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Circulatory modification in the blue crab Callinectes sapidus, during exposure and acclimation to low salinity
Comparative Biochemistry and Physiology Part A 121 (1998) 67 – 76
Circulatory modification in the blue crab Callinectes sapidus, during exposure and acclimation to low salinity I.J. McGaw *, C.L. Reiber Department of Biological Sciences, Uni6ersity of Ne6ada, Las Vegas, 4505 Maryland Parkway, Las Vegas, NV 89154 -4004, USA Received 5 March 1998; received in revised form 22 June 1998; accepted 1 July 1998
Abstract A pulsed-Doppler flowmeter was used to measure heart rate and haemolymph flow rates in each arterial system of the blue crab, Callinectes sapidus, enabling calculation of stroke volume and cardiac output. During exposure to a 6–6–12 h salinity cycle of 100–25–100% seawater, there was an immediate increase in heart rate upon dilution of the medium. After this initial increase it decreased steadily, but remained elevated above levels in 100% seawater. A smaller increase in heart rate occurred when the salinity was raised, declining thereafter and reaching pre-treatment levels after 6 h in 100% SW. There was a slight decrease in stroke volume of the heart, but overall this resulted in an increase in cardiac output when the salinity was lowered. Differential haemolymph flow through each major arterial system also occurred. There was an increase in flow rates through the anterior aorta, anterolateral arteries and sternal artery during the first 2 h of low salinity exposure and smaller increases occurred again when the salinity was raised to 100% seawater. No significant changes in flow were observed in the hepatic arteries or posterior aorta. During a 72 h acclimation period in low salinity, similar increases in cardiac parameters and flow rates were observed in the first 6 h. These values declined to levels comparable to those in 100% seawater, after 40 – 50 h acclimation in low salinity. The changes in cardiovascular parameters are not directly related to the osmoregulatory physiology of this species, but appear to be due to specific behaviours occurring in response to low salinity. The results obtained here for this efficient osmoregulator are compared and contrasted with similar studies on Cancer magister, which is classified as a weak hyperosmoregulator. © 1998 Elsevier Science Inc. All rights reserved. Keywords: Callinectes sapidus; Salinity; Cardiovascular; Circulation; Heart rate; Crab osmoregulation
1. Introduction The blue crab Callinectes sapidus, (Crustacea:Decapoda), is an efficient osmoregulator and can survive in a broad range of salinities, from freshwater to hypersaline lagoons [25,32]. Male crabs and larger individuals are reported to be more tolerant of hypo-osmotic conditions, compared with females and juvenile crabs [23]. The osmotic and ionic regulatory physiology of this species is well documented [53,6,52,31,9,16,15,19]. Abbre6iations: SW, seawater; ppt, parts per thousand. * Corresponding author. Tel.: +1 702 8953399; fax: + 1 702 8953956; e-mail: [email protected]
However, there are fewer reports on the cardiovascular physiology of decapod crustaceans exposed to dilute media and findings are controversial within and between species. Sabourin [47] reports an increase in the heart beat frequency of C. sapidus, exposed to a salinity cycle, but no significant change in stroke volume or cardiac output. However, after 2–4 days low salinity acclimation, there is no measurable change in cardiac parameters of C. sapidus [13,47]. Hume and Berlind [27] and Taylor [54] also report an increase in the heart rate of the green shore crab, Carcinus maenas, exposed to seawater in the 35–60% range. Spaargaren [49] reports an increase in heart rate and calculated cardiac output and gill blood flow of C. maenas, in low salinities (below 45% sea water), but observes a decrease in
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seawater of 45–80%. A similar decrease in heart rate occurs in the crab Libinia emarginata when transferred to 80% seawater [10,11]. In contrast, Taylor et al., [55] report that the heart rate of C. maenas remains stable when exposed to 50% sea water. Similar contradictions are seen in several shrimp species [48,14]. Recently we have developed a pulsed-Doppler technique for measurement of cardiac parameters in crustaceans, this has been used to study changes in the cardiac parameters and haemolymph redistribution in the Dungeness crab Cancer magister, during exposure and acclimation to low salinity [39]. When exposed to 50% sea water for short periods (6 h, duplicating a tidal cycle of salinity), C. magister responds with an increase in heart rate, however, the stroke volume decreases, which results in an overall decrease in cardiac output. Differential changes in haemolymph flow through each of the five major arterial systems leaving the heart occurs, but these changes in individual organ perfusion appear unrelated to osmo or ionoregulatory mechanisms. During chronic exposure to 50% seawater (4d), heart rate was still elevated, but stroke volume was depressed to a lower extent than observed following 6 h of exposure. On return to 100% seawater, heart rate decreases, but stroke volume increases, resulting in no significant change in cardiac output. Arterial flows are not significantly depressed, following 4d exposure to 50% seawater and with the exception of a transient increase in flow through the posterior aorta (which supplies the telson and hindgut), do not change significantly on return to 100% sea water [39]. Although C. magister occurs in estuaries, it is classified as a weak hyperosmoregulator [29,18,28] and cannot survive prolonged exposure to salinities below 12 ppt (36% seawater) seawater [8]. The aim of the present study was to investigate the cardiovascular physiology of the efficient hyperosmoregulator C. sapidus, during low salinity exposure and acclimation and to compare and contrast these changes with those occurring during hyposaline exposure in C. magister.
2. Material and methods Adult male blue crabs, Callinectes sapidus (Crustacea:Decapoda), of 12 – 16 cm carapace width (140– 260 g) were obtained from Gulf Specimens, Florida. Crabs were held in a recirculating seawater system (Instant Ocean) 1000 – 1050 mOsm l − 1 (considered as 100% sea water) at a temperature of 18 – 20°C. They were fed chopped liver twice weekly, but were not fed the day prior to experimentation. A 545C directional pulsed-Doppler flowmeter (University Of Iowa, Bioengineering) was used to measure haemolymph flow through each arterial system leaving the heart. Peizo-electric crystal probes (Iowa Doppler
products and Crystal Biotech) were either implanted in grooves abraded in the carapace directly above the artery (anterior and posterior aortae and left anterolateral artery), or guided to lie adjacent to the artery via catheter mounted probes (right hepatic artery and sternal artery). Probe implants were held in place with dental wax and super glue and maximal signal was obtained using the depth focus on the flowmeter. Calibration of the flowmeter and further details of the experimental procedure are detailed elsewhere [2,46]. The voltage output from the flowmeter was recorded as both phasic and mean signals on Sable Systems data acquisition package. Values were converted to ml min − 1 using a modified Doppler Equation [24]: Q= E*D2/(0.2712*cos A), where Q is volume flow in ml min − 1, E is the voltage output from the flowmeter, D is the arterial diameter (mm), 0.2712 is a constant accounting for velocity of sound in blood and cos A is the angle between the sound beam and blood velocity vector. Heart rate was obtained by counting peaks on the phasic traces. Summation of arterial flows (flows for paired arteries were doubled) gave total cardiac output and division of this value by heart rate yielded stroke volume of the heart. Following experimentation the animals were dissected for measurement of arterial diameters and verification of probe implants. The experimental chamber consisted of a covered plastic box (35×20×22 cm) with a recirculating aerated filter, the chamber was filled with sand to a depth of approximately 3 cm. A tap allowed water to be drained without aerially exposing the animal and salinity was changed by adding a known volume of distilled water. Crabs were held in a plastic mesh cage (22 × 14× 8 cm), inside the chamber, this allowed movement, but prevented the crab rolling or twisting and thus damaging probe implants. The crabs were allowed to settle for 12 h after instrumentation and recordings were carried out in constant dim light at a water temperature of 20°C. In a first series of experiments haemolymph flow and heart rates of ten crabs were monitored for 1 min at 15-min intervals over a 6 h period in 1000 mOsm water (considered as 100% sea water). The salinity was then dropped, over a 15 min period, to 25% sea water and animals were monitored for another 6 h. Finally salinity was raised again to 100% sea water, by addition of concentrated saline and animals were monitored for a further 12 h. In a second series of experiments eight animals were recorded for 1 min at 30-min intervals for 12 h in 100% seawater and then for a further 72 h acclimation period in 25% seawater. Salinity response curves were constructed for heart rate (beats min[1]) of eight crabs monitored for total period of 10 min in separate treatments of 100, 75, 50 and 25% sea water and in freshwater, after 15 min
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Fig. 1. Mean(9 S.E.): (a) Heart rate; (b) Stroke volume of the heart; and (c) Cardiac output of ten male Callinectes sapidus exposed to a 6–6–12 h salinity cycle of 100 – 25–100% seawater.
exposure to each condition. Between each treatment the water was raised to 100% seawater and animals allowed to settle for 6 h before exposure to the next test salinity. Finally haemolymph osmolality was measured at set intervals in eight crabs kept for 6 h in 100% seawater and thereafter for a further 72 h in 25% seawater and for a separate group of eight animals exposed to a 6 – 6 –12 h cycle of 100 – 25 – 100% seawater. Samples were collected at set intervals by withdrawing approximately 20 ml of haemolymph from the arthrodial membranes between the walking legs. Osmolality was measured using a vapor pressure osmometer (Wescor Incorporated Model 5100B). One-way ANOVA with repeated measures design [45] was used to test for significant differences in the measured variables, at each time point. The data showing a significant effect, were further analyzed by Fischers LSD multiple comparison test.
