Forster, M. E.

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HAEMOLYMPH OXYGENATION AND OXYGEN CONSUMPTION IN A HIGH SHORE CRAB, LEPTOGRAPSUS VARIEGATUS, BREATHING IN AIR AND WATER M.E. FORSTER Department of Zoology, University of Canterbury, Private Bag, Christchurch, New Zealand. (Received 2 September, 1991; revised and accepted 7 September, 1991) ABSTRACT Forster, M.E. (1991). Haemolymph oxygenation and oxygen consumption in a high shore crab, Leptograpsus variegatus, breathing in air and water. New Zealand Natural Sciences 18: 19-23. Post-branchial haemolymph was sampled in specimens of the high shore crab, Leptograpsus variegatus, held under two sets of conditions. The partial pressure of oxygen (Pao^ in crabs immersed for 24 h at 20-22°C in running sea water measured 15.9 ± 1.2 mmHg (2.1 kPa). After 24 h in air at the same temperature the Pao2 measured 24.9 ±1.6 mmHg. Pvo2 (pre-branchial) haemolymph measured 6.6 ±1.5 mmHg in sea water. The higher Pao2 in air is associated with the animal's observed preference for emersion. The low apparent diffusing capacity of the gas exchange system may minimise passive water losses in air. Resting oxygen consumptions (Vo^ were also measured in the two media and rates were similar. A 100 g crab would have a Vo2 ofSmlO^h"1. KEYWORDS: crustacean - haemolymph - oxygen - Vo2. INTRODUCTION Leptograpsus variegatus, the purple or large shore crab is one of the largest invertebrate ani-mals of the New Zealand sea shore, being found well above high-water marks. An active predator it shelters in rock crevices by day, emerging at night to forage on the shore (Trenery 1982). One indicator of its adaptation to a terrestrial existence is the acid-base regulating ability of L. variegatus, which is not dependent upon seawa-ter for restoration of its haemolymph pH following exhausting exercise (Forster et ai. 1989). This contrasts with the physiology of the smaller, sym-patric species Cyclograpsus lavauxi (Waldron etal 1986). When forced to exercise to exhaustion on a treadmill in air both species showed a pronounced acidosis, but only L. variegatus was able to restore pH to pre-exercise values when held in both air and seawater. L. variegatus is generally found emersed when placed in a container in which it has access to either sea water or air (Forster et al 1989). As a gill breathing animal it is of interest to know whether gas exchange is as efficient in air as it is in water. Gas exchange might be affected by several factors, including differences in the ventilation of the gills, their perfusion with haemolymph and their diffusive capacity in the two media. The partial pressure of oxygen in the post-branchial haemolymph (Pao^ is a good indicator of the efficiency of gas exchange. The more closely this approaches the Po2 of the branchial water or air, the more efficient an exchanger the gill is. Gener-ally, in crabs that use their gills for air-breathing, haemolymph Pao2 is lower on emersion than it is when immersed (Dejours & Truchot 1988). Gill lamellae may collapse or coalesce when unsup-ported by water, thus reducing the effective gill area available for gas exchange. Then, with time in air there can be a separation of the gill filaments allowing improved gas exchange (Innes et al 1986). In these experiments gas exchange in the two media was investigated by measuring both the Pao2 ofthe haemolymph and oxygen consumption 20 New Zealand Natural Sciences 18 (1991) (Vo^ of crabs held for 24 h either in air or submerged in seawater. Comparisons of these two parameters should indicate whether oxygen up-take in resting crabs is limited in either medium. MATERIALS AND METHODS Adult L. variegatus of both sexes were col-lected at night from rocky shores in the vicinity of Leigh (36°6'S, 174°48'E) and were taken to the Leigh laboratory and placed into containers sur-rounded by running seawater of relatively constant temperature (range 20 - 22°C). The crabs were either totally immersed in running seawater with-out access to air, or were placed in 151 containers in which there was no access to seawater. In both cases the animals