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Responses of adult mud crabs (Scylla serrata) (Forskal) to salinity and low oxygen tension
Camp. Biochem. Physiol. Vol. 86A, No. I, pp. 43-41, 1987 Printed in Great Britain
0300-9629/87 $3.00 + 0.00 Perpmoo Journals Ltd
RESPONSES OF ADULT MUD CRABS (SCYLLA SERRATA) (FORSKAL) TO SALINITY AND LOW OXYGEN TENSION J. DAVENPORT* and T. M. WONG~ *Animal Biology Group, Marine Science Laboratories (University College of North Wales), Gwynedd, North Wales, UK; tSchoo1 of Biological Sciences, Universiti Sains Malaysia, Penang, Malaysia (Received 24 Match 1986) Abstract-l. Scyllu serrutu tolerated all experimental salinities between 1 and 42%: the crabs survived for more than 6 hr in fresh water. 2. Mud crabs proved to be powerful osmoregulators in dilute media, producing copious isosmotic urine.
At high salinities they osmoconformed. 3. Scyllu showed no ability to discriminate between salinities. 4. Mud crabs were found to be oxyconformers with limited physiological capacity to survive hypoxic conditions. However they reconnized low 00, and climbed out of sea-water (at a mean ~0~ of 10.6 mm Hg) to breathe air. _ -
INTRODUCMON
shallow water which is liable to have a low pO* because of the high B.O.D. of the mud. S. serrutu is consequently likely to encounter hypoxic stress. Respiration in mud crabs has attracted some study. Rangneker and Madhyastha (1969) found that eyestalk removal resulted in an increase in oxygen uptake, while Veerannan (1972) found that the rate of oxygen uptake in Scyiia held out of water was less than 10% of the rate recorded in immersed crabs (although the measurement techniques used were deficient in some respects). Hill and Koopowitz (1975) demonstrated bradycardia in crabs exposed to air or to hypoxic water with a pOZ below 50 mm Hg, but no data concerned with respiratory or behavioural responses to progressive hypoxia appear to have been published. A study of such responses was the second aim of the investigation reported here.
mud crab Scyllu serruta (Forskal) is a large portunid crab, common in estuaries and mangrove swamps throughout the Indo-West Pacific region. It is of considerable commercial importance since it is both fished and cultured in many countries. The first major study on the species’ biology was carried out by Arriola (1940) in the Philippines, and he established that the adult crabs lived in brackish “often quite fresh” water, but showed that the females migrated to the mouths of estuaries after mating, so that their eggs and resultant zoeae were released into full strength sea-water. Hill (1974) later established that the early larval stages of S. serrufa show considerable mortality at salinities below 17.5960,and are unsuited to an estuarine existence. A later (largely ecological) study by Hill (1979) was performed on mud crabs inhabiting a lagoon system in South Africa. He mentioned that crabs survived a 4-month period of exposure to 2960,and showed that the crabs tolerated salinities as high as 60% before becoming moribund. Apart from these observations, there appear to have been no systematic studies of adult salinity tolerance, salinity preference or physiological responses to salinity. The present study was designed in part to remedy these deficiencies. Mud crabs exploit the intertidal zone, although much of the adult population retreats to subtidal regions as the tide ebbs (Hill er al., 1982). Young crabs, and the few adults left by the ebbing tide usually spend the period of tidal emersion in burrows dug into the mud. These burrows contain water, but it seems likely that oxygen within the water will be rapidly depleted by the crabs’ metabolic demands. On occasion crabs are stranded out of water by the receding tide, so they must be capable of withstanding aerial exposure. In addition, the mangroves and mud flats that the crabs inhabit are rich in organic material and microorganisms. On ebbing or flooding tides the mud may become covered with
The
MATERIALS
AND METHODS
Collection and muinrenunce
Adult mud crabs (60-100 mm carapace width, the normal commercial siiz in the area) were purchased from a local market in Penang, Malaysia; they had been collected early on the day of purchase using baited lift nets known locally as “bintoh”. The fishermen had substantially immobilized the chelipeds with lengths of raffia; these were left in place (except in behavioural experiments) as bites from the powerful claws are dangerous, and damaging to equipment; Scylla is also a markedly cannibalistic species. Only the chelipeds were restrained, any raAia around the walking legs was cut away to ensure that gill ventilation was not impaired. No attempt was made to separate the sexes, though females predominated. Crabs were held in aerated, flowing seawater (35%~)at laboratory temperature (28-30°C) until used in experiments (generally after l-2 days). They were not fed. Occasional failure of air or sea-water supply established that anoxic conditions were rapidly lethal. Salinity tolerance
Ten crabs were exposed to each of the following salinities: 0, 1, 2, 3,4, 5, 10, 20, 32 and 42%~.Salinities were made up 43
J. DAVENPORT and T. M. WONG
44
by mixing sea-water and rain water (pH 7.7), or, in the case of 42%, by adding coarse sea salt to sea-water. Salinities were checked with a Goldberg refractometer. Animals were held in 401. fiberglass tanks, whose contents were thoroughly aerated. Media were changed daily; experimental temperatures ranged between 28 and 30°C. No food was offered. Animals were inspected for mortality after 1,4 and 24 hr, and thereafter daily until 7 days had elapsed. Haemolymph
and urine osmolality
At the conclusion of the salinity tolerance experiments described above, haemolymph and urine samples were withdrawn from surviving crabs (which had therefore spent 7 days in a particular salinity). Medium samples were also collected. Haemolymph was extracted by hypodermic syringe, the needle being inserted into the body cavity via the proximal arthrodial membrane of one of the walking legs. Urine samples were collected by drying the body surfaces of the crab under investigation, paying particular attention to the area around the openings of the nephropores. The flap covering a nephropore was lifted slightly with a needle; this invariably caused urine to flow out, and allowed it to be collected with a Pasteur pipette. Osmolalities of medium, haemolymph and urine were measured with a Halbmikro freezing point depression osmometer. Triplicate 50 ~1 samples were analysed for each measurement; three crabs were studied from each salinity. Salinity preference
