean +SEM.

2.3. Burrow water values

Water samples were taken from a number of burrows in different sub-habitals, including forest and the plantation near the high tide mark, as well as from the creek. Selected burrows were enlarged and the depth of the burrow measured. A water sample was taken from the burrow and an infrabranchial haemolymph sample from any crab inhabiting the burrow. Water and haemolymph samples were analysed for osmotic pressure and ions as described above. In addition air and water temperature and water pH (temperature compensated) and P,> at the experimental site were determined on two

occasions at various depths. The pH was measured with an Activon 2 decimal place battery meter and probe and PO, with a radiometer electrode and a 781b meter

(Stratlikelvin).

3. Results

3.1. Burrow and creek conditions

From six burrows excavated, only burrows deeper than 90 cm had water in the bottom. The osmotic and ionic condition of water in burrows inhabited by C. carnifex varied considerably (Table 1). Water in burrows beneath the roots of Callophylum trees was 80 mOsm or less with low Na and Cl but with disproportionately high levels of Ca (2.7 mmol.1~‘; Ca:Na= 1:6.5) and Mg (4.7 mmol.l-‘) (Table 1). The water from sand flat burrows was brackish ranging from 200 to 371 mOsm but with a larger Ca:Na ratio of 1:27 (Table 1). The water in the creek was approximately 50% seawater and the Ca:Na ratio was higher at 1:63. The P,,, of the creek water was 126.5-C 1.5 torr with no

variation from surface to bottom. The pH measured at the same time was pH 8.2720.02 throughout the water column but in the water adjacent to the sediment increased to pH 8.95.

S. Morris, A.M. Adamczewska I J. Exp. Mar. Biol. Ecol. 206 (1996) 149-164 155

Table 1 Salt status and osmotic pressure (O.P.) of water taken from various sub-habitats of C. camifex on West Is. of

the Cocos Islands atoll

Sample site/type Depth 0.P [Nal Ul IKI [Cal [Mgl (cm) (mOsm) (mmol.1~‘)

Callophylum tree # 1 >lOO 66 15 11 0.8 4.0 5.0

Callophylum tree #2 >I00 80 21 17 0.8 1.5 4.5

Callophylum tree #3 50 _ _ no water _ _

Tree hollow _ 71 17 15 4.8 6.0 10.5

Sand flat burrow 1 >lOO 210 169 192 4.0 6.0 13.92

Sand flat burrow 2 90 371 153 207 4.4 5.8 19.4 Sand flat burrow 3 60 _ no water _

Creek water _ 415 216 219 4.5 7.5 20.9

Greek water 528 232 241.3 4.6 1.4 21.6

The samples tree #l and #2 were taken from burrows exceeding 1 m in depth between the roots of

Calophylum inophyllum L. The sand flat burrows were near the extreme high tide line. Creek samples refer to

water from a drainage channel from the lower flats to the open lagoon; in which the immersion experiments

were carried out. The ion concentration is in mmol .l~ ’

3.2. Haemolynzph oxygen

The haemolymph PO, of immersed C. carnzyex was significantly lower than in air-breathing crabs (P<O.OOl) (Fig. 2a), although this was dependent on the haemolymph type (p, a or v) examined (Fig. 2a). For example pulmonary haemolymph of air-breathing crabs had a PO? four-fold greater than in submerged crabs, although the venous values were very low m both cases (Fig. 2a). In air-breathing crabs PpO, was 84.9k15.3 torr while P,,

*

was considerably lower, at only 5.92 1.7 torr. In contrast immersed crabs had a PpO, (20.728.7 torr) which was not significantly elevated above the P,, of 8.6tl.l torr (Fig. 2a).

