Aerial and aquatic respiration in the ghost crab Ocypode saratan—I. Fine structure of respiratory surfaces, their ventilation and perfusion; oxygen consumption and carbon dioxide production

0300-9629189$3.00 + 0.00 ‘0 1989 Pergamon Press plc

Camp. Biochem. Physiol.Vol. 94A, NO. 4, pp. 755-764, 1989 Printed in Great Britain

AERIAL AND AQUATIC RESPIRATION IN THE GHOST CRAB OCYPODE SARATAN-I. FINE STRUCTURE OF RESPIRATORY SURFACES, THEIR VENTILATION AND PERFUSION; OXYGEN CONSUMPTION AND CARBON DIOXIDE PRODUCTION A. H. AL-WASSIA,* A. J. INN&P N. M. WHITELEYand E. W. TAYLOR~

School of Biological Telephone:

Sciences, University of Birmingham, Birmingham Bl5 2TT, UK. (021) 414-5472; *Department of Biology, King Abdulaziz University, Jeddah, Saudi Arabia; and tDepartment of Zoology, University of Aberdeen, Aberdeen, UK (Receiced

16 June 1989)

Abstract-l. Ucypode saratan has reduced gills and a branchiostegal wall specialized to function as a lung. 2. When submerged, or in humid air, ventilation was by means of the scaphognathites. Crabs inactive in dry air replaced scaphognathite beating by slow pressure variations in the branchial chambers. Increased levels of CO, in air caused elevated rates of scaphognathite beating. 3. Heart rate varied with changes in ventilation. 4. The gills were perfused both in submerged and in air breathing crabs, whilst the lungs were preferentially perfused in air, but not perfused in submerged crabs. 5. The respiratory quotient in air was about 0.55.

INTRODUCTION Land crabs have long been of interest to comparative physiologists (for reviews, see Bliss 1968; Powers and Bliss, 1983; Burggren and McMahon, 1988), due to the variety of adaptations they show for survival in air. The respiratory adaptations associated with the transition from water- to air-breathing have been identified by comparing the structure and function of the respiratory systems of a number of brachyurans, including primarily aquatic species, able to survive for varying periods in air, such as the shore crab Curcinus maenas (Truchot, 1975; Taylor and Butler, 1978; Taylor and Wheatly, 1979) and land crabs which are variously adapted for air-breathing including some species unable to survive prolonged submersion (Innes and Taylor, 1986a; Taylor and Innes, 1988; McMahon and Burggren, 1988). Land crabs typically possess a well perfused branchial chamber lining, modified to form a “lung”, specialized for oxygen uptake; having a lightly cuticularized surface layer of thin epithelial cell processes, separating air and haemolymph (Greenaway and Taylor. 1976) which in many species is either invaginated or evaginated to increase the area for gas exchange (Diaz and Rodriguez, 1977; Taylor and Innes, 1988; McMahon and Burggren, 1988). The reduced gills typically occupy the ventral portion of each branchial chamber and in amphibious species are bathed in a reservoir of water (e.g. Cardisoma carnifex, Wood and Randall, 1981a), which may act as an effective sink for CO, excretion (Burnett and McMahon, 1987). Respiration is typically bimodal in these species with oxygen taken up at the lung surface and CO, excreted at the gills into water. Even in fully $Author

to whom

all correspondence

should

be addressed.

terrestrial species such as Birgus lutro there is evidence of CO, excretion over the gills (Greenaway ef al., 1988). The scaphognathites, used to ventilate the branchial chambers in aquatic, water-breathing crabs (Borradaile, 1922; Arudpragasam and Naylor, 1964, Taylor er al., 1973) and in submerged land crabs (Taylor and Innes. 1988) are also effective in air (Taylor and Butler, 1978) and may be used for lung ventilation in land crabs (e.g. Burggren er al., 1985). Other species switch to lung ventilation by slow pressure variations generated by the branchiostegal muscles (e.g. Holthusiana transversa, Greenaway and Taylor, 1976) with the scaphognathites inactive in air. The species belonging to the family Ocypodidae are well adapted land crabs, although they are not completely independent of water. Hartnoll (1988) described five grades of terrestrial adaptation in land crabs, varying in their relative independence of water. Ocypode saratan was included in grade T3 characterized as: resident supratidally; active in air, usually at night; burrowing or cryptic in habit; requiring regular access to water in which they can immerse themselves, either by visits to the sea or to groundwater at the base of the burrow (Powers and Bliss, 1983); and dependent on water for the pelagic larval stages. The various species of 0. saratan are known as “ghost crabs” or “racing crabs” because of their pale colour and habit of running rapidly over sandy beaches at night; they are by far the fastest moving terrestrial crabs (Hartnoll, 1988). 0. saratan lives on sandy beaches around the Red Sea where their round burrow entrances, each accompanied by a pyramidal pile of excavated sand, are a common sight. By day the crabs are to be found deep within their often labyrinthine burrow systems, which can extend down to 1 m or more (Eshky, 1985). The 755