were held in these conditions for 22 to 24 h before blood sampling. Post-branchial haemolymph was sampled by piercing the carapace above the heart with a 21 gauge needle and withdrawing approximately 150 ul of haemolymph into a 1 ml syringe. The sample was then immediately introduced into a Strathkelvin MC100 microcell, with IL 1302 oxy-gen electrode and Strathkelvin oxygen meter (model 781). Samples were taken without remov-ing the animal from the medium in which it had been held. After sampling the puncture holes were sealed with vaseline. The crab was then placed into the alternative medium (sea water if previously emersed and vice versa) and was left for a further 22 to 24 h before a second set of haemo-lymph samples were taken. As a check that the cardiac puncture was indeed sampling post-bran-chial blood, a pre-branchial haemolymph sample was taken from seven immersed crabs, through a needle that punctured the arthrodial membranes at the bases of the legs. Crabs were weighed after cardiac puncture, but to minimise handling disturbance only super-ficial water was removed using paper towels. The weight changes in the two media are given as indicators of desiccatory stresses and to augment points in the discussion, but do not represent a definitive statement on water balance inL. variega-tus. Where Vo2 was to be measured the animals were held from between 30 and 36 h in the appro-priate medium. Vo2 was measured using the IL1302 electrodes and Strathkelvin meters and sealed, water-jacketed, perspex respirometers containing a crab in either seawater or air. For Vo2 in air the Po2 was measured directly in the cham-ber and the output from the oxygen meter was displayed on a Servoscribe recorder. For Vo2 in sea water, 250 ul samples of the medium were taken at intervals and injected into the microcell to determine the Po2 drop over time. The biological oxygen demand ofthe seawater was determined in respirometers without crabs amd this was sub-tracted from the values obtained with the animals in situ. There was no attempt to assess spontane-ous activity in these measurements of Vo2. A total of 49 animals (28 males) were used, ranging in body weight (wt.) from 26.6 to 202.4 g. The mean wt. was 86.1 ± 5.6g(± SE). Most ofthe female crabs carried eggs. After use, the animals were returned to the shore. RESULTS The Po2 of post-branchial haemolymph was higher in air than in sea water (Table 1). Sampling some individuals twice, once in each medium, confirms this difference. In immersed crabs the Po2 of pre-branchial haemolymph was lower than that of post-branchial haemolymph (Table 1), indicating that the cardiac puncture technique did indeed collect arterialised blood. Care was taken Table 1. Oxygen partial pressures in haemolymph samples taken from Leptograpsus variegatus maintained for 24 h in either seawater or air. "n" values in brackets. Po2 values in mmHg. Pao2 Pvo2 Emersed (23) 24.9 ± 1.6 Immersed (16) 15.9 ± 1.2* 6.6 ± 1.5§ (7) Emersed1 (12) 24.5 ± 2.1 Immersed1 (12) 15.4 ± 1.3* 1A subset of the larger group, where haemolymph was sampled in the same individual after 24 h in both media. * Significantly different from emersed crabs, P < 0.001 § Significantly different from arterial values in sea water, P < 0.001. M.E. Forster: Haemolymph oxygenation and Vo2 in Leptograpsus 21 to collect haemolymph without applying strong negative pressures and indeed once the pericar-dium was punctured the haemolymph generally flowed freely, particularly in water-breathing crabs. It is not likely that the samples were deoxy-genated as they were collected. Crabs transferred to air lost weight and those immersed in sea water after aerial exposure gained weight. The mean change in wt. (positive or negative) was 4.8 ± 0.4% (n = 24). Though much of this weight change might be attributed to water loss from the branchial chambers it could also include urinary losses. Oxygen consumption in air and seawater is graphed in Fig. 1. The size range of animals used in measurements of Vo2 in the emersed group was too restricted to fit a regression line. The regres-sion line for the