The Iklmonary and arterial Co, were both lowered after 30 min submersion (P<

0.001) (Fig. 2b). The haemolymph oxygen content (Co*) of air-breathing C. carnifex

was the same in both pulmonary and arterial haemolymph (Cpoz =0.83+0.07; CaO_, 0.8220.07 mmol.l-‘) but significantly more than venous haemolymph (C,o,= 0.37kO.05) (Fig. 2b). In contrast, neither Cpo, or C,, (0.49LO.08 and 0.4050.10 mmol.ll’ respectively) were significantly different from’ the C,, (0.27kO.05 mmol. 1-l) of submerged crabs. The same trends were evident in the haimocyanin bound 0, (Hc-0,) such that 30 min submersion of C. carnifenc elicited a general decrease in Hc-0, binding (P<O.OOl) although a substantial venous reserve persisted (Fig. 2~). By calculating the Hc-0, as a fraction of the Hc-0, max it is possible to derive 0, saturation values. In air-breathing C. carnifex the arterial and pulmonary haemolymph were 82t3% and 8829% saturated respectively and after passage through the tissues this was reduced to 45-+ 10% in the venous haemolymph. When submerged for 30 min C. carnifex were unable to saturate the pulmonary haemolymph (5629%) while arterial (43t9%) and venous haemolymph especially (3025%) were much lower.

156 S. Morris. A .M. Adamczewska I J. Exp. Mar. Biol. Ecol. 206 (1996) 149- 164

c 8 c

N

s”

120

100

80

60 1 0.8

-ti s 0.6

5.

N G 0.4

0.2

_ 0.8 :

-1 Z 3 0.6

2 0.4 I

C curnijk - haemolymph 0, and Hc-0,

I. 1 i

i

a

C

0.2 J , I’ulmonarj

I Arterial

I Venous

Fig. 2. Haemolymph 0, parameters of C. carnijex after sustained air-breathing (H) or 30 min submerged (0).

Data provided for efferent pulmonary, arterial and venous samples (n = 7), shared superscripted letters show

sample pairs as significantly different. Where ANOVA indicated effects, differences between selected means

were tested by post hoc CONTRAST. Means for pulmonary, arterial and venous samples were compared

within treatments. Between treatments the means of each type of sample (pulmonary, arterial or venous) were

compared.

3.3. Haemolymph carbon dioxide and pH

There was no difference in haemolymph CO, content (Ccoz between the different haemolymph types in each of the treatments (Fig. 3a). In air-breathing animals Cco, ranged between 18.6 and 19.1 mmol.l-’ and in submerged crabs between 19.8 and 21.1 mmol.l-‘. Thus submergence did not decrease haemolymph CO, content. There was no difference in pH between the different types of haemolymph within each treatment but submergence significantly reduced haemolymph pH from 7.569kO.020 to only 7.218t0.034 (P<O.OOl) (Fig. 3b). The calculated PcO, also did not vary between

haemolymph sampling site in air-breathing or submerged crabs (Fig. 3~). However, PC,?

S. Morris, A.M. Adamczewska I J. Exp. Mar. Biol. Ecol. 206 (1996) 149-164 157

C. curnifx - CO, and acid-base state

15 - I------

I

IO I I Pulmonary Arterial

I

I Venous

Fig. 3. Haemolymph COT/acid-base parameters of C. carnifa after sustained air-breathing (M) or 30 min

submerged (0). Data provided for efferent pulmonary, arterial and venous samples (n =7), * indicates

significant effect of submergence. Statistical analyses as for Fig. 2.

doubled from a mean 14.3k0.82 torr in air-breathing crabs to 28k2.5 torr in submerged C. carnifex (P<O.OOl).

3.4. Haemolymph metabolites

Haemolymph [L-lactate] measured in submerged crabs (4.38% 1.43 mmol.l-‘) was elevated above that in air-breathing crabs (1.34kO.42 mmol.l-‘) (P=O.O27). Haemolymph glucose levels were approximately four times greater in submerged (0.9320.22 mmol.lP’) than in air-breathing C. carnifex (0.25LO.03 mmol*l-‘) (PC 0.002). Haemolymph urate levels were low and did not alter with 30 min submergence (0.05+0.01 mmol*l-‘).

