GILL VENTILATION AND THE ROLE OF REVERSED RESPIRATORY CURRENTS IN CARCINUS MAENAS (L.)

J. Exp. Biol. (1964), 41, 299-307 299 With 7 text-figures Jointed in Great Britain GILL VENTILATION AND THE ROLE OF REVERSED RESPIRATORY CURRENTS IN CARCINUS MAENAS (L.) BY K. D. ARUDPRAGASAM* AND E. NAYLOR Department of Zoology, University College of Sioansea (Received 22 August 1963) INTRODUCTION Respiratory currents in decapod Crustacea are produced by the rhythmic action of the exopodite (scaphognathite) of the maxilla which, in Carcinus and other crabs, is a flattened structure lying in the pre-branchial chamber in a plane roughly parallel to the direction of current flow (Borradaile, 1922). In Carcinus the action of the scaphognathite normally draws water into the branchial chamber at the ventral edge of the branchiostegite, through openings located immediately anterior to the bases of the chelipeds (Milne-Edwards's openings) and through similar openings associated with the bases of the walking legs, whilst exhalant water passes out through paired openings situated between the antennae and the mouth parts (Bohn, 1897; Lim, 1918; Borradaile, 1922). Periodically, however, as in several decapods (Bohn, 1897), the respiratory current is reversed, apparently by the reversed action of the scaphognathite, and the role of these current reversals seems to be not fully understood. Borradaile (1922) considered that reversals of the respiratory current in Carcinus had no function other than those of cleaning the gills and of irrigating the gills with air when the crab was in very shallow foul water. This was perhaps reasonable in view of earlier observations which had demonstrated a predominantly reversed respiratory current in Corystes when buried in sand (Garstang, 1896). On the other hand, Bohn (1902) suggested that current reversals were highly regular and that they had a physiological advantage in resting the muscles of the scaphognathite. Present experiments were therefore designed to reconcile these two points of view. Previous observations on reversals of the respiratory current were of very short duration and involved the use of carmine or ink suspensions which might be expected to influence a possible spontaneous pattern of reversal. In the present investigation, therefore, continuous records of the normal pattern of gill ventilation in Carcinus maenas were obtained by recording changes of pressure within the gill chamber under a variety of experimental conditions. The results obtained make it possible to extend the interpretations of gill ventilation and the function of reversed respiratory currents given by Borradaile (1922). MATERIALS AND METHODS The method used to obtain continuous records of the direction offlowof respiratory currents in Carcinus is based upon the observation that reversal of the normal forward flow is associated with an abrupt increase in pressure within the branchial Present address: Department of Zoology, University of Ceylon, Colombo 3, Ceylon.

3 K. D. ARUDPRAGASAM AND E. NAYLOR chamber. This can very easily be demonstrated by drilling a small hole through the dorsal surface of the carapace of a crab to expose the branchial chamber and then placing the animal in a vessel with just sufficient sea water to cover the hole. Observations show that for most of the time the animal pumps water forwards and draws water into the branchial chamber through the hole. Periodically, however, the flow of the respiratory current is reversed and water rushes into the epibranchial space and out through the hole in the carapace. Each reversal, therefore, clearly causes a sudden increase in pressure within the branchial chamber and it is possible to record these increases using the apparatus illustrated in Fig. i. This consists of a plastic cannula fitted into the gill chamber and a tambour which records changes in pressure. Recording arm A Tambour r~?.-?y7?t?<.-,,*j&.~t?-.;-.---"- r - J - i v?-:*> Animal chamber I Ctnnula* Fig. i. Apparatus for recording pressure changes in the gill chamber of crabs. The cannula is about 25 mm. long with a stem diameter of about 3 mm. and a 2-5 mm. bore. At one end there are two projections which hook beneath the edge of the hole cut in the carapace of the crab. The stem of the cannula is threaded and carries two plastic nuts, and a length of polythene tubing of internal diameter about 5 mm. connects the free end of the cannula to the tambour. A paper rider rests upon the thin rubber diaphragm of the tambour and supports a recording arm which writes upon the smoked drum of a variable-speed kymograph. A T-piece, funnel, and pinch clips are included in the apparatus to facilitate filling the system with water and getting rid of air. When fitting the cannula to a crab, a hole is drilled through the dorsal surface of the carapace into the gill chamber at a point about two-thirds of the distance along the length of the crab and about half-way between the mid-line and the lateral edge. The hole is then notched slightly on one side so that the projections on the cannula can be worked in. After it has been introduced, the cannula is then rotated through 90 0 so that the projections fit firmly against the inner surface of the carapace and help to lock it in position. A tight seal is ensured by fitting a thin piece of rubber between the carapace and the nuts which are then screwed down. In most cases the cannula was fitted on to a crab at least 24 hr. before it was used in an experiment and could be