A. H. AL-WASSIA et al.

756

entrances to these burrows are above the water line but extend down to water level so that they contain damp sand. At night the crabs leave their burrows to forage on the shore line for food and it is then that they are most easily captured, when picked up in the beam of a flashlight. Their rapid running, however, often enables them to escape into a burrow or into the sea. They are clearly not adapted for immersion, as once in water they do not stay submerged for long and will re-emerge onto the beach within a few minutes, when they may be caught (S. Oakley, A. J. Innes and E. W. Taylor, personal communication). These field observations prompted the present investigation into the respiratory physiology of 0. saratan held in either humid or dry air or submerged in sea-water. These crabs have been the subject of numerous ecological studies (e.g. Bliss, 1968; Wolcott, 1978). Physiological studies have included respiratory structures and respiration @torch and Welsch, 1975; Burnett, 1979) the vascular system (Greenaway and Farrelly, 1984), oxygen affinity of haemocyanin in z+zro (Morris and Bridges, 1985) and the effects of temperature on oxygen consumption and heart rate (Eshky et ul.. 1988). The present study includes measurements of ventilation and perfusion of the respiratory organs in 0. saratan, when exposed in air and submerged in water, and of its rates of external respiration in air. An associated study (Whiteley ef al.. 1989) describes haemolymph oxygen and CO2 levels and acid-base regulation during aerial and aquatic respiration.

pressures were recorded simultaneously on either side ot’the crab. The cannulae were filled with saline and connected to pressure transducers (Elcomatic). Variations in hydrostatic or air pressure were recorded on a pen recorder with rectilinear coordinates (Devices M4). Rates of scaphognathite beating (ventilation rate, ,/k )< occurrence of reversals of scaphognathite beat, slow pressure changes associated with lung ventilation in air and ventilatory pauses were recorded continuously from crabs held on damp sand over shallow sea-water (humid airbreathers); from crabs exposed to air in the absence of sea-water (dry air-breathers) and from crabs submerged in aerated sea-water of 30cm depth, with no access to au (water-breathers). In this latter group of crabs. air was removed from the branchia! chambers when they were first submerged by holding each crab with the exhalent openings uppermost and checking that a!! air bubbles had escaped. Measu~ments were obtained from active crabs immediatelv after experimental manipuiation and after about 5 hr oi recovery, when they had become relatively inactive. Measurement @ ECG and impedence The ECG or heart movements and in some animals scaphognathite movements were recorded by inserting two varnished copper wires through the carapace to lie either side of the heart or scaphognathite. These wires were then led either to a preamplifier (Isleworth Electronics) and then the pen recorder for recording ECG or to an impedance coupler (Strathkelvin Instruments) for recording heart or scaphognathite movements. Simultaneous recordings of heart beat and ventilatory pressures were obtained during routine activity in air or water (n = 8) and following exposure to a gas mixture containing 5% CO, in air (a = 4). delivered from a gas mixing pump ~~sthoff). Distribution of huemvfymph .J‘?OW to the fungs and gi/k

MATERIALS

AND METHODS

Specimens of the ghost crab 0. sarafan (12 = 40) of either sex and weighing between 30 and 53 g, were obtained from local beaches cfose to King Abduiaziz University, Jeddah, Saudi Arabia and individually packed for transport in moist thermocontainers. The crabs were then air-freighted to Birmingham and kept in an aquarium room at 25’C. They were maintained for 2-3 weeks prior to experiments in tanks containing damp sand with access to shallow (I cm) seawater, and were provided with refuges made from lengths of plastic piping. The water was changed daily and the animals were fed with chopped ox heart and liver. They appeared to stay aggressively fit under these conditions and several survived for 2-3 months following experimentation. Examination of fine Structure Jndividua! gills and the lining of the branchiai chambers were fixed by perfusion with 2% glutaraidehyde in an isosmotic saline/sucrose solution and postfixed in osmium tetroxide for examination under the electron microscope. Whole mounts were critical point dried and viewed under a scanning electron microscope (I.S.I. IOOA) and thin sections were cut for examination under a transmission electron microscope (Phillips).