3. Results When C. sapidus was exposed to a salinity cycle there was an increase in heart rate from 85 beats min − 1 to 135 beats min − 1 (Fig. 1a), immediately upon salinity reduction. Thereafter, a decrease in heart rate occurred, stable levels of approximately 108 beats min − 1 were reached after 3 h of low salinity exposure, but these
were still significantly elevated above pre-treatment values (F= 3.51, PB0.000). Upon return to 100% seawater there was a slight, but significant increase in heart rate (F=3.23, PB 0.000), decreasing after the salinity changeover period, reaching values of 85 beats min − 1 after 6 h in 100% seawater (Fig. 1a). Stroke volume of the heart was more variable (Fig. 1b), there was a small decrease in mean values (F= 1.9, PB 0.01) which occurred after 2 h of reduction of salinity to 25% seawater. When the salinity was raised there was a slow increase in stroke volume and initial values were only regained towards the end of the experimental period. Changes in total cardiac output, were somewhat similar to those of heart rate. A significant increase in cardiac output (F= 5.79, PB0.000) occurred during the first 2 h (Fig. 1c) of low salinity exposure; rising from approximately 25 to 35 ml min − 1. Cardiac output declined steadily thereafter and reached values comparable to those in 100% seawater (23–28 ml min − 1) after 3 h. These levels of cardiac output were maintained for the remaining period of low salinity exposure and upon return to 100% seawater. There were also significant differences in haemolymph flow rates through each of the five arterial systems (Fig. 2). Although haemolymph flow was somewhat more variable than heart parameters there was no significant difference in haemolymph flow rates in any of the arteries during the initial 100% seawater control
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Fig. 2. Haemolymph flow rates (ml min − 1) (mean 9 S.E.) through the: (a) Anterior aorta; (b) Left anterolateral artery; (c) Right hepatic artery; (d) Posterior aorta; and (e) Sternal artery of C. sapidus (n =10), exposed to a 100 – 25 – 100% cycle of seawater.
period (P\ 0.05). There was a significant and rapid increase in haemolymph flow through the anterior aorta (F =2.41, PB 0.001) (Fig. 2a), anterolateral arteries (F=2.89, P B 0.000) (Fig. 2b) and sternal artery (F = 4.12, P B 0.000) (Fig. 2e) upon salinity reduction. These increased rates were, in each case, maintained for the first 2 h of exposure to 25% seawater. Flow rates in each of these three arteries (Fig. 2a, b, e) decreased after 2 h, reaching levels that were not significantly different from those in 100% seawater. Upon return to 100% seawater, there was a slight increase in flow through each of these arteries (Fig. 2a, b, e), however, this increase only proved to be statistically significant in the anterior aorta (F =1.86, P B 0.001) (Fig. 2a). There was no significant change in flow rates through the hepatic arteries, either during reduction of salinity to 25% seawater, or upon return to 100% seawater (F= 0.92, P\ 0.05) (Fig. 2c). Haemolymph flow through the posterior aorta, was routinely low and in some cases
barely detectable (Fig. 2d). This may have lead to failure to detect any significant changes with salinity (F= 1.44, P\ 0.05) (Fig. 2d). Changes in cardiovascular parameters were also followed continually during a 3 day acclimation period in 25% seawater (Figs. 3 and 4). There was no significant change (P\ 0.05) in heart rate, stroke volume or cardiac output during a 12 h control period in 100% seawater (Fig. 3a–c). During the first 6 h exposure to low salinity, changes in cardiac parameters were similar to those reported for salinity cycling (Fig. 1a–c). The heart rate of C. sapidus increased immediately upon salinity reduction (F= 4.85, PB 0.000), reaching maximal values of 149 beats min − 1, after 1.5 h of low salinity exposure (Fig. 3a). There was a slow and steady decline in heart rate, thereafter. Rates reached stable levels, which were not significantly different from those in 100% seawater, between 40 and 50 h after the initial salinity change-over.
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Fig. 3. (a) Heart rate; (b)Cardiac stroke volume; and (c) Cardiac output (Mean 9 S.E.) of eight C. sapidus, during 12 h in 100% seawater and 72 h of acclimation to 25% seawater.
Stroke volume of the heart decreased during the first 8 h of low salinity exposure (Fig. 3b) and increased steadily afterwards. However, due to the small magnitude of changes and the large inter-individual variability, these changes proved to be statistically insignificant (F =1.40, P\ 0.05). Cardiac output increased from around 30 to 45 ml min − 1 when the salinity was lowered (F = 2.23, PB 0.000) (Fig. 3c) followed by a short-term decrease in output after 2 h. A subsequent increase at 8 h was sustained for 50 h of low salinity acclimation, after which levels declined to those comparable in 100% seawater. Differential changes in flow rates through each arterial system were also observed during acclimation to low salinity (Fig. 4). There was a significant increase in flow through the anterior aorta (F =2.83, P B 0.000) (Fig. 4a) after 2 h exposure to low salinity, although flow rates decreased somewhat after this period, they still remained elevated for about 40 h afterwards. Haemolymph flow increased immediately in the anterolateral arteries (F= 2.60, P B0.000) (Fig. 4b) and remained elevated above control values for 7 h before declining steadily. Flow rates also increased significantly through the sternal artery (F =2.09, P B 0.000) (Fig. 4e) and remained elevated above control levels for the duration of the acclimation period. As with salinity cycling (Fig. 2) there was no significant change in flow through the hepatic arteries (F= 0.89, P \ 0.05) (Fig. 4c). There was a significant
decrease in flow through the posterior aorta (F=2.67, PB 0.000) upon salinity reduction (Fig. 4d), which was not observed during the salinity cycling experiment (Fig. 2d). The fact, however, that flow also changed significantly in this vessel during the control period (F= 6.93, PB 0.000) suggests that these changes were unrelated to salinity and were simply a result of the low and often intermittent flow observed in this artery. Heart rate (and presumably other cardiac parameters) changes inversely with salinity (Fig. 5). In 100% seawater, heart rate of the eight experimental animals varied between 60 and 140 beats min − 1 with a mean value of 929 8.5 beats min − 1. Heart rate increased significantly as the salinity decreased (F=4.51 PB 0.005) reaching a maximum of 1279 6 beats min − 1 in 25% seawater. Finally the haemolymph osmolality was monitored during salinity cycles and a 3 day acclimation period to 25% seawater (Fig. 6a, b). During a salinity cycle of 100–25–100% seawater, haemolymph osmolality decreased steadily from 10609 9 mOsm in 100% seawater, reaching the lowest level of 73697 mOsm after 6 h in 25% seawater (Fig. 6a). The osmolality of the haemolymph increased slowly when the salinity was raised and pre-treatment levels were only regained after 10 h in 100% seawater. Haemolymph osmolality fell rapidly during the first 10 h of long-term acclimation to 25% seawater (Fig. 6b). Stable levels of 500 mOsm were reached at around 18 h and maintained for the duration of the experimental period.
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Fig. 4. Mean ( 9 S.E.) haemolymph flow rates (ml min − 1) in eight C. sapidus exposed to 12 h of 100% seawater and 72 h of 25% seawater. (a) Anterior aorta; (b) Left anterolateral artery; (c) Right hepatic artery; (d) Hepatic artery; and (e) Sternal artery.