To assess whether mud crabs exhibited a salinity preference, 55 crabs were each offered, in turn, a choice between the following salinities: 0, 3.2,6.4,9.6, 12.8, 16.0, 19.2,22.4, 25.6, 28.8 and 32%0. The apparatus used consisted of 11 large, shallow dishes placed around the perimeter of an empty, 2 m-diameter glass fibre tank of 1 m depth (illuminated evenly from above). The 11 dishes were each filled with water (deep enough to allow crabs to immerse themselves) of one of the salinities mentioned above, the salinity being allocated by lot-drawing. A 12th shallow dish (empty) was placed in the centre of the glass fibre tank so that it was equidistant from all of the water-filled dishes. Each crab was placed in the empty dish and then allowed 30 min to make a choice. Some crabs (not included in the total of 55) did not make a choice in this time, but remained in air; they were discarded. Reallocation of salinities was carried out between each trial to eliminate systematic bias between dishes. The null hypothesis tested in this experiment was that crabs were incapable of discriminating between salinities, so that five animals would be expected to end the trials in each salinity. Respirometry
To investigate the effect of oxygen tension upon oxygen uptake in mud crabs, five crabs in turn were each placed in a closed respirometer (constructed largely of opaque ABS plastic so that the crabs were not disturbed during uptake measurements), filled initially with air-saturated sea-water, and having a volume of 2.061. The oxygen tension within the respirometer was continually monitored with a Radiometer ES046 Clark-type oxygen electrode, connected via a Radiometer PHM 71 MkII pH meter to a chart recorder. The respirometer was mounted in a constant temperature bath (held at 25°C) and its contents were magnetically stirred; the crab was separated from the magnetic follower by a coarse mesh. Experiments continued until each had ceased taking up oxygen. The respirometer and electrode were cleaned with sodium hypochlorite solution between experiments. Control trials without animals were performed between experiments; they always showed negligible background respiration. Behavioural responses to falling oxygen iension
To investigate the behavioural reactions of Scylla to falling oxygen tension, apparatus similar to that described by Davenport and Woolmington (1981) was used. A 101.
aquarium, half-tilled with sea-water, was equipped with a sloping mesh ramp which allowed a mud crab to climb out of water if it chose to do so. A Radiometer oxygen electrode was mounted in the sea-water of the aquarium, and the water was mixed by bubbles flowing from a diffuser stone. The diffuser stone could be supplied with either air or oxygen-free N2 . Five crabs were studied with this apparatus. Each crab was placed (alone) in the sea-water of the aquarium, which had been air-saturated. After a 20-min acclimation period, the flow of air to the diffuser stone was replaced by oxygen-free N, . The behaviour of the crab was observed until-ii climbed out of the sea-water. The behaviour of crabs held in shallow dishes of sea-water (each dish
containing several crabs, and therefore prone to oxygen depletion) was also observed. RESULTS Salinity tolerance S. serrata is a very euryhaline crab; pure freshwater was survived by all animals for over 6 hr and 10% survived for 24 hr (Table 1). At all other salinities tested (l-42%0) more than half of the animals tested survived for the full 7-day experimental period. Haemolymph and urine osmolality S. serrata is a powerful osmoregulator in salinities lower than those of the open sea (Fig. 1). At higher salinities Scyfla becomes an osmoconfonner like other euryhaline portunids (e.g. Car&us maenassee Krogh, 1939). At all salinities, the urine osmolality recorded was essentially identical to the haemolymph concentration, so it would appear that Scylla secretes an isosmotic urine. Urine flow rates were not recorded, but it is worth mentioning that emersed mud crabs often demonstrate a quite startling ability to eject strong streams of urine from the nephropores when disturbed. This phenomenon was seen in animals acclimated to high salinties, so it is not limited to animals likely to be water-loaded. Salinity preference S. serrata showed no significant preference for particular salinities in the medium surrounding them (Table 2). It should be stressed that most crabs climbed in and out of many or all of the salinities available to them during the experiment. Several pilot experiments were also carried out. If crabs were offered a range of low salinities (O-59&) many chose to remain out of water, and those which entered water did not show any significant preference. If mud crabs were placed in a bowl of freshwater with an island in the middle, they showed no tendency to climb out of the water. In two-chamber choice box trials with crabs being offered a choice between 0 and 32%0, crabs climbed regularly from one medium to the other, often staying for several hours in freshwater. It may be concluded, therefore, that Scylla are quite indifferent to the salinity around them. Responses to hypoxia
Figure 2 demonstrates the effects of declining oxygen tension upon oxygen uptake in mud crabs. From these data it would appear that Scylla is an oxyconformer (i.e. its oxygen uptake is progressively depressed by increasingly hypoxic external conditions), and appears to be unable to extract oxygen from sea-water with a mean oxygen tension below
Crab reqonses to salinity and low ~0,
45
Table 1. Salinity tolerance in ScyUrzserrata
4.2 mm Hg (SD 1.3 mm; r = 5). Crabs did not survive
for more than about 30min at this critical oxygen tension. Physiologically, therefore, the mud crab appears to be ill-adapted to low p02, since other crab species (e.g. Car&us, Pugwus ~r~~~) can take up oxygen in respirometers until none remains; Pagurus can also withstand several hours of totally anoxic conditions (Davenport et al., 1980). However, it is possible that the species is capable of oxygen regulation under less stressful conditions (e.g. when
0
700
1400
Medium osmolality (mOsm kg -‘)
Fig. 1. Osmolality of haemol~ph and urine samples taken from mud crabs acclimated to various salinities. Closed circles= haemolymph. Open circles=urine. Dashed line indicates equfity of osmoiality.