In an exploratory experiment the distribution of haemolymph to the lungs and gills of crabs in air and water was measured as the distribution of microspheres. I5 & I .5pm in diameter and labelled with 57Co (New England Nuclear, Stevenage). Approximately 250,O~ microspheres suspended in 100~1 of crab saline. containing 0.01% Tween-80 were injected unilaterally into the infrabranchia! sinus of the haemocoe! through the base of the second walking leg on the left side of each crab, as described by Taylor and Greenaway (1984). Two pairs of crabs were injected, one after several days in humid air and the other following 5d hr submersion in sea-water with the air expelled from their branchial chambers. After allowing a minimum of IO min for entrapment of microspheres in the vascular beds of the haemocoe!, the animals were killed and dissected individually and their gills and lungs were placed in separate gamma counting vials for assessment of the concentration of the radioactive microspheres. Counting was performed using an automatic gamma counter (Packard 5600). Details of this technique were described by Butler et al. (1988) but the relatively poor and variable degree of transport of microspheres in the crab’s open circulation precluded quantitative measurements and the data is restricted to the proportion of the injected load which was transported and entered the gills or the lungs (cf Taylor and Greenaway, 1984).

Measurement of ~e~ltilat~onand perfusion pressures Short lengths of poIypropylene cannula were inserted through holes drilled in the carapace at locations over the dorsal and ventral sides of the branchiostegites of individual crabs. These locations were chosen in order to record pressures immediately behind the exhalent chamber containing the scaphognathite, which opens anteriorly from the ventral region of each branchial chamber, and to record small pressure changes close to the surface of the lung in the dorsal region of each branchial chamber. Often these

~easureme~i

qf oxygen consumption and CO2 production

The rate of oxygen consumption (PO,)

was measured for individual, “active” and “inactive” crabs in air at 25”C, using a simple adaptation of the Barctoft constant pressure respirometer (Davis, 1966). Crabs were left 20-30 min in the respirometer to allow equilibration, prior to measuring their active PO, values. Inactive 30, measurements were obtained after S-6 hr in respirometer. During this period the respirometer was ventilated with humidified, CO: free air

Respiration

in the ghost

The same respirometer was used to measure CO, production (PCO,) of active and inactive crabs. This was estimated by substracting changes in volume in the respirometer in the absence of a CO, absorbing agent (20% KOH) from the values for li0, obtained in the presence of KOH. Measured values are given in the text as mean k SE of mean with the number of observations in parentheses. A two-tailed Student’s r-Test was used to test the significance of any apparent differences between the mean values of measured variables. RESULTS

Fine structure

of‘ respiratory

The surface lamellae from

structure and fine structure of the gill 0. saratan are illustrated in Fig. 1. 0.

gas exchange

surfaces

saratan. in common with other air-breathing land crabs, has reduced gills. There are seven gills in each branchial chamber, with the adjoining lamella of the

crab

757

fifth and sixth gill interdigitating at their tips (Greenaway and Farrelly, 1984). The gills are restricted to the ventral portion of each branchial chamber below a horizontal septum (Whiteley et al., 1989). The gills are phyllobranchiate with short, square-shaped lamellae having expanded margins (Fig. la). The lamellae are fairly widely spaced (approximately 50 pm apart). The TEM pictures of a lamella from gill 3 revealed that the gill epithelium has an outer layer of cuticle which varies in thickness from the expanded margin of each lamella (10 pm) to the sides (1-2 pm) (Fig. 1b). The epithelial cell processes enclosing the haemocoelic spaces are 3-5 pm thick. They contain complex membrane systems in contact with their outer, cuticularized surface, and are packed with mitochondria and vesicles of various sizes and staining properties (Fig. lb). The lining of the branchiostegite which comprises

a)

Fig. I, Scanning and transmission electron micrographs of the respiratory organs of 0. sarafun. (a) SEM of lamellae from gill 3, illustrating their blunt, squared-off shape and wide spacing. Epizootes can be identified coating the lamellae (scale bar, IOO~m). (b) TEM of gill lamella from gill 3 showing the cuticularised processes of two epithelial cells which contain abundant mitochondria. vesicles and complex membrane systems. The outer surface of the cuticle bears an adhering layer of micro-organisms (scale bar. 5 lm). (c) SEM of the surface of the branchiostegite or lung revealing prominent folds and the complex surface architecture (scale bar, 100 pm). (d) SEM of lung surface to show invaginations (scale bar. 10 nm).

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A. H. AL-WANA et al.

the respiratory surface of the lung in 0. saratan is thrown into a series of folds (Fig. lc) perforated by closely packed invaginations (Fig. Id). Examination of relatively poorly fixed material (not illustrated) revealed that in cross section the epithelial cells are covered in a thin layer of cuticle (0.5 pm) and are relatively simple in structure, extending as thin processes (0.2-0.5 pm) beneath the cuticle to enclose haemocoelic spaces close to the lung surface. Ventilation and heart rate Crabs breathing humid air. Active crabs showed continuous scaphognathite beating with regular switches from a forward to reversed direction, each for about 50% of the observed time (Fig. 2a). Mean ventilation frequency (&a) was 218 + 19 beats/min (7) and varied between 120 to 280 beats/min with the very rapid rates of scaphognathite beating occurring during and sometimes immediately after reversed ventilation. The pressures generated in the ventral region of the branchial chambers surrounding the gills (Whiteley et al., 1989) by scaphognathite beating