4. Discussion Variation in cardiovascular parameters between individual C. sapidus was high, this is common in decapod crustaceans maintained under identical experimental conditions [5,4,12,38]. In the present study we attempted to reduce some of this inherent variation by providing a layer of sand in the experimental chamber for crabs to bury into and minimally restraining them. Responses to external stimuli, as well as inter-individual differences in heart-rate can be reduced this way [21,12,36] The range of heart rates reported here, for C. sapidus, are similar to those in the literature [13,47]. The increase in heart rate observed in C. sapidus (Fig. 1a) in low salinity conforms with the majority of reports on crustaceans during acute exposure to dilute media [49,27,54,47,37,39]. However, DeFur and Mangum [13], find that the heart rate of C. sapidus is unchanged after
2–4 days exposure to similar salinities (8–20 ppt, 25– 60% SW). In the present study, after an initial increase in heart rate in 25% SW, it declines steadily (Fig. 3a) reaching levels similar to those in full strength seawater after 40–50 h. Therefore, it appears heart rate of blue crabs is only affected during acute exposure to low salinity and returns to normal levels after 2d in low salinity. In C. magister, although the heart rate also increases upon exposure to 50% SW, the changes are different to C. sapidus. Heart rate continues to rise during acute exposure to low salinity and remains elevated 25–30% above resting levels after 4–5 days low salinity acclimation [39]. Changes in stroke volume of the heart and cardiac output also differed between C. sapidus and C. magister. A small decrease in stroke volume occurred in C. sapidus in low salinity (Fig. 1b, Fig. 3b). This decrease probably results passively from a reduced filling time at elevated rates [21]. The decrease in stroke volume had
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little effect on total cardiac output, which tended to be governed by changes in heart rate and cardiac output therefore increased during low salinity exposure (Fig. 1c, Fig. 3c). In C. magister, stroke volume decreases significantly in 50% SW leading to an overall decrease in cardiac output [39]. In cancrid crabs stroke volume of the heart, rather than rate plays a greater role in determining cardiac output [41,1,3,38,39]. The opposite appears to be true in portunid crabs, where changes in heartbeat frequency largely determine total cardiac output ([49–51,42], Figs. 1 and 3). Ultimately increases in cardiac output will be followed by increased blood flow through the gills. This would aid oxygen uptake and active ion uptake and an increase in ventilation frequency and oxygen uptake is seen in C. sapidus in dilute seawater [30,17,20,47]. If heart rate and hence cardiac output of C. sapidus is representative of oxygen uptake, the linear increase in heart rate with decreasing salinity, shown in the salinity response data (Fig. 5), would reflect the increasing metabolic demand involved in osmotic and ionic regulation. Although both heart rate and oxygen uptake increase as soon as the medium is diluted, the time course for changes in heart rate and cardiac output during low salinity acclimation do not follow the same time course as changes in oxygen consumption [54,20,47]. It is possible that the changes in cardiac parameters are not closely coupled with the respiratory/ osmoregulatory physiology, but rather, reflect some other type of demand. Modulation of haemolymph flow also occurs upon lowering of the salinity. Different patterns of organ perfusion were seen in the blue crabs when compared with the weak hyperosmoregulator C. magister. There was an increase in flow through the anterior aorta during the first few hours in dilute seawater (Fig. 2a and Fig. 4a). This vessel supplies the supraoesophageal
Fig. 5. Salinity response curve of heart rate (mean 9 S.E.) of C. sapidus (n =8) in salinities of 100, 75, 50, 25 and 0% seawater.
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ganglion, eyestalks and antennae of the crab [44,40]. The antenna have been implicated in playing a chemosensory role in low salinity [22] and it is possible that increased flow to these organs could enhance this function. This response was the opposite to that observed for C. magister, where a decrease in flow was observed [39]. It is notable that the pattern of flow through the anterior aorta was very different between the two species. In C. magister haemolymph flow is usually undetectable and this artery is only perfused sporadically [38] or during periods of emersion [3]. In C. sapidus, the anterior aorta was more-or-less perfused continually and received a much higher percentage of total cardiac output. There was also an increase in flow through the anterolateral arteries during the first 2–3 h of low salinity exposure (Fig. 2b) and flow remained significantly elevated for about 8 h in low salinity (Fig. 4b). This vessel, supplies the foregut, some of the mouthparts and the antennal gland [44,40]. These increases could represent regulation of body fluids at the gut level, since the foregut has been implicated in osmotic and ionic regulation in some species of Crustacea [35]. The anterolateral artery also supplies blood to the antennal gland via its branches, thus increased flow could represent increased filtration and production of iso-osmotic urine, resulting from the increased water loading. The time period of the increase, however, is shorter than reported changes of urine production times in low salinity and thus increased flow may not represent urine production [33,7,57]. Pressure is the driving force for filtration, which was not measured in the present study and it is possible that this could vary independently of flow. In C. magister flow actually decreases through the anterolateral arteries [39], reasons for these discrepancies are unclear. The greatest increase in haemolymph flow occurred through the sternal artery (Fig. 2e, Fig. 4e), which supplies the legs, chelae and mouth parts [44,40]. Crabs are known to exhibit increased locomotor activity in low salinity defined as halokinesis [56], presumably acting as an escape response from unfavourable environments. However, in C. sapidus there is no significant difference in locomotor activity in low salinity, compared with 100% SW (McGaw et al, in prep). Indeed this species can survive in freshwater [32] and escape responses would therefore be inappropriate. The changes in haemolymph flow through the sternal artery are opposite to those for C. magister, where a decrease in flow through this vessel occurs [39], again reasons why this occurs are unclear, but overall decreases in flow lead to a decrease in cardiac output through the gills of C. magister and thus may reduce the gradient for inward water flux and diffusive ion loss. The time periods for changes in cardiac parameters or flow in C. sapidus, do not correlate with changes in
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Fig. 6. Mean haemolymph osmolality (mOsm) of eight male C. sapidus exposed to (a) 6 – 6 – 12 h cycle of 100 – 25 – 100% seawater; and (b) during acclimation over a 72 h period in 25% seawater.
osmolality of the blood, which decreased steadily in low salinity stabilizing after about 18 h (Fig. 6). Nor do they appear related to time course of changes in ATPase activity [43] or acid – base balance [26]. Recently we have categorized new behaviours of blue crabs in low salinity (McGaw et al., In prep). These behaviours include, increased movements of the mouthparts and cleaning of mouthparts with claws, these structures are supplied by the anterolateral arteries and sternal artery respectively and increased flicking and cleaning of the antennae, which are involved in osmoreception, [22] and are supplied by the anterior aorta. The duration and intensity of these behaviours correlate closely with those reported for blood flow and cardiac parameters. It therefore, appears that increased blood flow (which is
aided by increased heart beat frequency and differential contraction/relaxation of the cardioarterial valves) is not related directly to osmoregulatory function, but to increased demand due to the above mentioned behaviours, which were elicited by changes in salinity. The reason for the behaviours is not clear, but could be due to irritation of the membranes on the exopodites of the mouthparts, or the sensilla of the antenna, which are sloughed off in low salinity [22]. There was no significant change in flow, related to salinity, in the hepatic arteries or posterior aorta, (Figs. 2 and 4 c, d). This is as expected since these vessels supply the digestive gland and telson respectively, which are not involved in osmoregulation. In C. sapidus flow rates through the small posterior aorta were very
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low and intermittent, changing without obvious cause, this would have lead to detection of significant changes in flow in this vessel even during control periods (Fig. 4d). In C. magister flow through the posterior aorta actually increases [39]. Individuals of this species extend and retract the telson in dilute seawater, possibly exposing the membrane of the rectum for ion uptake (McGaw et al., In prep). This could account for extra blood delivery to this area and this behaviour was not observed in C. sapidus. In general flow rates through each vessel of C. sapidus, were somewhat higher than those reported for C. magister [39], despite the blue crab being about a third of the size. This is probably due to a number of factors: Callinectes is a much more active animal with a higher metabolic rate and cardiac output [34,36]; indeed considering its size, its vessels are greater in diameter (unpublished data). Clearly there are differences in the cardiovascular physiology of C. magister, which is classed as a weak osmoregulator and the efficient hyperosmoregulator C. sapidus. The changes reported here for blue crabs appear to be related to specific behaviours rather than directly to the osmoregulatory physiology. It is worthy to note that even under resting conditions in 100% SW there are interspecific differences in flow through each arterial system and control of cardiac output, as yet reasons are still unclear, but this area warrants further investigation. References [1] Airriess CN, McMahon BR. Cardiovascular adaptations enhance tolerance of environmental hypoxia in the crabs Cancer magister. J Exp Biol 1994;190:23–41. [2] Airriess CN, McMahon BR, McGaw IJ, Bourne GB. Application and in situ calibration of a pulsed-Doppler flowmeter for blood flow measurement in crustaceans. J Mar Biol Assoc UK 1994;74:445 – 58. [3] Airriess CN, McMahon BR. Short-term emersion effects cardiac function and regional haemolymph distribution in the crab Cancer magister. J Exp Biol 1996;199:3569–78. [4] Aldrich JC. On the relationship between oxygen consumption and feeding levels in decapods. In: Barnes H, editor. Proceedings of the Ninth European Marine Biology Symposium, Aberdeen, Aberdeen University Press, 1975:407–418. [5] Ansell AD. Changes in oxygen consumption, heart rate and ventilation accompanying starvation in the decapod Cancer pagurus. Neth J Sea Res 1973;7:455–75. [6] Ballard BS, Abbott JP. Osmotic accommodation in Callinectes sapidus Rathbun. Comp Biochem Physiol 1969;29:671–87. [7] Cameron JN, Batterton CV. Antennal gland function in the freshwater blue crab, Callinectes sapidus: water, electrolyte, acid base and ammonia excretion. J Comp Physiol 1978;123:143 – 8. [8] Cleaver FC. Preliminary results of the coastal (Cancer magister) investigation. Dept Fisheries Wash State Biol Rep 1957;49A:47 – 82. [9] Colvocoresses JA, Lynch MP, Webb KL. Variations in the serum constituents of the blue crab Callinectes sapidus: major cations. Comp Biochem Physiol 1974;49A:787–803.