buried in mud). It can be seen from Table 3 that Scylla responds behaviourally to oxygen depletion in the surrounding medium by climbing out of water at a mean oxygen tension of 10.6 mm Hg.
0 -Fm 0
60
160
Oxygen tension (mm Hg)
Fig. 2. Oxygen uptake in mud crabs exposed to falling oxygen tension. Above: weight specific uptake rates. Below: uptake in reiation to uptake in air saturated sea-water. Dashed line corresponds to oxygen conformer response.
J. DAVENF~RT andT. M. WONG Table 2. Salinity preference in .Scylh serrata
Offered
salinities
Observed
of
i’/OO)
number
entering
crabs
0
Expected of
number crabs
1
5
37._
b
5
6.4
2
ts
9.6
5
5
12.8
9
5
16.0
5
5
19.2
9
5
22.4
4
5
25.6
S
5
28.8
4
5
0.
52. Cl 5 l~~~~~~~~~~~~~l~~l.~l_--~1--1-1-~--_~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
x= =
12.0
(10”
of
freedom>:
Climbing out of water is the second stage of a two-part process. Like Carcinus (Taylor et ui., 1973; Taylor and Wheatly, 1979), Scy#u first responds to oxygen depletion (at a pOr of around 20-80 mm Hg) by moving to the water’s edge, raising the forepart of the cephalothorax out of water and bubbling air through the branchial ch8rnbers (presumably by
p
entering
5
> 0.05
reversing the direction of scaphogr&hite beat).Only at very low oxygen lions did cmbs le8ve the wr&r. Crabs held in crowded bowls of shallow sea-water showad repoat& bouts of cephlothorax raising and a’ir bubbling. DEXXJSSKBN
Table 3. Oxygen tensions causing mud crabs to leave sea-water
Animal
NQ.
cKl3
zit
emergence (mm Hg)
____-____I____________)__I__________c___~
1
16.0
2
9.3
3
10.4
4
16.9
J
4.5
mean Ci.D.
10.L 5. B
The observed euryhahnity of Scylla serrafa was even greater than that expected from the field observations of Menon and Raman (1977) and Hill (1979). The species will tolerate all salinities (including pure fresh-water) that it is likely to encounter in areas exposed to tidal salinity fluctuations, and survives down to 1%0for extended periods. Semiterrestrial, intertidal and brackish-water crustacearrs often demon&r& the ability to discrimimrte between salinities or to move along salinity gradients (e.g. Gross, 1955, 1957; L8gerspetz and Mattila, 1961; McLusky, 1970; Davenport, 1972; Davenport and Wankowski, 1973) and such abilities have adaptive significance in that they remove 8nimals from areas of suboptimal or deleterious salinity to more favourable conditions. Scylia does not respond behaviourally to salinity, presumably because its powerful osmoregulatory physiology allows it to cope efficiently with all environment81 salinities. It is probable that factors other than salinity (e.g. substratum type, food availability) determine the species’ dietribution in estuaries, mangroves and lagoons. No study of the osmotic physiology of the mud crab appear to have been published. Our study
Crab responses to salinity and low ~0,
reveals an osmoregulatory pattern similar to that of the much-studied European portunid Carcinus maenas, but effective to even lower salinities (Carcinus tolerates down to 56%). This osmoregulatory capacity has allowed Scylla to exploit the highly productive muddy areas of estuaries and mangroves in a manner denied to stenohaline portunids such as Portunas pelagicus, Thalamita prymna and Charybdis natator which live in neighbouring fully marine habitats. The large size and easy availability of Scylia would appear to make it an ideal experimental animal for further studies of osmotic and ionic regulation. The physiological response of Scylla to falling oxygen tension was not anticipated. A high proportion of aquatic invertebrates living in muddy areas are oxygen regulators, often possessing blood pigments which allow oxygen uptake to be sustained down to very low oxygen tensions. Many such animals are also capable of withstanding several hours of exposure to anoxic conditions by employing anaerobic biochemical pathways. Scylla is apparently poorly equipped to extract oxygen from oxygendepleted water, and dies rapidly under anoxic conditions. The respirometric methods available to us were somewhat suboptimal however and further work with throughflow respirometers, perhaps containing mud, would be desirable. On the other hand, mud crabs show a well developed ability (shared with some other portunids such as Car&us) to exploit the rich source of oxygen available in air. When partly immersed in shallow water the crabs respond to low oxygen tensions by bubbling