were reflected in pressure recordings obtained close to the “lung” (i.e. the dorsal region of the branchial chamber above the gills) (Fig. 2b). When allowed to settle for 4-5 hr, inactive crabs ventilated predominantly in the forward mode (60% of total time). Scaphognathite rate was markedly reduced to a mean of 43 + 3(8) beats/min (e.g. Fig. 3); which represents a significant reduction from the active rate of scaphognathite beating (P < 0.002). Reversed ventilation was less frequent (10% of the total time) but ventilation frequency was 131 k 21(8). which was significantly higher than the rate of forward beating (P < 0.01). Inactive crabs also showed ventilatory pauses, varying in duration from 30 set to 15 min. and occupying about 30% of total time, sometimes broken by brief periods of bilateral, reversed scaphognathite beating, at other times by the onset of slow pressure changes associated with lung ventilation in one branchial chamber whilst the other continued to show periodic scaphognathite beating. Mean heart rate in disturbed active animals was 102 + 4(6) beats/min and this reduced significantly to

a)

E

E 20

Fig. 2. Recording of pressure fluctuations in the branchial chambers of 0. saratan (male 52 g). (a) Simultaneous pressures recorded from the ventral region of the left and right branchial chambers of a crab exposed in air with access to shallow sea-water. Both scaphognathites were active, pumping alternately in a forwardly direction (subambient pressures) then a reversed direction (pressures above ambient) for approximately equal periods of time. These changes occurred simultaneously in both branchial chambers. (b) Pressures recorded from the left branchial chamber of a crab (male 45 g), in the ventral region of the chamber, immediately behind the scaphognathite (lower trace) and in the dorsal region of the chamber close to the wall of the branchiostegite (upper trace). The pumping action of the scaphognathite generated oscillating pressures in the ventral region of the chamber which were reflected in some similar oscillations and mean pressure variations in the dorsal region.

Respiration

in the ghost

crab

759

Fig. 3. 0. saratan (male 48 g). Heart rate, recorded as an ECG (upper trace), and pressures in the left (middle trace) and right (lower trace) branchial chambers of a crab exposed in air with access to sea-water. The scaphognathite in the right branchial chamber was continuously active but accelerated occasionally, often concurrently with a brief period of active ventilation of the left branchial chamber. These periods of increased ventilatory activity typically coincided with an acceleration of the ECG. Both pressure traces varied around zero, suggesting a combination of forward and reversed ventilation.

34 k 14(3) in settled, markedly arrythmic

inactive crabs. Heart rate was in association with the changes

in ventilation. During ventilatory pauses the heart slowed or stopped whilst at the onset of active ventilation there was characteristically a marked tachycardia with heart rate rising to 60-70 beats/min (Fig. 3). Exposure to high levels of CO, in the surrounding air caused the scaphognathites to commence beating at very high rates with a consequent increase in the standing pressure in the lung (Fig. 4). Heart rate was unaffected by exposure to high CO2 levels. Crabs breathing dry air. When crabs (n = 5) were exposed in dry air, the scaphognathites initially showed prolonged periods of reversed beating (from 5 to 75% of recorded time) then ceased to beat after 1-2 hr and showed prolonged ventilatory pauses. After IL4 hr in dry air without disturbance the scaphognathites ceased to beat. One crab showed no recorded ventilatory pressures for 70 min and was then disturbed when bilateral ventilatory pressures were immediately recorded, demonstrating that the pressure cannulae remained patent and that the scaphognathites were not operating in inactive crabs in dry air. During these pauses small, slow suprambient pressure changes were recorded from the branchial chambers (Fig. 5). These had a meanfR

of 9.5 f 3.4(6) beats/min which ranged between 24 and 1.5 beats/min. Samples of gas mixtures withdrawn from the left and right branchial chambers of a crab ventilating both chambers intermittently in air revealed that scaphognathite movements were more effective in ventilation than the slow pressure changes (Fig. 5b), as they generated higher partial pressures of oxygen. Submerged, water-breathing crabs. When submerged in aerated sea-water (n = 5) crabs showed predominant (95% of total time) forward ventilation using both scaphognathites with a mean fa of 53 f 13(5) beats/min, broken by infrequent reversals of beat (5% of total time). Rate of beating often varied on each side and ranged between 48 and 180 beats/min (e.g. Fig. 6). The highest rates (maximum 240 beats/min) were recorded during reversed beating. Heart rate was relatively high in a submerged crab at 132 beats/min but this regular rate was interrupted by periods of cardiac arrest of from 10 to 30 set, so that overall rate was 74 beats/min (Fig. 6). The distribution

qfmicrospheres

to the lungs and gills

The proportion of injected microspheres trapped in the lungs and gills of crabs (n = 4) in humid air and following 5-6 hr submersion are listed in Table 1. The lungs trapped the larger proportion of microspheres