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[10] Cornell JC. A reduction in water permeability in response to a dilute medium in the stenohaline crab Libinia emarginata (Brachyura, Majidae). Biol Bull 1973;145:430 – 1. [11] Cornell JC. A reduction in water exchange rates in an osmoconforming crab. Am Zool 1974;14:1259. [12] Cumberlidge N, Uglow RF. Heart and scaphognathite activity in the shore crab Carcinus maenas (L.). J Exp Mar Biol Ecol 1977;28:87 – 107. [13] DeFur PL, Mangum CP. The effects of environmental variables on the heart rates of invertebrates. Comp Biochem Physiol 1979;62A:283 – 94. [14] Dyer MF, Uglow RF. Cardiac and ventilatory responses associated with altered salinity in Crangon crangon. Ophelia 1980;19:117 – 25. [15] Engel DW. Comparison of the osmoregulatory capabilities of two portunid crabs Callinectes sapidus and C. similis. Mar Biol 1977;41:275 – 9. [16] Engel DW, Davis EM, Smith DE, Angelovic JW. The effect of salinity and temperature on the ion levels in the hemolymph of the blue crab Callinectes sapidus, Rathbun. Comp Biochem Physiol 1974;49A:259 – 66. [17] Engel DW, Ferguson RL, Eggert LD. Respiration rates and ATP concentrations in the excised gills of the blue crab as a function of salinity. Comp Biochem Physiol 1975;52A:669–73. [18] Engelhardt FR, Dehnel PA. Ionic regulation in the Pacific edible crab Cancer magister (Dana). Can J Zool 1973;51:735 –43. [19] Findley AM, Stickle WB. Effects of salinity fluctuation on the hemolymph composition of the blue crab Callinectes sapidus. Mar Biol 1978;46:9 – 15. [20] Findley AM, Belisle BW, Stickle WB. Effects of salinity fluctuations on the respiration rate of the southern oyster drill Thais haemastoma and the blue crab Callinectes sapidus. Mar Biol 1978;49:59 – 67. [21] Florey E, Kriebel ME. The effects of temperature, anoxia and sensory stimulation on the heart rate of unrestrained crabs. Comp Biochem Physiol 1974;48A:285 – 300. [22] Gleeson RA, Wheatly MG, Reiber CL. Perireceptor mechanisms sustaining olfaction at low salinities: insight from the euryhaline blue crab. J Exp Biol 1997;200:445 – 56. [23] Haefner PA, Shuster CN. Length increments during terminal molt of the female blue crab, Callinectes sapidus, in different salinity environments. Chesapeake Sci 1964;5(3):114 – 8. [24] Hartley CJ, Cole JS. An ultrasonic pulsed-Doppler system for measuring blood flow in small vessels. J Appl Physiol 1974;37:626 – 9. [25] Hedgpeth J. Ecological aspects of the Laguna Madre, a hypersaline estuary. In: Lauff GH, editor. Estuaries. Washington DC: Am Assoc Adv Sci, 1967:408 – 19. [26] Henry RP, Cameron JN. Acid – base balance in Callinectes sapidus during acclimation from high to low salinity. J Exp Biol 1982;101:255 – 64. [27] Hume RI, Berlind A. Heart and scaphognathite changes in a euryhaline crab Carcinus maenas exposed to a dilute environmental medium. Biol Bull 1976;150:241 – 54. [28] Hunter KC, Rudy PP. Osmotic and ionic regulation in the Dungeness crab Cancer magister (Dana). Comp Biochem Physiol 1975;51A:439 – 47. [29] Jones LL. Osmotic regulation in several crabs of the Pacific coast of North America. J Cell Com Physiol 1941;18:79 – 91. [30] King EN. The oxygen consumption of intact crab and excised gills as a function of decreased salinity. Comp Biochem Physiol 1965;15:93 – 102. [31] Lynch MP, Webb KL, Van Engel WA. Variations in the serum constituents of the blue crab Callinectes sapidus: chloride and osmotic concentration. Comp Biochem Physiol 1973;44A:719– 34.
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[32] Mangum CP, Amende LM. Blood osmotic concentration of blue crabs (Callinectes sapidus Rathbun) found in freshwater. Chesapeake Sci 1967;13:318–20. [33] Mangum CP, Silverthorn SU, Harris JL, Towle DW, Krall AR. The relationship between blood pH, ammonia excretion and adaptation to low salinity in the blue crab Callinectes sapidus. J Exp Zool 1976;195:129–36. [34] Mangum CP, Weiland AL. The function of hemocyanin in respiration of the blue crab Callinectes sapidus. J Exp Zool 1975;193:257 – 64. [35] Mantel LH. The foregut of Gecarcinus lateralis as an organ of salt and water balance. Am Zool 1968;8:433–42. [36] McDonald DG, McMahon BR, Wood CM. Patterns of heart and scaphognathite activity in the crabs Cancer magister. J Exp Zool 1977;202:33 – 44. [37] McGaw IJ, Behavioural responses of the shore crab Carcinus maenas to salinity variation. Ph.D. Thesis: University of Wales, 1991. [38] McGaw IJ, Airriess CN, McMahon BR. Patterns of haemolymph flow variation in decapod crustaceans. Mar Biol 1994;121:53 – 60. [39] McGaw IJ, McMahon BR. Cardiovascular responses resulting from variation in external salinity in the Dungeness crab Cancer magister. Physiol Zool 1996;69(6):1384–401. [40] McLaughlin PA, Internal anatomy. In: Mantel LH, editor. The biology of crustacea vol. 5. Internal Anatomy and Physiological Regulation. New York: Academic Press, 1983:1–52. [41] McMahon BR, Burnett LE. The crustacean open circulatory system: a reexamination. Physiol Zool 1991;63:35–71. [42] McMahon BR, Airriess CN, Airriess RA. Swimming in Charybdis feriatus: cardiovascular performance in a crustacean athlete. Am Zool 1996;36:63A. [43] Neufeld GJ, Holliday CW, Pritchard JB. Salinity adaption of gill Na,K-ATPase in the blue crab, Callinectes sapidus. J Exp Zool 1980;211:215 – 24. [44] Pearson J. Cancer (the edible crab). Proc Trans Liverpool Biol Soc 1908;22:291 – 499.
.
[45] Potvin C, Lechowicz MJ, Tardif S. The statistical analysis of ecophysiological response curves from experiments involving related measures. Ecology 1991;71:1389 – 400. [46] Reiber CL, McMahon BR, Burggren WW. Cardiovascular functions in two macruran decapod crustaceans (Procambarus clarkii and Homarus americanus) during periods of inactivity, tail flexion and cardiorespiratory pauses. J Exp Biol 1997;200:1103–13. [47] Sabourin TD. The relationship between fluctuating salinity and oxygen delivery in adult blue crabs. Comp Biochem Physiol 1984;78A:109 – 18. [48] Spaargaren DH. The effect of salinity and temperature on the heart rate of osmoregulating and osmoconforming shrimps. Comp Biochem Physiol 1973;45A:773 – 83. [49] Spaargaren DH. Measurements of the relative rate of blood flow in the shore crab Carcinus maenas. Neth J Sea Res 1974;8:398– 406. [50] Spaargaren DH. On stroke volume of the heart and cardiac output in aquatic animals. Neth J Sea Res 1976;10:131–9. [51] Spaargaren DH. Cardiac output in the shore crab Carcinus maenas in relation to solute exchange and osmotic stress. Mar Biol Lett 1982;3:231 – 40. [52] Tagatz ME. Osmoregulatory ability of blue crabs in different salinity-temperature combinations. Chesapeake Sci 1971;7:30–5. [53] Tan EC, Van Engel WA. Osmoregulation in the adult blue crab Callinectes sapidus. Chesapeake Sci 1966;7(1):30 – 5. [54] Taylor AC. Respiratory responses of Carcinus maenas to changes in environmental salinity. J Mar Biol Ecol 1977;29:197–210. [55] Taylor EW, Butler PW, Al-Wassia A. The effect of a decrease on salinity on respiration, osmoregulation and activity in the shore crab Carcinus maenas at different acclimation temperatures. J Comp Physiol 1977;119:155 – 70. [56] Thomas NJ, Lasiak TA, Naylor E. Salinity preference behaviour in Carcinus. Mar Behav Physiol 1981;7:277 – 82. [57] Wheatly MG. The role of the antennal gland in ion and acid– base balance regulation during hyposaline exposure of the Dungeness crab Cancer magister (Dana). J Comp Physiol 1985;155:445 – 54.