I58 S. Morris, A.M. Adamczewska I J. Exp. Mar. Biol. Ecol. 206 (1996) 149-164

Table 2

Mean (SEM in italics, n = 1 I) haemolymph salt concentration and osmotic pressure (O.P.) in Cardisorna

curnifvx excavated from the burrows (see Table I )

OP. (mOsm)

[Cl1 (mmol~l ‘)

[Nal (mmol~l~‘)

[Kl (mm0l.l~‘)

[Mgl (mmol~l~‘)

ICal (mm0l.l ‘)

742.6 326. I 362.2 7.8 7.6 12.7

+ 10.4 k6.5 26.6 kO.3 t 0.3 5 0.7

3.5. Haemolymph ion status

The osmotic pressure measured in crabs venturing onto the sand flats was 682+- 10 mOsm while that of C. curnifex extracted from burrows in the plantation and the forest was significantly higher (742-t 10 mOsm) (t-test, P = 0.001). The lowest values for measured elements in the haemolymph of crabs taken from burrows were, in mmol.1~‘: Cl, 290; Na, 337; K, 5.9; Mg, 5.9 and Ca, 8.1. Means and SEM are provided in Table 2.

4. Discussion

4. I. Oxygen and respiratory requirements

The PpO, value (80-90 torr) of air-breathing C. carnifex was comparable to that of C. hirtipes measured in the field (Adamczewska and Morris, 1996) suggesting that the lung and haemolymph were functioning in a comparable fashion. However, while the P,,, in air-breathing C. curnifex was similar to the 55 torr reported previously for this spelies (Wood and Randall, 198 lb) and other terrestrial crabs (e.g. G. nutulis, 45 torr: Adamczewska and Morris, 1994; B. latro, 43 torr: Greenaway et al., 1988) it was considerably less than P,, in C. hirtipes (-80 torr; Adamczewska and Morris, 1996). Thus, in C. curnifex, but not C. hirtipes, the post-branchial haemolymph must be of low P,, and mixes with efferent pulmonary haemolymph with a higher Po2.

Although the gills of C. curnlfex are apparently diffusion limited compared to the lungs this was not reflected in relatively decreased Caoz compared to CPoz since both

were between 80 and 90% saturated. The C,, of air-breathing C. curmfex was similar

to previous values (Burggren and McMahon, ‘198 1; Wood and Randall, 198 lb) and to those in C. hirtipes (Adamczewska and Morris, 1996). However, arterial O2 saturation in C. carncfex has previously been suggested to be both low (65-80%; Burggren and McMahon, 1981) and high ( >85%; Wood and Randall, 1981b). Similar arterial and pulmonary Co? despite different PO2 implies that the post-branchial haemolymph was either well saturated or that the contribution by the gills to arterial haemolymph flow was small. C. curnifex has been reported to have haemolymph with relatively high affinity for 02, Burggren and McMahon (1981) give a P,, value of 11 torr. The in vivo OZ equilibrium curves (Fig. 4) for air-breathing C. curnifex suggested Hc-02, affinity

S. Morris, A.M. Adamczewska I J. Exp. Mar. Biol. Ecol. 206 (1996) 149-164 159

C. carnifex - in viva 0, equilibrium curves

‘1

0.8 -

7 _) -: -

z

0.6

.E s- 0.4 -

0 20 40 60 80 100

PO, (toIT)

Fig. 4. The in viva oxygen equilibrium curves for the haemolymph from air-breathing (w) and from 30 min

submerged (0) C. carnifex; haemolymph Co, vs. haemolymph Po2. Pulmonary, arterial and venous samples

are indicated p, a and v for each respectively. The curves were fitted by iteration and the fits shown are for

P,,, = 6 torr and n,,, =2.2 for air-breathing and PC,, = 17 torr and n,,, =2.2 for water-breathing crabs. The

vertical bars indicate a-v 0, content differences for 3 different situations. Bar 1 is that for air breathing crabs

and bar 3 for water breathing crabs. Bar 2 is the theoretical a-v difference that would occur in water breathing

crabs in which the equilibrium curve had not been right shifted by the Bohr effect.

was higher still with a P,,, near 6 torr. Furthermore, this high affinity ensured that arterial haemolymph remained well saturated with 0,, even at a P, of 40 torr (Fig. 4).