Fig. 4. 0. saratan (female 38 g). Heart rate recorded as movements of the heart (upper trace), and pressures in the ventral region of the branchial chamber (lower trace) and dorsally close to the wall of the branchiostegite (middle trace). At the onset of the recording, the crab was exposed to a gas mixture containing 5% CO,. Scaphognathite rate accelerated markedly causing an increase in the standing pressure in the ventral and dorsal regions of the chamber. Removal of the stimulus half way through the recording resulted in recovery to the ventilatory pressures recorded prior to exposure. Heart rate was apparently unaffected.

A. H. AL-WASSIAet al

760

Fig. 5. 0. saman (male 52 g). (a) Heart rate recorded as the ECG (upper trace), and pressures recorded from the left branchial chamber (lower trace) of a crab settled in air for 3 hr without access to water. Brief periods of ventiiatory activity were accompanied by cessation of cardiac activity, followed by a period of increased heart rate. (b) Simultaneous recording of pressures from the right and left branchial chamber of a crab settled in air for 2 hr. Samples of lung air from these two branchial chambers revealed that the PO, in the right chamber was 144 mm Hg, while in the left chamber it was 73 mm Hg, indicating that scaphognathite movements ventilated the lung more effectively than the slow vcntilatory pressures.

Fig. 6. 0. .wratan(malt? 46 g), Heart rate recorded as movements of the heart (upper trace) and pressures recorded from the left (middle trace) and right (lower trace) branchial chambers of a crab submerged in aerated sea-water at 25°C. Both scaphognathites were continuously active in the forwardly pumping mode with & at 84 beats/min in the left chamber and 61 beats/min in the right chamber. Heart rate was high (I32 beats/min) but showed periods of cardiac arrest, resulting in an overall rate of 14 beatsimin.

(4 x that in gills) when crabs were in air. Following 5% hr submersion, there was a 99% reduction in the fraction of injected microspheres trapped by the lungs. The gills, however, did not show a substantial change in the proportion of trapped microspheres Table I. Proportion of microsphcres trapped in the respiratory organs of 0. SCWCIIG~ (as 9%of injected load) following their injection into the infrabranchial sinus at the base of the second walking leg on the left side of each crab (n = 4) Air/water-breathing (5-6 hr over damp sand) giI,s Left Right

7.21 0.27

Left Right

10 0.04

@ = *)

lunas 28.14 6.22 40 4

Water-breathing (5-6 hr submersion) eil,s fn = 2,

hmas

6.48 0.42

0.46 0.17

14.92 2.56

1.Q4 0.45

following 5-6 hr submersion. There was a marked unilaterial distribution of microspheres to the injected side of the animal, indicating that they were trapped on their first circuit following injection. Although the number of observations is small the results seem unequivocal with the only variation being in the proportion of injected microspheres entering the circulation. Oxygen

uptake and CO2 production

The rates of oxygen uptake (PO,) and COz production ($YXI,) of individual disturbed and settled crabs were measured in air at 25°C and are plotted against their live mass in Fig. 7. Mean values for active crabs immediately after insertion in the respirometer were: for I%,, 4.99 -t_0.35 ml O,/hr and for pCOz, 2.70 f 0.40 ml CO,&, Following 5-6 hr