air through the branchial chambers; they probably use this behaviour when left in their burrows by the tide. Under more severe conditions of hypoxia, mud crabs leave water and breathe air. It is well known by commercial fishermen that Scylla may be kept out of water for long periods, and Hill and Koopowitz (1975) demonstrated that mud crabs survived for more than a week in air of 95% relative humidity (the humidity of air above wet muddy substrata will always be high). We feel that Scylfa serrata is a more amphibious species than generally recognized, and that mud crabs regularly exploit air as a respiratory medium, though probably for short periods since Veerrannan (1972) indicated that aerial respiration was rather inefficient (as it is in Carcinus). From an aquaculture (or laboratory maintenance) viewpoint these findings may be of some value; it would seem sensible to ensure that all tanks/ponds containing Scylia should feature shallow areas to allow crabs to exploit atmospheric air in the event of failure of artificial aeration. authors wish to thank Prof. D. R. Fielder. Denartment of Zoology, _ __ University of Queensland, and Dr. B. Hill, CSIRO, Cleveland laborator& QueensAcknowledgements-The
land, Australia for their help in making literature concerning Scyllu available to us. One of us (J.D.) wishes to thank
47
the authorities of the Universiti Sains Malaysia for inviting him to work in Penang, and the Royal Society of London for the provision of a travel grant. We are also grateful to Mr. Burhanuddin for his technical assistance. REFERENCES Arriola F. J. (1940) A preliminary study of the life history of Scyila serrata (Forskal). Philip. J. Sci. 73, 437456.
Davenport J. (1972) Salinity tolerance and preference in the por&lain
c;abs Porcellhna plarycheles -and Porcellana lonnicornis. Mar. Behao. Phvsiol. 1, 123-138.
Davenport J., Busschots, P. C.-M. F. gnd Cawthorne D. F. (1980) The influence of salinity on behaviour and oxygen uptake of the hermit crab Pagurus bernhardus L. J. mar. biol. Ass. U.K. 60, 127-134.
Davenport J. and Wankowski J. (1973) Pre-immersion salinity choice behavior in Porcellana platycheles. Mar. Biol. 22, 3 13-3 16. Davenport J. and Woohnington A. D. (1981) Behavioural responses of some rocky shore fish exposed to adverse environmental conditions. Mar. Behav. Physiol. 8, 1-12. Gross W. J. (1955) Aspects of osmotic regulation in crabs showing the terrestrial habit. Am. Nat. 89, 205-222. Gross W. J. (1957) A behavioral mechanism for osmotic regulation in a semiterrestrial crab. Biol. Bull. mar. biol. Lab., Wooh
Hole 113, 268-274.
Hill B. J. (1974) Salinity and temperature tolerance of zoeae of the portunid crab Scylla serrata. Mar. Biol. 25, 21-24. Hill B. J. (1979) Biology of the crab Scylla serrata (Forskal) in the St. Lucia system. Trans. R. Sot. S. Ajiica. 44, 55-62.
Hill B. J. and Koopowitz H. (1975) Heart-rate of the crab Scylla serrata (Forskal) in air and in hypoxic conditions. Camp. Biochem. Physiol. 52A, 385-387.
Hill B. J., Williams M. J. and Dutton P. (1982) Distribution of juvenile, subadult and adult Scylla serrata on tidal flats in Australia. Mar. Biol. 69, 117-120. Krogh A. (1939) Osmotic Regulation in Aquatic Animals. Cambridge University Press, Cambridge (reprinted by Dover Publications, New York, 1965). Lagerspetz K. and Mattila M. (1961) Salinity reactions of some ‘fresh and brackish water crustaceans. Biol. Bull. mar. biol. L.ab., Woo& Hole 120, 44-53.
McLusky D. S. (1970) Salinity preference in Corophium volutator.
J. mar. biol. Ass. U.K. 50, 747-152.
Menon A. G. K. and Raman K. (1977) Ecology of some marine lagoons along utilization of their resources. Mar. Res. Indonesia. 20, 131-138. Rangneker P. V. and Madhyastha M. N. (1969) Effect ofeyestalk removal on the -rate of oxygen consumption in the crab Scvlla serrata (Forskal). J. Anim. Morph. Physiol.
16, 8&88.
Taylor E. W., Butler P. J. and Sherlock P. J. (1973) The respiratory and cardiac changes associated with the emersion response of Curcinus maenas (L.) during environmental hypoxia at three different temperatures. J. camp. Physiol. 86, 95-l 15. Taylor E. W. and Wheatly M. G. (1979) The behaviour and respiratory physiology of the shore crab Carcinus maenas (L.) at moderately high temperatures. J. camp. Physiol. 130, 309-316. Veerannan K. M. (1972) Respiratory metabolism of crabs from marine and estuarine habitats. I. Scyllu serratn. Mar. Biol. 17, 284-290.