Circulatory modification in the blue crab Callinectes sapidus, during exposure and acclimation to low salinity I.J. McGaw *, C.L. Reiber Department of Biological Sciences, Uni6ersity of Ne6ada, Las Vegas, 4505 Maryland Parkway, Las Vegas, NV 89154 -4004, USA Received 5 March 1998; received in revised form 22 June 1998; accepted 1 July 1998
Abstract A pulsed-Doppler flowmeter was used to measure heart rate and haemolymph flow rates in each arterial system of the blue crab, Callinectes sapidus, enabling calculation of stroke volume and cardiac output. During exposure to a 6–6–12 h salinity cycle of 100–25–100% seawater, there was an immediate increase in heart rate upon dilution of the medium. After this initial increase it decreased steadily, but remained elevated above levels in 100% seawater. A smaller increase in heart rate occurred when the salinity was raised, declining thereafter and reaching pre-treatment levels after 6 h in 100% SW. There was a slight decrease in stroke volume of the heart, but overall this resulted in an increase in cardiac output when the salinity was lowered. Differential haemolymph flow through each major arterial system also occurred. There was an increase in flow rates through the anterior aorta, anterolateral arteries and sternal artery during the first 2 h of low salinity exposure and smaller increases occurred again when the salinity was raised to 100% seawater. No significant changes in flow were observed in the hepatic arteries or posterior aorta. During a 72 h acclimation period in low salinity, similar increases in cardiac parameters and flow rates were observed in the first 6 h. These values declined to levels comparable to those in 100% seawater, after 40 – 50 h acclimation in low salinity. The changes in cardiovascular parameters are not directly related to the osmoregulatory physiology of this species, but appear to be due to specific behaviours occurring in response to low salinity. The results obtained here for this efficient osmoregulator are compared and contrasted with similar studies on Cancer magister, which is classified as a weak hyperosmoregulator. © 1998 Elsevier Science Inc. All rights reserved. Keywords: Callinectes sapidus; Salinity; Cardiovascular; Circulation; Heart rate; Crab osmoregulation
1. Introduction The blue crab Callinectes sapidus, (Crustacea:Decapoda), is an efficient osmoregulator and can survive in a broad range of salinities, from freshwater to hypersaline lagoons [25,32]. Male crabs and larger individuals are reported to be more tolerant of hypo-osmotic conditions, compared with females and juvenile crabs [23]. The osmotic and ionic regulatory physiology of this species is well documented [53,6,52,31,9,16,15,19]. Abbre6iations: SW, seawater; ppt, parts per thousand. * Corresponding author. Tel.: +1 702 8953399; fax: + 1 702 8953956; e-mail: [email protected]
However, there are fewer reports on the cardiovascular physiology of decapod crustaceans exposed to dilute media and findings are controversial within and between species. Sabourin [47] reports an increase in the heart beat frequency of C. sapidus, exposed to a salinity cycle, but no significant change in stroke volume or cardiac output. However, after 2–4 days low salinity acclimation, there is no measurable change in cardiac parameters of C. sapidus [13,47]. Hume and Berlind [27] and Taylor [54] also report an increase in the heart rate of the green shore crab, Carcinus maenas, exposed to seawater in the 35–60% range. Spaargaren [49] reports an increase in heart rate and calculated cardiac output and gill blood flow of C. maenas, in low salinities (below 45% sea water), but observes a decrease in
1095-6433/98/$ - see front matter © 1998 Elsevier Science Inc. All rights reserved. PII S1095-6433(98)10103-4
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seawater of 45–80%. A similar decrease in heart rate occurs in the crab Libinia emarginata when transferred to 80% seawater [10,11]. In contrast, Taylor et al., [55] report that the heart rate of C. maenas remains stable when exposed to 50% sea water. Similar contradictions are seen in several shrimp species [48,14]. Recently we have developed a pulsed-Doppler technique for measurement of cardiac parameters in crustaceans, this has been used to study changes in the cardiac parameters and haemolymph redistribution in the Dungeness crab Cancer magister, during exposure and acclimation to low salinity [39]. When exposed to 50% sea water for short periods (6 h, duplicating a tidal cycle of salinity), C. magister responds with an increase in heart rate, however, the stroke volume decreases, which results in an overall decrease in cardiac output. Differential changes in haemolymph flow through each of the five major arterial systems leaving the heart occurs, but these changes in individual organ perfusion appear unrelated to osmo or ionoregulatory mechanisms. During chronic exposure to 50% seawater (4d), heart rate was still elevated, but stroke volume was depressed to a lower extent than observed following 6 h of exposure. On return to 100% seawater, heart rate decreases, but stroke volume increases, resulting in no significant change in cardiac output. Arterial flows are not significantly depressed, following 4d exposure to 50% seawater and with the exception of a transient increase in flow through the posterior aorta (which supplies the telson and hindgut), do not change significantly on return to 100% sea water [39]. Although C. magister occurs in estuaries, it is classified as a weak hyperosmoregulator [29,18,28] and cannot survive prolonged exposure to salinities below 12 ppt (36% seawater) seawater [8]. The aim of the present study was to investigate the cardiovascular physiology of the efficient hyperosmoregulator C. sapidus, during low salinity exposure and acclimation and to compare and contrast these changes with those occurring during hyposaline exposure in C. magister.
2. Material and methods Adult male blue crabs, Callinectes sapidus (Crustacea:Decapoda), of 12 – 16 cm carapace width (140– 260 g) were obtained from Gulf Specimens, Florida. Crabs were held in a recirculating seawater system (Instant Ocean) 1000 – 1050 mOsm l − 1 (considered as 100% sea water) at a temperature of 18 – 20°C. They were fed chopped liver twice weekly, but were not fed the day prior to experimentation. A 545C directional pulsed-Doppler flowmeter (University Of Iowa, Bioengineering) was used to measure haemolymph flow through each arterial system leaving the heart. Peizo-electric crystal probes (Iowa Doppler
products and Crystal Biotech) were either implanted in grooves abraded in the carapace directly above the artery (anterior and posterior aortae and left anterolateral artery), or guided to lie adjacent to the artery via catheter mounted probes (right hepatic artery and sternal artery). Probe implants were held in place with dental wax and super glue and maximal signal was obtained using the depth focus on the flowmeter. Calibration of the flowmeter and further details of the experimental procedure are detailed elsewhere [2,46]. The voltage output from the flowmeter was recorded as both phasic and mean signals on Sable Systems data acquisition package. Values were converted to ml min − 1 using a modified Doppler Equation [24]: Q= E*D2/(0.2712*cos A), where Q is volume flow in ml min − 1, E is the voltage output from the flowmeter, D is the arterial diameter (mm), 0.2712 is a constant accounting for velocity of sound in blood and cos A is the angle between the sound beam and blood velocity vector. Heart rate was obtained by counting peaks on the phasic traces. Summation of arterial flows (flows for paired arteries were doubled) gave total cardiac output and division of this value by heart rate yielded stroke volume of the heart. Following experimentation the animals were dissected for measurement of arterial diameters and verification of probe implants. The experimental chamber consisted of a covered plastic box (35×20×22 cm) with a recirculating aerated filter, the chamber was filled with sand to a depth of approximately 3 cm. A tap allowed water to be drained without aerially exposing the animal and salinity was changed by adding a known volume of distilled water. Crabs were held in a plastic mesh cage (22 × 14× 8 cm), inside the chamber, this allowed movement, but prevented the crab rolling or twisting and thus damaging probe implants. The crabs were allowed to settle for 12 h after instrumentation and recordings were carried out in constant dim light at a water temperature of 20°C. In a first series of experiments haemolymph flow and heart rates of ten crabs were monitored for 1 min at 15-min intervals over a 6 h period in 1000 mOsm water (considered as 100% sea water). The salinity was then dropped, over a 15 min period, to 25% sea water and animals were monitored for another 6 h. Finally salinity was raised again to 100% sea water, by addition of concentrated saline and animals were monitored for a further 12 h. In a second series of experiments eight animals were recorded for 1 min at 30-min intervals for 12 h in 100% seawater and then for a further 72 h acclimation period in 25% seawater. Salinity response curves were constructed for heart rate (beats min[1]) of eight crabs monitored for total period of 10 min in separate treatments of 100, 75, 50 and 25% sea water and in freshwater, after 15 min
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Fig. 1. Mean(9 S.E.): (a) Heart rate; (b) Stroke volume of the heart; and (c) Cardiac output of ten male Callinectes sapidus exposed to a 6–6–12 h salinity cycle of 100 – 25–100% seawater.
exposure to each condition. Between each treatment the water was raised to 100% seawater and animals allowed to settle for 6 h before exposure to the next test salinity. Finally haemolymph osmolality was measured at set intervals in eight crabs kept for 6 h in 100% seawater and thereafter for a further 72 h in 25% seawater and for a separate group of eight animals exposed to a 6 – 6 –12 h cycle of 100 – 25 – 100% seawater. Samples were collected at set intervals by withdrawing approximately 20 ml of haemolymph from the arthrodial membranes between the walking legs. Osmolality was measured using a vapor pressure osmometer (Wescor Incorporated Model 5100B). One-way ANOVA with repeated measures design [45] was used to test for significant differences in the measured variables, at each time point. The data showing a significant effect, were further analyzed by Fischers LSD multiple comparison test.