Respiration in the ghost crab

761

1976; Taylor and Greenaway, 1979; Taylor, Greenaway and Farrelley, 1984; Farrelly and 7Greenaway, 1987). In 0. saratan this surface elabor5ation takes the form of numerous small folds, as _ 3revealed by the present study and the earlier work by 0 Diaz and Rodriguez (1977). The surface of these folds is covered with invaginations. Sections of poorly fixed : material revealed that the diffusion distance was l; r around I pm with about haif this distance comprised of cuticle, resembling the surface of the “lung” in 5 0.5 Holthuisana transversa (Taylor and Greenaway, 1979) and Pseudothelphusa (Innes et al., 1987). With its large surface area and short diffusion distances this lung has the physical properties of an effective respiratory organ and measurement of blood gases in 0 30 40 5060 0 30 40x)60 crabs breathing humid air revealed that it was capable of generating postpulmonary PO2 levels as mass (9) mass tgl high as 17.3 kPa (Whiteley er al., 1989). Fig. 7. Rate of oxygen uptake (P02. ()/a) and CO1 The branchial chambers containing the gills and production (liCO,, U/w) for individual male or female 0. “lungs” were ventilated with either air or water by sararan. related to their live mass using logged axes. Meameans of the scaphognathites. These bailing organs surements were taken from each crab soon after insertion generated oscillatory, sub-ambient pressures in their into the respirometer (filled symbols) and after 4-5 hr with no disturbance (open symbols). Initial disturbed rates were normal, forwardly directed pumping mode, which higher than settled rates and PO? was higher than the predominated in submerged crabs. Regular reversals equivalent I)O, in each case. of beat, which caused branchial chamber pressures to rise above ambient, as previously described in a range of crustaceans (McMahon and Wilkens, 1983) were in the ventilated respirometer, ri0, fell to recorded during ventilation in air and rates of 1.84~0.20ml O,/hr and PCO, to 1.03~0.16ml scaphognathite beating were higher in the reversed CO,/hr. Both of these inactive values were signifimode in settled 0. saratan. Similar increased rates of cantly lower than the active values (P < 0.01). The reversed ~aphognathite beating in air were recorded mean respiratory quotient (lie) was 0.54 (range in Card&ma guanhumi (Burggren et al., 1985). In 0.39-0.65) for active crabs and 0.56 (range 0.38-0.70) humid air, ventilation was predominantly by means of the scaphognathites, with prolonged alternate for inactive crabs. periods of forward and reversed pumping similar to those recorded from Cardisoma by Burggren ef al. DISCUSSION (I 985). These generated equivalent pressure changes The gills in 0. saratan have short widely-spaced in the dorsal areas of the branchial chambers close lamellae with relatively long diffusion distances that the to the “lung” surface demonstrating through cuticularized epithelial cell processes of scaphognathite is able to pump air as well as water, 4-6pm (cf. McMahon and Burggren, 1988). The as described in the shore crab Carcinus maenas crabs observed in the present study had large num(Taylor and Butler, 1978) and in Cardisoma bers of microorganisms attached to their gill surfaces (Burggren et al., 1985). which may further limit respiratory gas exchange, as When crabs were allowed to settle undistur~d in their presence is likely to increase the effective diffuhumid air the scaphognathites became less active and sion distance for oxygen and they may themselves eventually pressures generated by them were replaced consume some of the available oxygen, Examination by slow pressures of low amplitude. These slow of the fine-structure of the epithelial cells from gill 3 positive pressure waves must be generated by a revealed the membrane systems and high mitochonmusculo-skeletal system inserted on the branchial chamber lining, possibly the epimeral or dorsodrial densities typical of surfaces specialised for active ion transport and it is probable that their primary ventral muscles. A role in ventilation and perfusion of the lungs was attributed to these muscles in role is ionoregulation, together with the associated Holthuisana (Greenaway and Taylor, 1976) and processes of CO1 and ammonia excretion (Mantel and Farmer, 1983; Cameron, 1986). The reduced gills Pseudothelphusa (Innes et al., 1987). Simultaneous recordings of pressures from both of land crabs are poor respiratory gas exchange branchial chambers of Ocypode, coupled with samorgans with respect to oxygen uptake, being strongly pling of the gas space, revealed that higher PO, levels diffusion limited (Innes and Taylor, 1986a). 0. were generated when the scaphognathites were intersaratan submerged in aerated water were internally hypoxic with an a-v 0, content difference of only mittently active, than were generated by the slow 0.14 mmoljl and resorted to anaerobic metabolism, positive pressures. As these latter pressure oscillations accumulating lactate (Whiteley et al., 1989). Coupled were all suprambient, they will not result in air with this loss of effective gill surface area, the dorsal flowing in and out of the branchial chambers. Howregion of the branchial chamber in land crabs is ever, some pressure and flow changes may occur over enlarged (Diaz and Rodriguez. 1977) and the surface the invaginations in the walls of the chambers and elaborated into a “lung” for uptake of oxygen from these slow pressure variations may demand less resair @torch and Welsch, 197.5, 1984; Greenaway and piratory effort than scaphognathite movements. As qp-