immersed group indicates that a 100 g crab would have had an oxygen consumption of c. 5 ml h1. The mass exponent was 0.63 for the water breathers, whose wt.s ranged from 28.3 to 170.8 g. DISCUSSION The higher Pao2 values in emersed L. variega-tus might be expected from its observed prefer-ence for this medium, rather than sea water. The difference in Pao2 values between L. variegatus breathing in air and sea water could reflect a difference in the relative importance of alternative sites of oxygen uptake. Methods of aerial ventila-tion in L. variegatus have not been described, but the appearance of the branchiostegal membranes suggests that these might function as a lung (H.H. Taylor, Department of Zoology, University of wt.(g) Figure 1. Oxygen consumption in either sea water (•) or air (#) in different sized individuals of the high shore crab Leptograp-sus variegatus. The regression line for the group immersed in sea water is given by the formula: log Vo2 = -0.56 + 0.625 log wt. (r2 = 0.85) where Vo2 is expressed as ml 0 2 (STPD).h"1 and wt. is the wet weight of the animal in grams. 22 New Zealand Natural Sciences 18 (1991) Canterbury, pers. comm.). Another amphibious grapsid crab, Hemigrapsus nudus, which occurs lower on the shore than L. variegatus, has a sub-stantial venous circulation in its branchiostegal lining (Greenaway & Farrelly 1990). What is noteworthy is that Pao2 values in both media are low. Pao2 tends to fall in intertidal decapods when emersed (DeFur 1988). In the intertidal shore crab Carcinus maenas Pao2 in emersed animals was only 40% of the value when immersed in seawater, but even the Pao2 in emersed crabs measured 41 mmHg (Dejours & Truchot 1988). Higher Pao2 values are reported for species that are primarily either aquatic or air breathing (McMahon 1981, Taylor & Innes 1988). The Pao2 values inL. variegatus are slightly higher than those reported for Coenobita clypeatus, an anomuran crab also using Phyllobranchiate gills for aerial gas exchange (McMahon & Burggren 1979, Innes & Taylor 1986). It is possible that the mechanisms that result in the low Pao2 in L. variegatus also minimise passive water loss in air in the species, which is found well above the high-tide mark. Crustaceans generally are suscep-tible to desiccatory water losses (Innes et al 1986). The c. 5% weight change after 24 h of emersion suggests that L. variegatus might also be suscep-tible to dehydration, although in its natural habitat it wedges itself into rock crevices, a behaviour which may limit water losses. As mean water content has not been determined for L. variegatus, it is not possible to convert the measured weight losses and gains to a change in total body water (see Innes et al. 1986). For immersed L. variegatus it is possible to calculate the apparent diffusion limitation index for gas exchange (Piiper 1982). This is given by the formula: V = ( P i 0 2- . P a 0 2) / ( P i °2 - P V 0 2) -For immersed gill-breathing crustaceans, Ldiff values range from 0.5 to 0.8 (Taylor & Innes 1988). The value for immersed L. variegatus, based on mean arterial and venous Po2s is 0.937, similar to the values reported for gill-breathing land crabs in air and indicating a marked diffusion limitation. We cannot calculate Ldiff for emersedL. variegatus, as Pvo2 was not measured in air. However, as Pao2 values are also low, Ldiff must be high inL. variega-tus in air. We should note that the Ldi£f values referred to above may overestimate the true diffu-sion limitation if the branchial chambers are not well ventilated. The low diffusing capacity in the gas exchange system of L. variegatus may not compromise oxy-gen uptake at rest. If the haemolymph contains significant amounts of the respiratory pigment, haemocyanin, and its affinity for oxygen is high, then it may well be that oxygen transport by the haemolymph is not disadvantaged by the low Pao2 values. However the Po2 gradient from haemo-lymph to myocyte mitochondria, for example, must be low, which implies a constraint upon aerobic metabolism. Measurements of Pao2 in active individuals might indicate whether the ap-parent