0300-9629/87 $3.00 + 0.00 Perpmoo Journals Ltd
RESPONSES OF ADULT MUD CRABS (SCYLLA SERRATA) (FORSKAL) TO SALINITY AND LOW OXYGEN TENSION J. DAVENPORT* and T. M. WONG~ *Animal Biology Group, Marine Science Laboratories (University College of North Wales), Gwynedd, North Wales, UK; tSchoo1 of Biological Sciences, Universiti Sains Malaysia, Penang, Malaysia (Received 24 Match 1986) Abstract-l. Scyllu serrutu tolerated all experimental salinities between 1 and 42%: the crabs survived for more than 6 hr in fresh water. 2. Mud crabs proved to be powerful osmoregulators in dilute media, producing copious isosmotic urine.
At high salinities they osmoconformed. 3. Scyllu showed no ability to discriminate between salinities. 4. Mud crabs were found to be oxyconformers with limited physiological capacity to survive hypoxic conditions. However they reconnized low 00, and climbed out of sea-water (at a mean ~0~ of 10.6 mm Hg) to breathe air. _ -
INTRODUCMON
shallow water which is liable to have a low pO* because of the high B.O.D. of the mud. S. serrutu is consequently likely to encounter hypoxic stress. Respiration in mud crabs has attracted some study. Rangneker and Madhyastha (1969) found that eyestalk removal resulted in an increase in oxygen uptake, while Veerannan (1972) found that the rate of oxygen uptake in Scyiia held out of water was less than 10% of the rate recorded in immersed crabs (although the measurement techniques used were deficient in some respects). Hill and Koopowitz (1975) demonstrated bradycardia in crabs exposed to air or to hypoxic water with a pOZ below 50 mm Hg, but no data concerned with respiratory or behavioural responses to progressive hypoxia appear to have been published. A study of such responses was the second aim of the investigation reported here.
mud crab Scyllu serruta (Forskal) is a large portunid crab, common in estuaries and mangrove swamps throughout the Indo-West Pacific region. It is of considerable commercial importance since it is both fished and cultured in many countries. The first major study on the species’ biology was carried out by Arriola (1940) in the Philippines, and he established that the adult crabs lived in brackish “often quite fresh” water, but showed that the females migrated to the mouths of estuaries after mating, so that their eggs and resultant zoeae were released into full strength sea-water. Hill (1974) later established that the early larval stages of S. serrufa show considerable mortality at salinities below 17.5960,and are unsuited to an estuarine existence. A later (largely ecological) study by Hill (1979) was performed on mud crabs inhabiting a lagoon system in South Africa. He mentioned that crabs survived a 4-month period of exposure to 2960,and showed that the crabs tolerated salinities as high as 60% before becoming moribund. Apart from these observations, there appear to have been no systematic studies of adult salinity tolerance, salinity preference or physiological responses to salinity. The present study was designed in part to remedy these deficiencies. Mud crabs exploit the intertidal zone, although much of the adult population retreats to subtidal regions as the tide ebbs (Hill er al., 1982). Young crabs, and the few adults left by the ebbing tide usually spend the period of tidal emersion in burrows dug into the mud. These burrows contain water, but it seems likely that oxygen within the water will be rapidly depleted by the crabs’ metabolic demands. On occasion crabs are stranded out of water by the receding tide, so they must be capable of withstanding aerial exposure. In addition, the mangroves and mud flats that the crabs inhabit are rich in organic material and microorganisms. On ebbing or flooding tides the mud may become covered with
The
MATERIALS
AND METHODS
Collection and muinrenunce
Adult mud crabs (60-100 mm carapace width, the normal commercial siiz in the area) were purchased from a local market in Penang, Malaysia; they had been collected early on the day of purchase using baited lift nets known locally as “bintoh”. The fishermen had substantially immobilized the chelipeds with lengths of raffia; these were left in place (except in behavioural experiments) as bites from the powerful claws are dangerous, and damaging to equipment; Scylla is also a markedly cannibalistic species. Only the chelipeds were restrained, any raAia around the walking legs was cut away to ensure that gill ventilation was not impaired. No attempt was made to separate the sexes, though females predominated. Crabs were held in aerated, flowing seawater (35%~)at laboratory temperature (28-30°C) until used in experiments (generally after l-2 days). They were not fed. Occasional failure of air or sea-water supply established that anoxic conditions were rapidly lethal. Salinity tolerance
Ten crabs were exposed to each of the following salinities: 0, 1, 2, 3,4, 5, 10, 20, 32 and 42%~.Salinities were made up 43
J. DAVENPORT and T. M. WONG
44
by mixing sea-water and rain water (pH 7.7), or, in the case of 42%, by adding coarse sea salt to sea-water. Salinities were checked with a Goldberg refractometer. Animals were held in 401. fiberglass tanks, whose contents were thoroughly aerated. Media were changed daily; experimental temperatures ranged between 28 and 30°C. No food was offered. Animals were inspected for mortality after 1,4 and 24 hr, and thereafter daily until 7 days had elapsed. Haemolymph
and urine osmolality
At the conclusion of the salinity tolerance experiments described above, haemolymph and urine samples were withdrawn from surviving crabs (which had therefore spent 7 days in a particular salinity). Medium samples were also collected. Haemolymph was extracted by hypodermic syringe, the needle being inserted into the body cavity via the proximal arthrodial membrane of one of the walking legs. Urine samples were collected by drying the body surfaces of the crab under investigation, paying particular attention to the area around the openings of the nephropores. The flap covering a nephropore was lifted slightly with a needle; this invariably caused urine to flow out, and allowed it to be collected with a Pasteur pipette. Osmolalities of medium, haemolymph and urine were measured with a Halbmikro freezing point depression osmometer. Triplicate 50 ~1 samples were analysed for each measurement; three crabs were studied from each salinity. Salinity preference