762

A. H.

AL-WASSA ef

they will cause little ventilatory air-flow they may reduce convective loss of water vapour. This ventilatory mode was characteristic of crabs in dry air, when it may serve to reduce the rate of water loss as described in Pse~dothelphusa (Innes et al., 1987). Introduction of elevated levels of PCQ into the branchial chambers of Ocypode caused a pronounced ventiiatory response. A similar response was noted in H~~~thuisana(Greenaway et al., 1983) and in Pseudothelphusa (E. W. Taylor and A. J. Innes, unpublished data). Respiratory drive from chemosensory areas sensitive to CO, or the associated changes in pH are characteristic of air-breathing animals, with water-breathers responding primarily to stimulation of peripheral oxygen receptors (Greenaway et al., 1983a,b; Taylor, 1985). However, sensitivity to elevated COZ levels has been described in the aquatic, freshwater crayfish (Massabuau and Dejours, 1981), so that this adaptation for air-breathing, with the associated accumulation of CO,, is foreshadowed in water-breathing crustaceans. Greenaway et al., (1983b) suggested that the slow mode of lung ventilation may be primarily CO? sensitive whilst scaphognathite beating was primarily sensitive to variations in P02. However, in 0. saratan both elevated levels of PCO,, and increased activity levels caused increased rates of due to disturbance. scaphognathite beating and a similar response was noted in Pseudothelphusa (E. W. Taylor and A. J. Innes, unpublished data). Ventilation in submerged 0. saratun was generated by continuous, rapid scaphognathite beating. The apparent lack of ventilatory pauses characteristic of water-breathing crabs such as Carcinus (Taylor et al.. 1973), may relate to the fact that 0. saratan experiences internal hypoxia and is hypercapnic, when submerged in normoxic water (Whiteley et al., 1989). This is commonly the case in submerged land crabs. presumably due to their reduced gill areas (Taylor and Davies, 1982; Greenaway er al., 1983a; Taylor and Innes. 1988). The resultant hypoxic and hypercapnic drives may account for the continuously active ventilation of the branchial chambers with water. Heart rate in 0. saratan varied with ventilation, showing arrhythmias and slow rates of beating during ventilatory pauses and tachycardia at the onset of periods of active ventilation, The functional correlates of these relationships are clearly the effective delivery of oxygen to the tissues, with haemolymph flow related to ventilation of the respiratory surfaces, The close coordination implies controlling central nervous interactions (Wilkens, 1981; Taylor, 1982). In contrast, Burggren et al. (1985) recorded reductions in heart rate associated with bouts of active reversed ventilation in Curd~soma and attributed in intracardiac associated rise these to an haemolymph pressure. The exploratory experiments in which microspheres vvere injected into the haemocoel indicated that the lungs are preferentially perfused when 0. saratan is breathing air, while perfusion of the lungs ceases when the crab is submerged. This may be due merely to the physical presence of water in the branchial chambers exerting pressure on the lung lining and increasing the resistance to the flow of haemolymph through the sinuses in the branchioste-

al.

gites. Alternatively. there may be active control 01 lung perfusion relative to gill perfusion. Perfusion of the gills was unaffected by aerial exposure as the proportion of injected microsphercs trapped in the gills was the same for air and water-breathing crabs. This contrasts with the results of a study using microspheres on the land crab Nolt/ruisurzu tranwersu (Taylor and Greenaway, 1984). ~~~)/?~~~4j,~~~~z[~ when breathing either water or air could silnultaneo~lsly direct a proportion of the haemolymph via both respiratory routes. which operate in parallel (Innes and Taylor. 1986a) but crabs breathing water were capable of directing almost the entire flow via the gills and air-breathers almost the entire flow via the lungs. This variable response infers active control rather than the effects of passive changes in the peripheral resistance of the pill and lung circuits in the two respiratory media. Some directional flow may be obtained by the operation of valves in either circuit and valves which rectify haemolymph flow through the vessels in the gills have been described in C’arcinus (Taylor and Taylor. 1986). The constant proportion of haemolymph perfusing the gills in 0. .saratufz suggests that their role in ionoregulation, acid -base regulation, NH3 and CO; excretion is rctaincd in air. This may relate to then characteristic habitat of burrowing in damp sand on the sea shore where the high humidity and proximity of water enable retention of branchial ion exchange, as a route for excretion This suggests a reliance on bimodal (lung/gill) exchange in air with oxygen taken up over the lung surface and CO1 excreted over the gills as described for many other land crabs (e.g. Curdisoma. Wood and Randall, 1981a.b; Birgus. Greenaway et al.. 198X: for review, see Taylor and Innes. 1988). In contrast the arid-zone crab Ho~t~l~~i~~(~n~~ ~tlay r-tly entirely on exchange via the lungs when deprived of water (Greenaway et ul.. 1983b), as described for P.wdothelphusa, when inactive in burrows during the dry season (Innes and Taylor. 1986~ Taylor and innes. 1988). Measurement of the rate of oxygen uptake of 0. saratan in air, either when active immediately after insertion into the respirometcr, or inactive following 5-6 hr undisturbed to allow them to settle. revealed that it varied from around 5 ml 0, :hr in active crabs down to about half this value in inactive crabs. These values fall either side of the weight specific t0, measured at 25 C on 0. .YUJYJ~(UI by Eshky c’f O, values for acttve 0. ,~f~~~~~~~~ fill on the regression lines constructed through the measured variations in PO, with mass for nine species of tropical air-breath&g crabs, described by lnnes and Taylor (1986b). The settled values for 0 .wratatI. however. are approximate1~ double the value ohtained by interpolation on their regression line for settled animals, indicating that the present data was taken from crabs which were either more active than these previous species (which included the closely related 0. quadrata) or that PO, in these other species was depressed, possibly by hypoxic conditions in the closed unventilated respirometers. In the present study, both active and settled 0. saratan had a respiratory quotient {&)) for CO,:O, exchange of about 0.55. This value is unusually low in comparison to the values of 0.7 -1 0 normally