Submergence of C. carrzifex in the field induced a clear intern:1 hypoxia since the P pO1 decreased from more than 80 torr in air-breathing crabs to near 20 torr in crabs immersed in well oxygenated water (P, = 127 torr) for 30 min. Internal hypoxia was

also observed for submerged C. hirtipes IAdamczewska and Morris, 1996), Gecarcinus

later&is (Taylor and Davies, 1982) and Pseudothelphusa garmani (Innes and Taylor, 1986). In C. carnifex the decreased Pp02 was accompanied by lowered Hc-0, (post-

pulmonary saturation 56%) resulting in a non-significant arterial-venous 0, content difference. Unless cardiac output was increased to more than 300% this reflects reduced 0, transport, similar to that in immersed C. hirtipes, suggesting that Curdisoma

generally suffer a significant hypoxia during submergence. Both C. carnifex and C. guanhumii are suggested to retain air within their branchial

chamber while immersed (Cameron, 1981; O’Mahoney and Full, 1984). Voluntarily immersing C. carnifex released only few air bubbles while submerged. In submersion studies of C. carnifex Cameron (1981) found it necessary to artificially remove branchial air and thus expose the respiratory surfaces to aerated water, and reported submersion to cause stress or even death. In C. guanhumi in which the branchial chambers were also deliberately flooded O’Mahoney and Full (1984) measured a similar MO, in water and in air. However, 0, extraction efficiency from water was 80% lower and ventilation 300% that in air (O’Mahoney and Full, 1984). It is difficult to determine what portion of the respiratory surfaces (gills and/or lungs) of C. carnifex might be bathed in the immersed crabs in the field but a combination of trapped hypoxic air, reduced ventilatory

160 S. Morris, A.M. Adamczewska I J. Exp. Mar. Biol. Ecol. 206 (1996) 149- 164

flow and relatively poor extraction efficiency could explain the internal hypoxia after 30 min of immersion. In laboratory studies quiescent C. carnifex trapped 94% of the branchial air in which the PO, decreased to 22 torr after 30 min submersion at 25°C

(Dela-Cruz and Morris, unpublished). This low air PO, is close to the 21 torr in

pulmonary haemolymph of field immersed crabs suggesting an air-haemolymph PO9

equilibrium and thus minimal 0, transfer from outside of the‘branchial chamber after 3d min immersion.

High Hc-0, affinity during hypoxia would assist 0, loading at the gas exchange organs. However, when the a-v 0, partial pressure difference becomes very small it is

imperative that this difference be translated into as large as possible a-v 0, content

difference by maintaining arterial and venous P,, on the steep part of the equilibrium

curve. The in vivo 0, equilibrium curves indicated that after 30 min of immersion the

affinity of Hc for 0, decreased, with the Pso increasing to 18 tot-r (Fig. 4), as a result of a large respiratory acidosis (see below). The lowered affinity reduced 0, loading and

decreased the a-v content difference (Fig. 4). Extrapolating the PuO, and P.+ of

submerged crabs to the equilibrium curve for air-breathing crabs showed the a-v 0, content difference that would have occurred in the absence of a Bohr shift (Fig. 4). Therefore, if there were no increase in cardiac output the hypoxia and acidosis reduce 0,

delivery. The low PuOz is necessary in Curdisomu species (- 12 torr in C. carnifex;

Burggren and McMahon, 198 1; Wood and Randall, 198 lb; 5 tot-r in C. hirtipes;

Adamczewska and Morris, 1996) to unload O2 from the high affinity Hc (Fig. 4) (Morris, 1991; Truchot, 1992 for review of Hc-0, affinity). During the large internal

hypoxia venous haemolymph remained 30% 0, saturated (approx. 0.3 mmol.l- ‘) and it may be that P,, never declines sufficiently to unload this OZ reserve.