Respiratory currents in Carcinus maenas (L.) 301 left in situ for long periods of time without any apparent ill effects on the animal. In most cases the crabs were kept in aquaria for some days before being used in the experiments but in some long-term experiments freshly collected crabs were used. (a) '. I I I L TTTTTTTT Minutes Fig. 2. Pressure changes in the gill chamber of Carcinus at three stages during a prolonged experiment: (a) 0-15 min., (6) 120-135 min., (c) 195-210 min. Spikes indicate reversals of the respiratory current. RESULTS Ventilation pattern in clean sea water Representative sections of a kymograph trace obtained using a crab kept in about 2-5 1. of fresh sea water are illustrated in Fig. 2. This shows that for most of the time a fairly steady pressure level was maintained within the branchial chamber apparently associated with the normal forward flow of respiratory water. At fairly regular intervals, however, there were marked but short-lived increases in pressure appearing on the trace as spikes and indicating regular reversals of the respiratory current, each lasting for only a few seconds. The return to forward flow was occasionally accompanied by a drop in pressure to below the 'normal' level, presumably owing to the temporary partial evacuation of the gill chamber by the sudden vigorous forward action of the scaphognathite. The normal forward pressure level was, however, quickly reestablished. In the early stages of each such experiment, reversals occurred about once every minute, falling off to about once every 6 min. at the end of 6 or 7 hr. (see also Fig. 3 a). Moreover, comparison of the heights of the spikes on the traces at different stages of the experiment (see Fig. 2) indicated a gradual weakening in the force of reversals which paralleled the drop in frequency. Since the progressive fall in the rate of current reversal in crabs kept in a limited volume of water was possibly related to the accumulation of carbon dioxide or depletion of oxygen, further experiments were carried out on crabs kept in water covered by a film of liquid paraffin and in others kept in continuously circulating sea water. In crabs kept in water covered by liquid paraffin (Fig. 3 b) the drop in reversal

K. D. ARUDPRAGASAM AND E. NAYLOR rate was even more marked than in crabs kept in open water (Fig. 3 a), and there was a correspondingly greater decrease in the strength of the reversals. Moreover, reversals ceased altogether after 6 or 7 hr., by which time there was also no evidence of respiratory pumping, which can normally be deduced from movements of the antennules and mouth parts. The suspension of reversals was always accompanied by the cessation of pumping and the animals usually soon died under these conditions. On the other hand, if at the end of 6 hr. the paraffin layer was removed and the water aerated, the animals soon became active again and established a high rate of reversal. 30r 30r o 1 10 (e) 20 It W Hlw 10 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Duration of experiment (hr.) Fig. 3. Changes in the frequency of reversal of the respiratory current of aquarium crabs kept (a) in 2 1. of sea water, (b) in 2 1. of sea water covered with liquid paraffin, and (c) in flowing sea water, and (d) of a freshly collected crab kept in flowing sea water, (HW indicates the times of high tide on the shore outside.) In crabs kept in running sea water the pattern of current reversal varied according to whether the specimens were freshly collected or whether they had been kept previously in an aquarium. In crabs from an aquarium (Fig. 3 c) reversal frequency remained fairly high for long periods and, though in some experiments reversals were completely suspended for up to 2 hr., the crabs usually resumed a high rate of reversal. This was in contrast to the apparent irreversibility of the fall-off in reversal rate in crabs kept in a small volume of un-aerated water.

Respiratory currents in Carcinus maenas (L.) 303 The significance of these periodic suspensions of pumping activity can perhaps be explained since they seem to occur under natural conditions according to the state of the tide on the shore from which the crabs were collected. Freshly collected crabs showed a rhythm of variation in reversal frequency during the first 24 hr. or so of an experiment (Fig. 3 d), with highest reversal rates occurring at about the times of high tide and with the lowest rates occurring about low tide. 10 mln. M i^u u tt^^ 30 min. Fig. 4. Pressure changes in the gill chamber of Carcinus (a) when partially exposed to air in very shallow sea water, and (6) when partially buried in sand. Gill vejitilation pattern when partially exposed to air In their natural environment crabs are often stranded in shallow pools which may become rapidly deoxygenated, but they are able to survive by raising the front of the body above water and bubbling air into the gill chamber. This can also be observed in crabs kept in shallow tanks in the laboratory and a section of a typical trace obtained from a crab under these conditions is shown in Fig. 4 a. The lower pressure levels indicate periods during which the scaphognathite beat forwards, for if the front of the body was raised water could usually be observed to stream out of the exhalant openings at those times. The periods of higher pressure and higher reversal frequency were always observed to be associated with 'bubbling' by the crab. At these times the animal often drew in air through the normally exhalant openings and force d it through the water retained in the branchial chamber to emerge as a stream of bubbles from Milne-Edwards's openings and other normally inhalant openings. Thus when the animal is partially exposed to air, the reversed action of the scaphognathite apparently plays a major part in aerating water retained in the branchial chamber. 20 Exp. Biol. 41, 2