3. Results When C. sapidus was exposed to a salinity cycle there was an increase in heart rate from 85 beats min − 1 to 135 beats min − 1 (Fig. 1a), immediately upon salinity reduction. Thereafter, a decrease in heart rate occurred, stable levels of approximately 108 beats min − 1 were reached after 3 h of low salinity exposure, but these
were still significantly elevated above pre-treatment values (F= 3.51, PB0.000). Upon return to 100% seawater there was a slight, but significant increase in heart rate (F=3.23, PB 0.000), decreasing after the salinity changeover period, reaching values of 85 beats min − 1 after 6 h in 100% seawater (Fig. 1a). Stroke volume of the heart was more variable (Fig. 1b), there was a small decrease in mean values (F= 1.9, PB 0.01) which occurred after 2 h of reduction of salinity to 25% seawater. When the salinity was raised there was a slow increase in stroke volume and initial values were only regained towards the end of the experimental period. Changes in total cardiac output, were somewhat similar to those of heart rate. A significant increase in cardiac output (F= 5.79, PB0.000) occurred during the first 2 h (Fig. 1c) of low salinity exposure; rising from approximately 25 to 35 ml min − 1. Cardiac output declined steadily thereafter and reached values comparable to those in 100% seawater (23–28 ml min − 1) after 3 h. These levels of cardiac output were maintained for the remaining period of low salinity exposure and upon return to 100% seawater. There were also significant differences in haemolymph flow rates through each of the five arterial systems (Fig. 2). Although haemolymph flow was somewhat more variable than heart parameters there was no significant difference in haemolymph flow rates in any of the arteries during the initial 100% seawater control
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Fig. 2. Haemolymph flow rates (ml min − 1) (mean 9 S.E.) through the: (a) Anterior aorta; (b) Left anterolateral artery; (c) Right hepatic artery; (d) Posterior aorta; and (e) Sternal artery of C. sapidus (n =10), exposed to a 100 – 25 – 100% cycle of seawater.
period (P\ 0.05). There was a significant and rapid increase in haemolymph flow through the anterior aorta (F =2.41, PB 0.001) (Fig. 2a), anterolateral arteries (F=2.89, P B 0.000) (Fig. 2b) and sternal artery (F = 4.12, P B 0.000) (Fig. 2e) upon salinity reduction. These increased rates were, in each case, maintained for the first 2 h of exposure to 25% seawater. Flow rates in each of these three arteries (Fig. 2a, b, e) decreased after 2 h, reaching levels that were not significantly different from those in 100% seawater. Upon return to 100% seawater, there was a slight increase in flow through each of these arteries (Fig. 2a, b, e), however, this increase only proved to be statistically significant in the anterior aorta (F =1.86, P B 0.001) (Fig. 2a). There was no significant change in flow rates through the hepatic arteries, either during reduction of salinity to 25% seawater, or upon return to 100% seawater (F= 0.92, P\ 0.05) (Fig. 2c). Haemolymph flow through the posterior aorta, was routinely low and in some cases
barely detectable (Fig. 2d). This may have lead to failure to detect any significant changes with salinity (F= 1.44, P\ 0.05) (Fig. 2d). Changes in cardiovascular parameters were also followed continually during a 3 day acclimation period in 25% seawater (Figs. 3 and 4). There was no significant change (P\ 0.05) in heart rate, stroke volume or cardiac output during a 12 h control period in 100% seawater (Fig. 3a–c). During the first 6 h exposure to low salinity, changes in cardiac parameters were similar to those reported for salinity cycling (Fig. 1a–c). The heart rate of C. sapidus increased immediately upon salinity reduction (F= 4.85, PB 0.000), reaching maximal values of 149 beats min − 1, after 1.5 h of low salinity exposure (Fig. 3a). There was a slow and steady decline in heart rate, thereafter. Rates reached stable levels, which were not significantly different from those in 100% seawater, between 40 and 50 h after the initial salinity change-over.
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Fig. 3. (a) Heart rate; (b)Cardiac stroke volume; and (c) Cardiac output (Mean 9 S.E.) of eight C. sapidus, during 12 h in 100% seawater and 72 h of acclimation to 25% seawater.
Stroke volume of the heart decreased during the first 8 h of low salinity exposure (Fig. 3b) and increased steadily afterwards. However, due to the small magnitude of changes and the large inter-individual variability, these changes proved to be statistically insignificant (F =1.40, P\ 0.05). Cardiac output increased from around 30 to 45 ml min − 1 when the salinity was lowered (F = 2.23, PB 0.000) (Fig. 3c) followed by a short-term decrease in output after 2 h. A subsequent increase at 8 h was sustained for 50 h of low salinity acclimation, after which levels declined to those comparable in 100% seawater. Differential changes in flow rates through each arterial system were also observed during acclimation to low salinity (Fig. 4). There was a significant increase in flow through the anterior aorta (F =2.83, P B 0.000) (Fig. 4a) after 2 h exposure to low salinity, although flow rates decreased somewhat after this period, they still remained elevated for about 40 h afterwards. Haemolymph flow increased immediately in the anterolateral arteries (F= 2.60, P B0.000) (Fig. 4b) and remained elevated above control values for 7 h before declining steadily. Flow rates also increased significantly through the sternal artery (F =2.09, P B 0.000) (Fig. 4e) and remained elevated above control levels for the duration of the acclimation period. As with salinity cycling (Fig. 2) there was no significant change in flow through the hepatic arteries (F= 0.89, P \ 0.05) (Fig. 4c). There was a significant
decrease in flow through the posterior aorta (F=2.67, PB 0.000) upon salinity reduction (Fig. 4d), which was not observed during the salinity cycling experiment (Fig. 2d). The fact, however, that flow also changed significantly in this vessel during the control period (F= 6.93, PB 0.000) suggests that these changes were unrelated to salinity and were simply a result of the low and often intermittent flow observed in this artery. Heart rate (and presumably other cardiac parameters) changes inversely with salinity (Fig. 5). In 100% seawater, heart rate of the eight experimental animals varied between 60 and 140 beats min − 1 with a mean value of 929 8.5 beats min − 1. Heart rate increased significantly as the salinity decreased (F=4.51 PB 0.005) reaching a maximum of 1279 6 beats min − 1 in 25% seawater. Finally the haemolymph osmolality was monitored during salinity cycles and a 3 day acclimation period to 25% seawater (Fig. 6a, b). During a salinity cycle of 100–25–100% seawater, haemolymph osmolality decreased steadily from 10609 9 mOsm in 100% seawater, reaching the lowest level of 73697 mOsm after 6 h in 25% seawater (Fig. 6a). The osmolality of the haemolymph increased slowly when the salinity was raised and pre-treatment levels were only regained after 10 h in 100% seawater. Haemolymph osmolality fell rapidly during the first 10 h of long-term acclimation to 25% seawater (Fig. 6b). Stable levels of 500 mOsm were reached at around 18 h and maintained for the duration of the experimental period.
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Fig. 4. Mean ( 9 S.E.) haemolymph flow rates (ml min − 1) in eight C. sapidus exposed to 12 h of 100% seawater and 72 h of 25% seawater. (a) Anterior aorta; (b) Left anterolateral artery; (c) Right hepatic artery; (d) Hepatic artery; and (e) Sternal artery.