Respiration in the ghost crab described for animals metabolising aerobically using fat, carbohydrate and/or lipid as a source of metabolic energy. Similar low values for crustaceans have been reported previously (Bliss, 1953; Wood and Randall, 1981b; Wood and Boutilier, 1985; Wheatly et al., 1986) and it has been suggested that they could arise from a portion of excreted CO* being diverted to deposition of carbonates in the exoskeleton (Wood and Randali. 1981a; Wood and Boutilier, 1985). Certainly, CO, exchange is affected by the calcification of the exoskeleton at the time of the moult, with net COZ uptake reported in CaNinectes several days after ecdysis (Cameron and Wood, 1985). However, it seems less likely that CO, will be retained between moults and McMahon and Burggren (1988) considered these low values for RQ to be abnormal, possibly resulting from the build-up of CO, in closed respirometry systems. In the present study this problem was avoided by ventilation of the respirometer with CO: free air prior to readings and we consider that the low values for RQ obtained indicate a real depression of the rate of CO, excretion into the surrounding air in 0. saratan. Their low RQ is further supported by the directly measured PO, and PC02 levels in samples of air withdrawn from the branchial chambers of 0. saratan (Whiteley er al., 1989). These indicated a mean respiratory exchange ratio (R) in the branchial chamber air of 0.42 It 0.06(6) which compares closely in value with the range of RQ values measured for the whole animals in the present study. The value for R is towards the bottom of the range of RQ possibly due to excretion of some CO* via a branchial route over the gills. into water retained in the branchial chambers. In conclusion, 0. saratan seems well adapted for air-breathing in humid air, with the lungs taking over from the gills as the primary site for oxygen uptake but some CO2 output probably still occurring over the reduced gills, which are perfused with haemolymph in submerged and air-exposed crabs. Ventilation of gills and lungs is by means of the scaphognathites in active or submerged crabs but these are replaced by slow, positive pressures when the crabs are settled in air, such as would be the case when in their burrows during the day. Under these circumstances the lungs are preferentially perfused with haemolymph. Ventilation in air is sensitive to CO2 levels, with increased rates of scaphognathite beating in response to high COZ. These patterns of ventilation and perfusion can result in high PO, levels in the haemolymph in air. but the crabs are hypoxic when submerged and relying on their reduced gills for respiratory gas exchange (Whiteley et al., 1989). In common with other crustaceans, 0. saratan has a low RQ when breathing air, indicating some internal retention of CO,, possibly associated with the formation and maintenance of the exoskeleton, as well as excretion of some COZ by the branchial route into water bathing the gills. SUMMARY

Ocypode saratan is a typical air-breathing land crab, having a vascularized branchiostegal wall specialized to function as a lung for gas exchange in air, with a folded surface bearing invaginations and a

763

minimum diffusion distance of 1 pm. The reduced gills, which have a diffusion distance of about 5 pm, may be specialized for ionoregulation and the excretion of CO2 and ammonia. When submerged, or in humid air, ventilation was by means of the scaphognathites. These beat at mean rates of 53 beats/min in submerged crabs, at 218 beats/min when active in air, and at 43 beats/min, with occasional ventilatory pauses, when inactive in air. Reversals were associated with rapid scaphognathite beating (mean & = 131 beatsimin). Crabs inactive in dry air, replaced scaphognathite beating by slow pressure variations (mean rate, 9.5 beats/min) in the branchial chambers. Exposure to increased levels of CO, during air-breathing caused eIevated rates of scaphognathite beating (235 beatsimin). Mean heart rate was 102 beats/min in active crabs in humid air, but fell to 34 beats/min in inactive crabs. Heart rate varied with changes in ventilation. An exploratory experiment on the distribution of radiolabeiled microspheres indicated that the gills were perfused both in water and in air whilst the lungs were preferentially perfused in air, with a lung to gill ratio of about 4: I, but were not perfused in submerged crabs. Measurement of oxygen uptake and CO, production of individual crabs in air revealed that mean C’O1 fell from 5.0 to 1.8 ml O,/hr and VCO, from 2.7 to 1.O ml CO,/hr . after 5-6 hr in the respirometer. Mean RQ was 0.54 in active crabs and 0.56 in settled crabs, indicating CO2 retention. Acknowledgements---The

authors wish to thank Dr Steve

Oakley, Department of Marine Sciences, King Abdulaziz University, Jeddah, for catching the crabs and despatching them safely to Birmingham. N.M.W. was supported by an SERC research studentship. REFERENCES

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