To assess whether mud crabs exhibited a salinity preference, 55 crabs were each offered, in turn, a choice between the following salinities: 0, 3.2,6.4,9.6, 12.8, 16.0, 19.2,22.4, 25.6, 28.8 and 32%0. The apparatus used consisted of 11 large, shallow dishes placed around the perimeter of an empty, 2 m-diameter glass fibre tank of 1 m depth (illuminated evenly from above). The 11 dishes were each filled with water (deep enough to allow crabs to immerse themselves) of one of the salinities mentioned above, the salinity being allocated by lot-drawing. A 12th shallow dish (empty) was placed in the centre of the glass fibre tank so that it was equidistant from all of the water-filled dishes. Each crab was placed in the empty dish and then allowed 30 min to make a choice. Some crabs (not included in the total of 55) did not make a choice in this time, but remained in air; they were discarded. Reallocation of salinities was carried out between each trial to eliminate systematic bias between dishes. The null hypothesis tested in this experiment was that crabs were incapable of discriminating between salinities, so that five animals would be expected to end the trials in each salinity. Respirometry
To investigate the effect of oxygen tension upon oxygen uptake in mud crabs, five crabs in turn were each placed in a closed respirometer (constructed largely of opaque ABS plastic so that the crabs were not disturbed during uptake measurements), filled initially with air-saturated sea-water, and having a volume of 2.061. The oxygen tension within the respirometer was continually monitored with a Radiometer ES046 Clark-type oxygen electrode, connected via a Radiometer PHM 71 MkII pH meter to a chart recorder. The respirometer was mounted in a constant temperature bath (held at 25°C) and its contents were magnetically stirred; the crab was separated from the magnetic follower by a coarse mesh. Experiments continued until each had ceased taking up oxygen. The respirometer and electrode were cleaned with sodium hypochlorite solution between experiments. Control trials without animals were performed between experiments; they always showed negligible background respiration. Behavioural responses to falling oxygen iension
To investigate the behavioural reactions of Scylla to falling oxygen tension, apparatus similar to that described by Davenport and Woolmington (1981) was used. A 101.
aquarium, half-tilled with sea-water, was equipped with a sloping mesh ramp which allowed a mud crab to climb out of water if it chose to do so. A Radiometer oxygen electrode was mounted in the sea-water of the aquarium, and the water was mixed by bubbles flowing from a diffuser stone. The diffuser stone could be supplied with either air or oxygen-free N2 . Five crabs were studied with this apparatus. Each crab was placed (alone) in the sea-water of the aquarium, which had been air-saturated. After a 20-min acclimation period, the flow of air to the diffuser stone was replaced by oxygen-free N, . The behaviour of the crab was observed until-ii climbed out of the sea-water. The behaviour of crabs held in shallow dishes of sea-water (each dish
containing several crabs, and therefore prone to oxygen depletion) was also observed. RESULTS Salinity tolerance S. serrata is a very euryhaline crab; pure freshwater was survived by all animals for over 6 hr and 10% survived for 24 hr (Table 1). At all other salinities tested (l-42%0) more than half of the animals tested survived for the full 7-day experimental period. Haemolymph and urine osmolality S. serrata is a powerful osmoregulator in salinities lower than those of the open sea (Fig. 1). At higher salinities Scyfla becomes an osmoconfonner like other euryhaline portunids (e.g. Car&us maenassee Krogh, 1939). At all salinities, the urine osmolality recorded was essentially identical to the haemolymph concentration, so it would appear that Scylla secretes an isosmotic urine. Urine flow rates were not recorded, but it is worth mentioning that emersed mud crabs often demonstrate a quite startling ability to eject strong streams of urine from the nephropores when disturbed. This phenomenon was seen in animals acclimated to high salinties, so it is not limited to animals likely to be water-loaded. Salinity preference S. serrata showed no significant preference for particular salinities in the medium surrounding them (Table 2). It should be stressed that most crabs climbed in and out of many or all of the salinities available to them during the experiment. Several pilot experiments were also carried out. If crabs were offered a range of low salinities (O-59&) many chose to remain out of water, and those which entered water did not show any significant preference. If mud crabs were placed in a bowl of freshwater with an island in the middle, they showed no tendency to climb out of the water. In two-chamber choice box trials with crabs being offered a choice between 0 and 32%0, crabs climbed regularly from one medium to the other, often staying for several hours in freshwater. It may be concluded, therefore, that Scylla are quite indifferent to the salinity around them. Responses to hypoxia
Figure 2 demonstrates the effects of declining oxygen tension upon oxygen uptake in mud crabs. From these data it would appear that Scylla is an oxyconformer (i.e. its oxygen uptake is progressively depressed by increasingly hypoxic external conditions), and appears to be unable to extract oxygen from sea-water with a mean oxygen tension below
Crab reqonses to salinity and low ~0,
45
Table 1. Salinity tolerance in ScyUrzserrata
4.2 mm Hg (SD 1.3 mm; r = 5). Crabs did not survive
for more than about 30min at this critical oxygen tension. Physiologically, therefore, the mud crab appears to be ill-adapted to low p02, since other crab species (e.g. Car&us, Pugwus ~r~~~) can take up oxygen in respirometers until none remains; Pagurus can also withstand several hours of totally anoxic conditions (Davenport et al., 1980). However, it is possible that the species is capable of oxygen regulation under less stressful conditions (e.g. when
0
700
1400
Medium osmolality (mOsm kg -‘)
Fig. 1. Osmolality of haemol~ph and urine samples taken from mud crabs acclimated to various salinities. Closed circles= haemolymph. Open circles=urine. Dashed line indicates equfity of osmoiality.