4. Discussion Variation in cardiovascular parameters between individual C. sapidus was high, this is common in decapod crustaceans maintained under identical experimental conditions [5,4,12,38]. In the present study we attempted to reduce some of this inherent variation by providing a layer of sand in the experimental chamber for crabs to bury into and minimally restraining them. Responses to external stimuli, as well as inter-individual differences in heart-rate can be reduced this way [21,12,36] The range of heart rates reported here, for C. sapidus, are similar to those in the literature [13,47]. The increase in heart rate observed in C. sapidus (Fig. 1a) in low salinity conforms with the majority of reports on crustaceans during acute exposure to dilute media [49,27,54,47,37,39]. However, DeFur and Mangum [13], find that the heart rate of C. sapidus is unchanged after
2–4 days exposure to similar salinities (8–20 ppt, 25– 60% SW). In the present study, after an initial increase in heart rate in 25% SW, it declines steadily (Fig. 3a) reaching levels similar to those in full strength seawater after 40–50 h. Therefore, it appears heart rate of blue crabs is only affected during acute exposure to low salinity and returns to normal levels after 2d in low salinity. In C. magister, although the heart rate also increases upon exposure to 50% SW, the changes are different to C. sapidus. Heart rate continues to rise during acute exposure to low salinity and remains elevated 25–30% above resting levels after 4–5 days low salinity acclimation [39]. Changes in stroke volume of the heart and cardiac output also differed between C. sapidus and C. magister. A small decrease in stroke volume occurred in C. sapidus in low salinity (Fig. 1b, Fig. 3b). This decrease probably results passively from a reduced filling time at elevated rates [21]. The decrease in stroke volume had
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little effect on total cardiac output, which tended to be governed by changes in heart rate and cardiac output therefore increased during low salinity exposure (Fig. 1c, Fig. 3c). In C. magister, stroke volume decreases significantly in 50% SW leading to an overall decrease in cardiac output [39]. In cancrid crabs stroke volume of the heart, rather than rate plays a greater role in determining cardiac output [41,1,3,38,39]. The opposite appears to be true in portunid crabs, where changes in heartbeat frequency largely determine total cardiac output ([49–51,42], Figs. 1 and 3). Ultimately increases in cardiac output will be followed by increased blood flow through the gills. This would aid oxygen uptake and active ion uptake and an increase in ventilation frequency and oxygen uptake is seen in C. sapidus in dilute seawater [30,17,20,47]. If heart rate and hence cardiac output of C. sapidus is representative of oxygen uptake, the linear increase in heart rate with decreasing salinity, shown in the salinity response data (Fig. 5), would reflect the increasing metabolic demand involved in osmotic and ionic regulation. Although both heart rate and oxygen uptake increase as soon as the medium is diluted, the time course for changes in heart rate and cardiac output during low salinity acclimation do not follow the same time course as changes in oxygen consumption [54,20,47]. It is possible that the changes in cardiac parameters are not closely coupled with the respiratory/ osmoregulatory physiology, but rather, reflect some other type of demand. Modulation of haemolymph flow also occurs upon lowering of the salinity. Different patterns of organ perfusion were seen in the blue crabs when compared with the weak hyperosmoregulator C. magister. There was an increase in flow through the anterior aorta during the first few hours in dilute seawater (Fig. 2a and Fig. 4a). This vessel supplies the supraoesophageal
Fig. 5. Salinity response curve of heart rate (mean 9 S.E.) of C. sapidus (n =8) in salinities of 100, 75, 50, 25 and 0% seawater.
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ganglion, eyestalks and antennae of the crab [44,40]. The antenna have been implicated in playing a chemosensory role in low salinity [22] and it is possible that increased flow to these organs could enhance this function. This response was the opposite to that observed for C. magister, where a decrease in flow was observed [39]. It is notable that the pattern of flow through the anterior aorta was very different between the two species. In C. magister haemolymph flow is usually undetectable and this artery is only perfused sporadically [38] or during periods of emersion [3]. In C. sapidus, the anterior aorta was more-or-less perfused continually and received a much higher percentage of total cardiac output. There was also an increase in flow through the anterolateral arteries during the first 2–3 h of low salinity exposure (Fig. 2b) and flow remained significantly elevated for about 8 h in low salinity (Fig. 4b). This vessel, supplies the foregut, some of the mouthparts and the antennal gland [44,40]. These increases could represent regulation of body fluids at the gut level, since the foregut has been implicated in osmotic and ionic regulation in some species of Crustacea [35]. The anterolateral artery also supplies blood to the antennal gland via its branches, thus increased flow could represent increased filtration and production of iso-osmotic urine, resulting from the increased water loading. The time period of the increase, however, is shorter than reported changes of urine production times in low salinity and thus increased flow may not represent urine production [33,7,57]. Pressure is the driving force for filtration, which was not measured in the present study and it is possible that this could vary independently of flow. In C. magister flow actually decreases through the anterolateral arteries [39], reasons for these discrepancies are unclear. The greatest increase in haemolymph flow occurred through the sternal artery (Fig. 2e, Fig. 4e), which supplies the legs, chelae and mouth parts [44,40]. Crabs are known to exhibit increased locomotor activity in low salinity defined as halokinesis [56], presumably acting as an escape response from unfavourable environments. However, in C. sapidus there is no significant difference in locomotor activity in low salinity, compared with 100% SW (McGaw et al, in prep). Indeed this species can survive in freshwater [32] and escape responses would therefore be inappropriate. The changes in haemolymph flow through the sternal artery are opposite to those for C. magister, where a decrease in flow through this vessel occurs [39], again reasons why this occurs are unclear, but overall decreases in flow lead to a decrease in cardiac output through the gills of C. magister and thus may reduce the gradient for inward water flux and diffusive ion loss. The time periods for changes in cardiac parameters or flow in C. sapidus, do not correlate with changes in
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Fig. 6. Mean haemolymph osmolality (mOsm) of eight male C. sapidus exposed to (a) 6 – 6 – 12 h cycle of 100 – 25 – 100% seawater; and (b) during acclimation over a 72 h period in 25% seawater.
osmolality of the blood, which decreased steadily in low salinity stabilizing after about 18 h (Fig. 6). Nor do they appear related to time course of changes in ATPase activity [43] or acid – base balance [26]. Recently we have categorized new behaviours of blue crabs in low salinity (McGaw et al., In prep). These behaviours include, increased movements of the mouthparts and cleaning of mouthparts with claws, these structures are supplied by the anterolateral arteries and sternal artery respectively and increased flicking and cleaning of the antennae, which are involved in osmoreception, [22] and are supplied by the anterior aorta. The duration and intensity of these behaviours correlate closely with those reported for blood flow and cardiac parameters. It therefore, appears that increased blood flow (which is
aided by increased heart beat frequency and differential contraction/relaxation of the cardioarterial valves) is not related directly to osmoregulatory function, but to increased demand due to the above mentioned behaviours, which were elicited by changes in salinity. The reason for the behaviours is not clear, but could be due to irritation of the membranes on the exopodites of the mouthparts, or the sensilla of the antenna, which are sloughed off in low salinity [22]. There was no significant change in flow, related to salinity, in the hepatic arteries or posterior aorta, (Figs. 2 and 4 c, d). This is as expected since these vessels supply the digestive gland and telson respectively, which are not involved in osmoregulation. In C. sapidus flow rates through the small posterior aorta were very
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low and intermittent, changing without obvious cause, this would have lead to detection of significant changes in flow in this vessel even during control periods (Fig. 4d). In C. magister flow through the posterior aorta actually increases [39]. Individuals of this species extend and retract the telson in dilute seawater, possibly exposing the membrane of the rectum for ion uptake (McGaw et al., In prep). This could account for extra blood delivery to this area and this behaviour was not observed in C. sapidus. In general flow rates through each vessel of C. sapidus, were somewhat higher than those reported for C. magister [39], despite the blue crab being about a third of the size. This is probably due to a number of factors: Callinectes is a much more active animal with a higher metabolic rate and cardiac output [34,36]; indeed considering its size, its vessels are greater in diameter (unpublished data). Clearly there are differences in the cardiovascular physiology of C. magister, which is classed as a weak osmoregulator and the efficient hyperosmoregulator C. sapidus. The changes reported here for blue crabs appear to be related to specific behaviours rather than directly to the osmoregulatory physiology. It is worthy to note that even under resting conditions in 100% SW there are interspecific differences in flow through each arterial system and control of cardiac output, as yet reasons are still unclear, but this area warrants further investigation. References [1] Airriess CN, McMahon BR. Cardiovascular adaptations enhance tolerance of environmental hypoxia in the crabs Cancer magister. J Exp Biol 1994;190:23–41. [2] Airriess CN, McMahon BR, McGaw IJ, Bourne GB. Application and in situ calibration of a pulsed-Doppler flowmeter for blood flow measurement in crustaceans. J Mar Biol Assoc UK 1994;74:445 – 58. [3] Airriess CN, McMahon BR. Short-term emersion effects cardiac function and regional haemolymph distribution in the crab Cancer magister. J Exp Biol 1996;199:3569–78. [4] Aldrich JC. On the relationship between oxygen consumption and feeding levels in decapods. In: Barnes H, editor. Proceedings of the Ninth European Marine Biology Symposium, Aberdeen, Aberdeen University Press, 1975:407–418. [5] Ansell AD. Changes in oxygen consumption, heart rate and ventilation accompanying starvation in the decapod Cancer pagurus. Neth J Sea Res 1973;7:455–75. [6] Ballard BS, Abbott JP. Osmotic accommodation in Callinectes sapidus Rathbun. Comp Biochem Physiol 1969;29:671–87. [7] Cameron JN, Batterton CV. Antennal gland function in the freshwater blue crab, Callinectes sapidus: water, electrolyte, acid base and ammonia excretion. J Comp Physiol 1978;123:143 – 8. [8] Cleaver FC. Preliminary results of the coastal (Cancer magister) investigation. Dept Fisheries Wash State Biol Rep 1957;49A:47 – 82. [9] Colvocoresses JA, Lynch MP, Webb KL. Variations in the serum constituents of the blue crab Callinectes sapidus: major cations. Comp Biochem Physiol 1974;49A:787–803.