304 K. D. ARUDPRAGASAM AND E. NAYLOR Gill ventilation pattern when partially buried in sand Cardnus is sometimes found partially buried in sand on the shore and it might be expected to show a different pattern of gill ventilation at these times. The animals did not always bury themselves in sand provided in the animal chamber but the pattern obtained in one of several successful experiments is shown in Fig. \b. Immediately after burying there was a lowering of pressure within the branchial chamber, presumably since little water entered directly into the posterior parts of the gill chamber. Moreover, since the posterior gills would be deprived of water in this way one would also expect the increases in the frequency of reversals which are apparent in Fig. 4b since these would facilitate irrigation of the posterior gills. Thus when buried Cardnus clearly ventilates its gills by alternately drawing in and evacuating water through the normally exhalant openings, though it apparently continues to take in some water through Milne-Edwards's openings. 60 Carmine added 50 40 S. 30 20 10 0 1 2 3 4 5 Time (hr.) Fig. 5. Effects of carmine on the frequency of reversal of the respiratory current (6, c and d) compared with a control (a). Effects of suspended material on ventilation pattern Fig. $b, c, d shows variations in reversal frequency in three crabs after the addition of carmine particles to the water, and clearly the overall pattern was very similar to that in a control animal kept in clean water (Fig. 5 a). There were occasional shortlived increases in reversal rate but no lasting responses to the presence of suspensions of carmine, charcoal or fine sand. Effects of oxygen depletion and increased carbon dioxide Throughout a number of experiments estimations of oxygen tension and total carbon-dioxide concentrations (by ph) were made using methods described (Arudpragasam & Naylor, 1964) and in most cases reversals of the respiratory current were

Respiratory currents in Carcinus maenas (L.) 305 suspended by the time oxygen concentrations had fallen from 6 to about 2 c.c./l. and carbon-dioxide concentrations had risen from 46 to about 51-5 c.c./l. More precise observations on the relationship between current reversal and external gas tensions were made by bubbling nitrogen through the water to reduce abruptly the oxygen tension and, in other cases, by adding water saturated with carbon dioxide to increase the carbon-dioxide concentration. The results of typical experiments are illustrated in Fig. 6 which suggests that in stagnant water the rate of reversal of the respiratory current appears to be inhibited by carbon-dioxide accumulation rather than by oxygen depletion (Fig. 6b). Oxygen depletion in the absence of excess carbon dioxide clearly enhanced the rate of reversal (Fig. 6 a). Fig. 6. Effects of (a) reduced oxygen, and (b) increased carbon dioxide on the frequency of reversal of the respiratory current. The path of water through the gill chamber The flow of water through the gill chamber during forward and reversal phases appears to be as illustrated in Fig. 7. It is deduced from an examination of the gills of crabs kept in a suspension of carmine particles and also from observations on crabs in which the gills were exposed by removing part of the branchiostegite (see p. 300). In Fig. 7 it should be noted that the gills are attached ventrally and are reflexed upwards and inwards so that the outer (ventral) surfaces are convex and the inner (dorsal) surfaces are concave. Gills 1-5 are smaller and are situated medial to gill 6 (see Fig. 7). During forward flow most water appears to enter the gill chamber at the bases of the chelipeds and irrigates gills 1-6. Of the water which enters at the bases of the peraeopods some flows immediately forwards across the lower parts of the outer surfaces of gills 7-9 but most enters the interbranchial spaces and flows through the gill lamellae.