buried in mud). It can be seen from Table 3 that Scylla responds behaviourally to oxygen depletion in the surrounding medium by climbing out of water at a mean oxygen tension of 10.6 mm Hg.
0 -Fm 0
60
160
Oxygen tension (mm Hg)
Fig. 2. Oxygen uptake in mud crabs exposed to falling oxygen tension. Above: weight specific uptake rates. Below: uptake in reiation to uptake in air saturated sea-water. Dashed line corresponds to oxygen conformer response.
J. DAVENF~RT andT. M. WONG Table 2. Salinity preference in .Scylh serrata
Offered
salinities
Observed
of
i’/OO)
number
entering
crabs
0
Expected of
number crabs
1
5
37._
b
5
6.4
2
ts
9.6
5
5
12.8
9
5
16.0
5
5
19.2
9
5
22.4
4
5
25.6
S
5
28.8
4
5
0.
52. Cl 5 l~~~~~~~~~~~~~l~~l.~l_--~1--1-1-~--_~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
x= =
12.0
(10”
of
freedom>:
Climbing out of water is the second stage of a two-part process. Like Carcinus (Taylor et ui., 1973; Taylor and Wheatly, 1979), Scy#u first responds to oxygen depletion (at a pOr of around 20-80 mm Hg) by moving to the water’s edge, raising the forepart of the cephalothorax out of water and bubbling air through the branchial ch8rnbers (presumably by
p
entering
5
> 0.05
reversing the direction of scaphogr&hite beat).Only at very low oxygen lions did cmbs le8ve the wr&r. Crabs held in crowded bowls of shallow sea-water showad repoat& bouts of cephlothorax raising and a’ir bubbling. DEXXJSSKBN
Table 3. Oxygen tensions causing mud crabs to leave sea-water
Animal
NQ.
cKl3
zit
emergence (mm Hg)
____-____I____________)__I__________c___~
1
16.0
2
9.3
3
10.4
4
16.9
J
4.5
mean Ci.D.
10.L 5. B
The observed euryhahnity of Scylla serrafa was even greater than that expected from the field observations of Menon and Raman (1977) and Hill (1979). The species will tolerate all salinities (including pure fresh-water) that it is likely to encounter in areas exposed to tidal salinity fluctuations, and survives down to 1%0for extended periods. Semiterrestrial, intertidal and brackish-water crustacearrs often demon&r& the ability to discrimimrte between salinities or to move along salinity gradients (e.g. Gross, 1955, 1957; L8gerspetz and Mattila, 1961; McLusky, 1970; Davenport, 1972; Davenport and Wankowski, 1973) and such abilities have adaptive significance in that they remove 8nimals from areas of suboptimal or deleterious salinity to more favourable conditions. Scylia does not respond behaviourally to salinity, presumably because its powerful osmoregulatory physiology allows it to cope efficiently with all environment81 salinities. It is probable that factors other than salinity (e.g. substratum type, food availability) determine the species’ dietribution in estuaries, mangroves and lagoons. No study of the osmotic physiology of the mud crab appear to have been published. Our study
Crab responses to salinity and low ~0,
reveals an osmoregulatory pattern similar to that of the much-studied European portunid Carcinus maenas, but effective to even lower salinities (Carcinus tolerates down to 56%). This osmoregulatory capacity has allowed Scylla to exploit the highly productive muddy areas of estuaries and mangroves in a manner denied to stenohaline portunids such as Portunas pelagicus, Thalamita prymna and Charybdis natator which live in neighbouring fully marine habitats. The large size and easy availability of Scylia would appear to make it an ideal experimental animal for further studies of osmotic and ionic regulation. The physiological response of Scylla to falling oxygen tension was not anticipated. A high proportion of aquatic invertebrates living in muddy areas are oxygen regulators, often possessing blood pigments which allow oxygen uptake to be sustained down to very low oxygen tensions. Many such animals are also capable of withstanding several hours of exposure to anoxic conditions by employing anaerobic biochemical pathways. Scylla is apparently poorly equipped to extract oxygen from oxygendepleted water, and dies rapidly under anoxic conditions. The respirometric methods available to us were somewhat suboptimal however and further work with throughflow respirometers, perhaps containing mud, would be desirable. On the other hand, mud crabs show a well developed ability (shared with some other portunids such as Car&us) to exploit the rich source of oxygen available in air. When partly immersed in shallow water the crabs respond to low oxygen tensions by bubbling