On the basis ‘that C. carnijex contains Hc at approximately 0.8 mmol.lK’ and maintains OZ consumption at 2.1 ,umol.g-’ .h-’ (O’Mahoney and Full, 1984) the Hc-0, would provide a maximum of 7.5 min 0, to a 300 g crab. Using the trapped air

volume of 18.6 ml and a AP,, of 95 torr (Dela-Cruz and Morris, unpublished), the crabs utilized 110 pmol from 0, stored in the branchial chamber, which would support a

further 10 min of respiration. These derivations assume negligible 0, transfer from the water. The 50% reduction in pulmonary and arterial O? loading suggests that 0, uptake

is diffusion limited at the gas exchange organs rather than perfusion limited. The 68% decrease in a-v 0, content difference could be coupled with increased cardiac output but

the increased metabolic demand would be counter-adaptive and quickly exhaust 0, in a

limited volume of air. Therefore, C. curnifex submerged for 30 min must either reduce metabolism by -30% to remain aerobic or rely increasingly on anaerobiosis. The accumulation of L-lactate and an increase in haemolymph glucose after 30 min submersion were consistent with anaerobiosis elevating glycolytic flux and suggest that metabolic rate is sustained. The negligible a-v 0, content difference after 30 min submergence reflects the near exhaustion of 0, stores and the near equilibrium between haemolymph PO, and that of the branchial chamber. This response is different to that of submerged C. hirtipes (Adamczewska and Morris, 1996), in which a hypometabolic state (-20% aerial rate) was postulated to be a part of the submersion response (Adamczew- ska and Morris, 1996; Dela-Cruz and Morris, 1997). Therefore, while C. carnifex like C.

S. Morris, A.M. Adamczewska I J. Exp. Mar. Biol. Ecol. 206 (1996) 149-164 161

hirtipes appears reluctant to breathe water under natural conditions, it seems to lack the same facility to reduce metabolic demand while submerged.

4.2. Carbon dioxide and acid-base state

The P,, in air-breathing C. carnifex on the Cocos Islands (14.3 torr) compares well

with resting values of 14.5 torr for C. carnifex (Randall and Wood, 1981) and 12-14 torr in C. hirtipes (Farrelly and Greenaway, 1994; Adamczewska and Morris, 1996). A crucial difference between the field studies of C. hirtipes (Adamczewska and Morris, 1996) and now of C. carnifex, compared to previous studies is the importance of retained branchial air. Previously, laboratory studies used animals in which the chambers were deliberately water filled. This difference would have marked effects on the functioning of the lung and gills in gas and ion exchange.

Normally, submersion of amphibious crabs results in a respiratory alkalosis (McMahon and Burggren, 1988) since the increased ventilation requirement to extract 0, from water compared to air promotes CO, excretion in water. This was not the case for submersed C. hirtipes (Adamczewska and Morris, 1996) nor for C. carnifex in this study. C. carnifex in the field exhibited a respiratory acidosis of -0.4 pH units which promoted a large internal hypercapnia compared to air-breathing crabs (Fig. 5). The small metabolic component of the acidosis was equivalent to a base loss of 1.5

C. carnifex - pH - HCO,. diagram

25.0

; _) = 22.5

:

L- Om 20.0 u + ’ 0 8 17.5 z

15.0

7.1 1.2 7.3 7.4 7.5 7.6 7.1

PH

Fig. 5. The pH/HCO; diagram for C. carnifex haemolymph for analysis of acid-base perturbations comparing air-breathing (W) and submerged (0) crabs. The Pc,, isopleths were constructed for haemolymph of

air-breathing crabs using (YCO, = 0.037 1 mmol .l~ ’ and pK=6.03. The slightly different isopleths for

water-breathing crabs have been omitted for clarity. The nonbicarbonate buffer line was fitted using a value for

A[HCO;]/ApH of -6.4 (Adamczewska and Morris, 1996; Dela-Cruz and Morris, 1997). The letters p. a and v indicate pulmonary, arterial and venous haemolymph respectively.