306 K. D. ARUDPRAGASAM AND E. NAYLOR This water thenflowsforwards, either across the inner surfaces of the gills or above the gills. The inner (dorsal) surfaces of the gills are also irrigated by the forward flow of water entering at the base of the last peraeopod. The upper parts of the outer surfaces of the gills appear to be irrigated mainly during the reversed flow by water which is diverted from the stream entering at the bases of the chelipeds and water which enters through the anterior ' exhalant' opening. Reversed currents Forward currents Fig. 7. Paths of water through the gill chamber of Carcimu. (Branchiostegite and limbs removed: c, cheliped; g 6-9, gills 6-9; m, 3rd maxillipede; p 1-4, peraeopods 1-4; s, scaphognathite.) DISCUSSION From the results presented above, it seems fairly evident that reversals of the respiratory current in Carcinus primarily serve to irrigate the dorsal aspects of the more posterior gills by pumping water into the epibranchial chamber. The presence of suspended particles in water often results in a temporary increase in the rate of current reversal as was also noted by Lim (1918) and Borradaile (1922), but reversal does not seem to be primarily brought about by the presence of detritus as was suggested by Borradaile (1922). Indeed, current reversal appears to be a spontaneously rhythmic phenomenon, as was suggested by Bohn (1897, 1902), and appears to form an integral part of a rhythm of gill ventilation. This is further borne out by the fact that reversal frequency seems to increase spontaneously at the times of high tide (see p. 303) in a manner which parallels a rhythm of respiration described elsewhere (Arudpragasam & Naylor, 1964). Moreover, the rate of reversal of the respiratory current varies consistently with the gas tensions of the water in which an animal is kept (p. 305). In addition, this interpretation of current reversal seems to be reasonable in view of the confirmation by present experiments of earlier observations which have already shown that reversal plays a considerable part in aerating the gill chamber in Carcinus which are partly buried in sand (Lim, 1918) or partly exposed to air (Bohn, 1897; Borradaile, 1922).

Respiratory currents in Carcinus maenas (L.) 307 Further evidence of a relationship between current reversal and gill ventilation has been demonstrated in experiments in which it has been shown that strong and frequent reversals are associated with rapid forward pumping of respiratory water whilst a weakening and slowing of the reversals is correlated with a reduction in ventilation volume (Arudpragasam & Naylor, 1964). Those experiments have also shown that as much as 80% of the total volume pumped through the gill chamber enters through Milne-Edwards's openings, anterior to gill 7, which itself might suggest that there is a mechanism involved in passing some of this water backwards across the large posterior gills. Indeed, some authors have previously supposed this to be so and have suggested that water is deflected passively backwards during the normal forward flow (Milne- Edwards, 1839; Claus, 1885; Pearson, 1908). Present results indicate, however, that water is not passively deflected backwards across the gills, but is actively pumped into the dorsal part of the gill chamber at each reversal of the respiratory current. SUMMARY 1. A method is described for obtaining continuous records of the direction of flow of the respiratory current of Carcinus by recording changes of pressure in the gill chamber. 2. Frequent and rhythmic reversals of the normally forward-flowing respiratory current appear to irrigate the upper surfaces of the posterior gills and do not serve primarily to clean the gills. 3. The rate of reversal of this respiratory current increases spontaneously at times of high tide. It decreases under conditions of carbon-dioxide accumulation and increases under conditions of oxygen depletion in the absence of excess carbon dioxide. 4. The pattern of gill ventilation varies according to whether crabs are totally in water, partially buried in sand, or in very shallow water. REFERENCES ARUDPRAGASAM, K. D. & NAYLOR, E. (1964). Gill ventilation volumes, oxygen consumption and respiratory rhythms in Carcinus maenas (L.). J. Exp. Biol. 41, 309-321. BOHN, G. (1897). Sur la respiration du Carcinus maenas Leach. C. R. Acad. Sci., 135, 441. BOHN, G. (1902). Des mecanismes respiratoires chez les Crustacea Decapodes. Bull. Sci. Fr. Belg. 36, I78-554- BORRADAILE, L. A. (1922). On the mouthparts of the shore crab. J. Linn. Soc. (Zool.), 35, 115-42. CLAUS, C. (1885). Neue BeitrSge zur Morphologic der Crustaceen. Arb. zool. Inst. Univ. Wien, 6(i), 1-108. GARSTANG, W. (1896). The habits and respiratory mechanism of Coryttes castivelaunus.j. Mar. biol. Ass. U.K. 4, 223-32. LIM, R. K. S. (1918). Experiments on the respiratory mechanism of the shore crab (Carcinus maenas). Proc. Roy. Soc. Edinb. 38, 48-56. MILNE-EDWARDS, H. (1839). Recherches sur le mecanisme de la respiration chez lea Crustacea. Ann. Sci. Nat. II, 129-42. PEARSON, J. (1908). Cancer. Liverpool Mar. Biol. Comrn. Mem. 16, 209 pp. London.