limitation to the diffusion of oxygen at the gas exchange surfaces can be lessened. Activity can change Pao2 in other crustaceans, but some species show a fall, rather than a rise (Wood & Randall 1981). Vo2 at rest in Leptograpsus at 20-22°C was similar to that recorded for other brachyurans at these temperatures (McMahon 1981). The high shore crab Cyclograpsus lavauxi also consumes oxygen at rest at a similar rate when emersed and immersed (Innes et al 1986). Resting Vo2 de-creased in two intertidal species on emersion (Burnet & McMahon 1987). Vo2max values would be informative and would indicate the animal's aerobic scope. Certainly L. variegatus is an active predator, foraging widely over the shore (Trenery 1982 and pers, obs.), which suggests that at such times it has a high rate of expenditure of metabolic energy. ACKNOWLEDGEMENTS These experiments were performed at the University of Auckland's Leigh Marine Labora-tory. I would like to thank Dr. Bill Balantyne for his help and hospitality in making the work pos-sible and Professor Rufus Wells for the loan of an oxygen meter and recorder. The University of Canterbury provided financial assistance for the project. REFERENCES Burnet, L.E. & McMahon, B.R. (1987). Gas exchange, hemolymph acid-base status, and the role of branchial water stores during air M.E. Forster: Haemolymph oxygenation and Vo2 in Leptograpsus 23 exposure in three littoral crab species. Phys-iol Zool. 60: 27-36. DeFur,P.L. (1988). Systemic respiratory adapta-tions to air exposure in intertidal decapod crustaceans. Amer. Zool 28:115-124. Dejours, P. & Truchot, J-P. (1988). Respiration ofthe emersed shore crab at variable ambient oxygenation. /. Comp. Physiol. 158: 387-391. Forster, M.E., Waldron, F.M. & Taylor, H.H. (1989). Recovery from exhausting exercise in a bimodally breathing crab, Leptograpsus vari-egatus (Decapoda: Grapsidae). /. Exp. Mar. Biol Ecol 127:165-173. Greenaway, P. and Farrelly, C. (1990). Vascula-ture of the gas exchange organs in air-breath-ing brachyurans. Physiol. Zool. 63:117-139. Innes, AJ., Forster, M.E., Jones, M.B., Marsden, LD. & Taylor, H.H. (1986). Bimodal respira-tion, water balance and acid-base regulation in a high-shore crab, Cyclograpsus lavauxi H. Milne Edwards. /. Exp. Mar. Biol Ecol 100: 127-145. Innes, AJ . & Taylor, E.W. (1986). The evolution of air-breathing in crustaceans: a functional analysis of branchial, cutaneous and pulmo-nary gas exchange. Comp. Biochem. Physiol. 85A: 621-637. McMahon, B.R. (1981). Oxygen uptake and acid-base balance during activity in decapod crus-taceans. In Locomotion and energetics in ar-thropods (ed. CF. Herreid & C.R. Fourtner), pp. 299-335. Plenum Press, New York. McMahon, B.R. & Burggren, W.W. (1979). Res-piration and adaptation to the terrestrial habi-tat in the land hermit crab Coenobita clypeatus. J. Exp. Biol. 79: 265-281. Piiper, J. (1982). A model for evaluating diffusion limitation in gas-exchange organs of verte-brates. In^4 Companion to Animal Physiology (ed. C.R. Taylor, K. Johansen & L. Bolis). pp. 49-64. Cambridge University Press, Cam-bridge Taylor, E.W. & Innes, AJ. (1988). A functional analysis of the shift from gill- to lung-breath-ing during the evolution ofland crabs. Biol. J. Linn. Soc. 34: 229-247. Trenery, D. (1982). The feeding ecology of the rocky shore crab, Leptograpsus variegatus. M.Sc. Thesis, Auckland University, New Zealand. Waldron, F.M., Taylor, H.H. & Forster, M.E. (1986). Acid-base disturbances following exercise in a high shore crab, Cyclograpsus lavauxi. N.Z. J. Mar. Freshwater Res. 20: 479-487. Wood, CM. & Randall, DJ. (1981). Haemo-lymph gas transport, acid-base regulation, and anaerobic metabolism during exercise in the land crab (Cardisoma carnifex). J. Exp. Zool. 218: 23-35. 

Haemolymph oxygenation and oxygen consumption in a high shore crab, Leptograpsus variegatus, breathing in air and water

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Haemolymph oxygenation and oxygen consumption in a high shore crab, Leptograpsus variegatus, breathing in air and water

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