75
[10] Cornell JC. A reduction in water permeability in response to a dilute medium in the stenohaline crab Libinia emarginata (Brachyura, Majidae). Biol Bull 1973;145:430 – 1. [11] Cornell JC. A reduction in water exchange rates in an osmoconforming crab. Am Zool 1974;14:1259. [12] Cumberlidge N, Uglow RF. Heart and scaphognathite activity in the shore crab Carcinus maenas (L.). J Exp Mar Biol Ecol 1977;28:87 – 107. [13] DeFur PL, Mangum CP. The effects of environmental variables on the heart rates of invertebrates. Comp Biochem Physiol 1979;62A:283 – 94. [14] Dyer MF, Uglow RF. Cardiac and ventilatory responses associated with altered salinity in Crangon crangon. Ophelia 1980;19:117 – 25. [15] Engel DW. Comparison of the osmoregulatory capabilities of two portunid crabs Callinectes sapidus and C. similis. Mar Biol 1977;41:275 – 9. [16] Engel DW, Davis EM, Smith DE, Angelovic JW. The effect of salinity and temperature on the ion levels in the hemolymph of the blue crab Callinectes sapidus, Rathbun. Comp Biochem Physiol 1974;49A:259 – 66. [17] Engel DW, Ferguson RL, Eggert LD. Respiration rates and ATP concentrations in the excised gills of the blue crab as a function of salinity. Comp Biochem Physiol 1975;52A:669–73. [18] Engelhardt FR, Dehnel PA. Ionic regulation in the Pacific edible crab Cancer magister (Dana). Can J Zool 1973;51:735 –43. [19] Findley AM, Stickle WB. Effects of salinity fluctuation on the hemolymph composition of the blue crab Callinectes sapidus. Mar Biol 1978;46:9 – 15. [20] Findley AM, Belisle BW, Stickle WB. Effects of salinity fluctuations on the respiration rate of the southern oyster drill Thais haemastoma and the blue crab Callinectes sapidus. Mar Biol 1978;49:59 – 67. [21] Florey E, Kriebel ME. The effects of temperature, anoxia and sensory stimulation on the heart rate of unrestrained crabs. Comp Biochem Physiol 1974;48A:285 – 300. [22] Gleeson RA, Wheatly MG, Reiber CL. Perireceptor mechanisms sustaining olfaction at low salinities: insight from the euryhaline blue crab. J Exp Biol 1997;200:445 – 56. [23] Haefner PA, Shuster CN. Length increments during terminal molt of the female blue crab, Callinectes sapidus, in different salinity environments. Chesapeake Sci 1964;5(3):114 – 8. [24] Hartley CJ, Cole JS. An ultrasonic pulsed-Doppler system for measuring blood flow in small vessels. J Appl Physiol 1974;37:626 – 9. [25] Hedgpeth J. Ecological aspects of the Laguna Madre, a hypersaline estuary. In: Lauff GH, editor. Estuaries. Washington DC: Am Assoc Adv Sci, 1967:408 – 19. [26] Henry RP, Cameron JN. Acid – base balance in Callinectes sapidus during acclimation from high to low salinity. J Exp Biol 1982;101:255 – 64. [27] Hume RI, Berlind A. Heart and scaphognathite changes in a euryhaline crab Carcinus maenas exposed to a dilute environmental medium. Biol Bull 1976;150:241 – 54. [28] Hunter KC, Rudy PP. Osmotic and ionic regulation in the Dungeness crab Cancer magister (Dana). Comp Biochem Physiol 1975;51A:439 – 47. [29] Jones LL. Osmotic regulation in several crabs of the Pacific coast of North America. J Cell Com Physiol 1941;18:79 – 91. [30] King EN. The oxygen consumption of intact crab and excised gills as a function of decreased salinity. Comp Biochem Physiol 1965;15:93 – 102. [31] Lynch MP, Webb KL, Van Engel WA. Variations in the serum constituents of the blue crab Callinectes sapidus: chloride and osmotic concentration. Comp Biochem Physiol 1973;44A:719– 34.
76
I.J. McGaw, C.L. Reiber / Comparati6e Biochemistry and Physiology, Part A 121 (1998) 67–76
[32] Mangum CP, Amende LM. Blood osmotic concentration of blue crabs (Callinectes sapidus Rathbun) found in freshwater. Chesapeake Sci 1967;13:318–20. [33] Mangum CP, Silverthorn SU, Harris JL, Towle DW, Krall AR. The relationship between blood pH, ammonia excretion and adaptation to low salinity in the blue crab Callinectes sapidus. J Exp Zool 1976;195:129–36. [34] Mangum CP, Weiland AL. The function of hemocyanin in respiration of the blue crab Callinectes sapidus. J Exp Zool 1975;193:257 – 64. [35] Mantel LH. The foregut of Gecarcinus lateralis as an organ of salt and water balance. Am Zool 1968;8:433–42. [36] McDonald DG, McMahon BR, Wood CM. Patterns of heart and scaphognathite activity in the crabs Cancer magister. J Exp Zool 1977;202:33 – 44. [37] McGaw IJ, Behavioural responses of the shore crab Carcinus maenas to salinity variation. Ph.D. Thesis: University of Wales, 1991. [38] McGaw IJ, Airriess CN, McMahon BR. Patterns of haemolymph flow variation in decapod crustaceans. Mar Biol 1994;121:53 – 60. [39] McGaw IJ, McMahon BR. Cardiovascular responses resulting from variation in external salinity in the Dungeness crab Cancer magister. Physiol Zool 1996;69(6):1384–401. [40] McLaughlin PA, Internal anatomy. In: Mantel LH, editor. The biology of crustacea vol. 5. Internal Anatomy and Physiological Regulation. New York: Academic Press, 1983:1–52. [41] McMahon BR, Burnett LE. The crustacean open circulatory system: a reexamination. Physiol Zool 1991;63:35–71. [42] McMahon BR, Airriess CN, Airriess RA. Swimming in Charybdis feriatus: cardiovascular performance in a crustacean athlete. Am Zool 1996;36:63A. [43] Neufeld GJ, Holliday CW, Pritchard JB. Salinity adaption of gill Na,K-ATPase in the blue crab, Callinectes sapidus. J Exp Zool 1980;211:215 – 24. [44] Pearson J. Cancer (the edible crab). Proc Trans Liverpool Biol Soc 1908;22:291 – 499.
.
[45] Potvin C, Lechowicz MJ, Tardif S. The statistical analysis of ecophysiological response curves from experiments involving related measures. Ecology 1991;71:1389 – 400. [46] Reiber CL, McMahon BR, Burggren WW. Cardiovascular functions in two macruran decapod crustaceans (Procambarus clarkii and Homarus americanus) during periods of inactivity, tail flexion and cardiorespiratory pauses. J Exp Biol 1997;200:1103–13. [47] Sabourin TD. The relationship between fluctuating salinity and oxygen delivery in adult blue crabs. Comp Biochem Physiol 1984;78A:109 – 18. [48] Spaargaren DH. The effect of salinity and temperature on the heart rate of osmoregulating and osmoconforming shrimps. Comp Biochem Physiol 1973;45A:773 – 83. [49] Spaargaren DH. Measurements of the relative rate of blood flow in the shore crab Carcinus maenas. Neth J Sea Res 1974;8:398– 406. [50] Spaargaren DH. On stroke volume of the heart and cardiac output in aquatic animals. Neth J Sea Res 1976;10:131–9. [51] Spaargaren DH. Cardiac output in the shore crab Carcinus maenas in relation to solute exchange and osmotic stress. Mar Biol Lett 1982;3:231 – 40. [52] Tagatz ME. Osmoregulatory ability of blue crabs in different salinity-temperature combinations. Chesapeake Sci 1971;7:30–5. [53] Tan EC, Van Engel WA. Osmoregulation in the adult blue crab Callinectes sapidus. Chesapeake Sci 1966;7(1):30 – 5. [54] Taylor AC. Respiratory responses of Carcinus maenas to changes in environmental salinity. J Mar Biol Ecol 1977;29:197–210. [55] Taylor EW, Butler PW, Al-Wassia A. The effect of a decrease on salinity on respiration, osmoregulation and activity in the shore crab Carcinus maenas at different acclimation temperatures. J Comp Physiol 1977;119:155 – 70. [56] Thomas NJ, Lasiak TA, Naylor E. Salinity preference behaviour in Carcinus. Mar Behav Physiol 1981;7:277 – 82. [57] Wheatly MG. The role of the antennal gland in ion and acid– base balance regulation during hyposaline exposure of the Dungeness crab Cancer magister (Dana). J Comp Physiol 1985;155:445 – 54.