air through the branchial chambers; they probably use this behaviour when left in their burrows by the tide. Under more severe conditions of hypoxia, mud crabs leave water and breathe air. It is well known by commercial fishermen that Scylla may be kept out of water for long periods, and Hill and Koopowitz (1975) demonstrated that mud crabs survived for more than a week in air of 95% relative humidity (the humidity of air above wet muddy substrata will always be high). We feel that Scylfa serrata is a more amphibious species than generally recognized, and that mud crabs regularly exploit air as a respiratory medium, though probably for short periods since Veerrannan (1972) indicated that aerial respiration was rather inefficient (as it is in Carcinus). From an aquaculture (or laboratory maintenance) viewpoint these findings may be of some value; it would seem sensible to ensure that all tanks/ponds containing Scylia should feature shallow areas to allow crabs to exploit atmospheric air in the event of failure of artificial aeration. authors wish to thank Prof. D. R. Fielder. Denartment of Zoology, _ __ University of Queensland, and Dr. B. Hill, CSIRO, Cleveland laborator& QueensAcknowledgements-The
land, Australia for their help in making literature concerning Scyllu available to us. One of us (J.D.) wishes to thank
47
the authorities of the Universiti Sains Malaysia for inviting him to work in Penang, and the Royal Society of London for the provision of a travel grant. We are also grateful to Mr. Burhanuddin for his technical assistance. REFERENCES Arriola F. J. (1940) A preliminary study of the life history of Scyila serrata (Forskal). Philip. J. Sci. 73, 437456.
Davenport J. (1972) Salinity tolerance and preference in the por&lain
c;abs Porcellhna plarycheles -and Porcellana lonnicornis. Mar. Behao. Phvsiol. 1, 123-138.
Davenport J., Busschots, P. C.-M. F. gnd Cawthorne D. F. (1980) The influence of salinity on behaviour and oxygen uptake of the hermit crab Pagurus bernhardus L. J. mar. biol. Ass. U.K. 60, 127-134.
Davenport J. and Wankowski J. (1973) Pre-immersion salinity choice behavior in Porcellana platycheles. Mar. Biol. 22, 3 13-3 16. Davenport J. and Woohnington A. D. (1981) Behavioural responses of some rocky shore fish exposed to adverse environmental conditions. Mar. Behav. Physiol. 8, 1-12. Gross W. J. (1955) Aspects of osmotic regulation in crabs showing the terrestrial habit. Am. Nat. 89, 205-222. Gross W. J. (1957) A behavioral mechanism for osmotic regulation in a semiterrestrial crab. Biol. Bull. mar. biol. Lab., Wooh
Hole 113, 268-274.
Hill B. J. (1974) Salinity and temperature tolerance of zoeae of the portunid crab Scylla serrata. Mar. Biol. 25, 21-24. Hill B. J. (1979) Biology of the crab Scylla serrata (Forskal) in the St. Lucia system. Trans. R. Sot. S. Ajiica. 44, 55-62.
Hill B. J. and Koopowitz H. (1975) Heart-rate of the crab Scylla serrata (Forskal) in air and in hypoxic conditions. Camp. Biochem. Physiol. 52A, 385-387.
Hill B. J., Williams M. J. and Dutton P. (1982) Distribution of juvenile, subadult and adult Scylla serrata on tidal flats in Australia. Mar. Biol. 69, 117-120. Krogh A. (1939) Osmotic Regulation in Aquatic Animals. Cambridge University Press, Cambridge (reprinted by Dover Publications, New York, 1965). Lagerspetz K. and Mattila M. (1961) Salinity reactions of some ‘fresh and brackish water crustaceans. Biol. Bull. mar. biol. L.ab., Woo& Hole 120, 44-53.
McLusky D. S. (1970) Salinity preference in Corophium volutator.
J. mar. biol. Ass. U.K. 50, 747-152.
Menon A. G. K. and Raman K. (1977) Ecology of some marine lagoons along utilization of their resources. Mar. Res. Indonesia. 20, 131-138. Rangneker P. V. and Madhyastha M. N. (1969) Effect ofeyestalk removal on the -rate of oxygen consumption in the crab Scvlla serrata (Forskal). J. Anim. Morph. Physiol.
16, 8&88.
Taylor E. W., Butler P. J. and Sherlock P. J. (1973) The respiratory and cardiac changes associated with the emersion response of Curcinus maenas (L.) during environmental hypoxia at three different temperatures. J. camp. Physiol. 86, 95-l 15. Taylor E. W. and Wheatly M. G. (1979) The behaviour and respiratory physiology of the shore crab Carcinus maenas (L.) at moderately high temperatures. J. camp. Physiol. 130, 309-316. Veerannan K. M. (1972) Respiratory metabolism of crabs from marine and estuarine habitats. I. Scyllu serratn. Mar. Biol. 17, 284-290.