162 S. Morris, A.M. Adamczewska I J. Exp. Mar. Biol. Ecol. 206 (1996) 149-164

mmol.l-’ (Fig. 5) and was due to H+ accompanying lactate production. The internal hypercapnic acidosis was further evidence that submerged C. carnifex ventilated either little or not at all and that gas exchange was primarily with a progressively hypoxic- hypercapnic air bubble.

4.3. Haemolymph ion and osmotic status

The concentrations of Na, Cl and K measured in the haemolymph of C. carnifex were similar to previous values for this species (Kormanik and Harris, 198 1; Wood and Boutilier, 1985; Wood et al., 1986). However, the values for [Cal and [Mg] in C. carnifex appear variable and in the Moorea (French Polynesia) population ranged between 15 and 22, and between 9 and 19 mmol + 1~ ’ , (Wood and Boutilier, 1985; Wood et al., 1986, respectively). The values for Cocos Island crabs were at the lower end of this range but this was not a function of burrow water salinity since on Cocos the forest burrows were 66-80 mOsm and on Moorea 16-41 mOsm (Wood and Boutilier, 1985). However, in this latter study the water Ca:Na ratio was 1: 1.6 providing relatively Ca rich water and even more so for Mg (Wood and Boutilier, 1985). These salt levels easily exceed those in the freshwater habitat of C. hirtipes (Greenaway, 1989; Adamczewska and Morris, 1996) which consequently have a much lower osmotic pressure than C. carnifex. Thus C. carnifex differs from C. hirtipes in both ionic status as well as metabolic, CO,, and acid-base changes in response to submergence.

Adamczewska and Morris (1996) suggest that immersion in freshwater could present a special energetic expense for salt regulation in C. hirtipes, which could be mitigated by reducing contact between the water and permeable gill epithelium. Salt regulation by C. carnifex on the Cocos (Keeling) Islands with access to brackish water should be less of a problem. However, C. carnifex has a haemolymph osmotic pressure 200 mOsm greater than C. hirtipes and the vast majority of the crabs normally remained in the forest with access to only burrow water, rain water or dew. The osmotic pressure measured in crabs moving on the sand flats was 60 mOsm lower than those from the forest suggesting that these crabs were attempting to replenish ions by saltwater emersion. If so, this is a clear indication that C. carnifex is different from C. hirtipes, which have access to freshwater only.

C. carnifex become both hypoxic and hypercapnic during voluntary submergence in situ. Increasingly, Cardisoma appear to respire during long-term natural immersion to the extent that blood and branchial chamber O2 stores permit. Thus Cardisoma are probably not amphibious species, as previously thought. The extent to which Curdisoma

utilize burrow water for ion and water balance is still unclear and while C. carnifex voluntarily immerses in salt water, it is a relatively rare occurrence. Ongoing laboratory studies will determine respiratory gas changes in the branchial chamber during air bubble retention and the extent to which gas exchange is reduced. Further field work must establish the dependency of Cardisoma on immersion for salt and water balance.

Acknowledgments

We would like to express our appreciation to the ANCA, their conservator, Geoff

S. Morris, A.M. Adamczewska I J. Exp. Mar. Biol. Ecol. 206 (1996) 149-164 163

Tranter for their assistance and hospitality during the visits to the Cocos (Keeling) Islands. We thank Prof. Peter Greenaway for useful discussion of the ecophysiology of Curdisoma, and Jocelyn Dela-Cruz for analysing the ionic composition of the water samples. This work was carried out while AMA was in receipt of an Australian Post-graduate Award and the study was funded by an Australian Research Council grant to SM.

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