- Email: [email protected]
An estimation of the haemolymph volume in Gammarus pulex
Coral. Biochem. PhydoL, 1968, Vol. 26, pp. 1123 to 1125. PergamonPress. Printedin Great Britain
SHORT COMMUNICATION AN ESTIMATION OF THE HAEMOLYMPH VOLUME IN G A M M A R U S PULEX P E N E L O P E E. B U T T E R W O R T H The Molteno Institute, University of Cambridge
(Received 5 March 1968) Abmtra©t--1. The haemolymph volume of Gammarus pulex was measured by means of isotope dilution using C 14 labelled inulin in a physiological saline. 2. The haemolymph volume was found to be 26 ± 5 per cent wet weight of
G.pu/~. 3. The dry weight of G. pulex was found to be 20"45 per cent of the total wet weight. INTRODUCTION AN ESTIMATIONof the volume of h a e m o l y m p h in Gammarus pulex was required for a physiological study of the parasite Polymorphgs minutus (Goeze, 1782) (Acanthocephala), during its development in the haemocoele of this amphipod. MATERIALS AND METHODS Haemolyph volume of G. pulex was measured by a dilution method involving the use of inulin labelled with C 14. After washing and drying the outer surfaces of the amphipods, they were conf.ned in an apparatus designed to facilitate the introduction of materials into the haemocoele of G. pulex (Crompton, 1967). A physiological saline was prepared for G. pulex; this was derived from Van Harrevald's basic salt solution (Lockwood, 1961), but contained glucose and amino acids. One pl of saline containing a known amount of C!4 inulin, was injected into the haemocoele of G. pule~ with a Hamilton microsyringe inserted through an abdominal intersegmental membrane slightly to the right or left of the mid-dorsal line. The syringe was held in this position for 1 or 2 rain to reduce haemorrhage. It was then removed and the haemolyph taken up by capillarity using 1 pl Drummond micro-pipettes. Small samples of haemolyph (up to 3 pl) were recovered; squeezing the amphipods was avoided as this would have damaged the tissues and introduced impurities. The Drummond pipettes were discharged into counting vials containing 7 ml of scintillation fluid. This consisted of P.P.O.--2,5-diphenyloxazole--and P.O.P.O.P.--1,4-di-[2-(5-phenyloxazolyl)]-benzene--in toluene and ethanol 7 : 3 v/v (Hall & Cocking, 1965). Each pipette was washed three times in this fluid. Standards to estimate the initial radioactivity of the saline were aho prepared. All determinations of radioactivity were made with a Panax A.C. 300/6 water-cooled liquid scintillation counter. The radioactivity in each vial was determined five times for 100 s and the background count was subtracted. The dead time with this instrument was only 20 Vts, which amounts to less than 0"25 per cent of all counts below 2000; therefore, a dead-time correction was made only when the counts exceeded 2000. 1123
1124
PENELOPEE. BUTT~WORTH
The volume of haemolymph in each Gammarus was calculated from the equation: X
(1/y).n where x is the number of counts in 1 ~1 of saline injected, y is the volume of haemolymph recovered from the Garamarus in izl and n is the average number of counts for y pl. After removing the haemolymph the amphipods were placed in tin-foil trays, dried at 60°C for 24 hr, and weighed on an electro-microbalance (R.I.I.C. EMB 1). The wet weight and dry weight of ten fresh Gammarus were measured and from these figures the dry weight was calculated as a percentage of the wet weight. RESULTS AND DISCUSSION T h e relationship between haemolymph volume and dry weight of Gammarus pu/ex is shown in Fig. 1. An average haemolymph volume of 1.27 + 0.25 ~I/mg dry weight was found, but it may be less in the smaller shrimps. T h e dry weight of 28
7
o/
24
2o
E
>o
E6
E --~
2
.,r
0
I 4
I I 8 12 Dry w e i g h t ,
I 16
I 20
mg
FIG. 1. Ratio between haemolymph volume and dry weight of Gammarus pulex. O, C 14 inulin circulated in amphipod 1-2 min; l , C x4 inulin circulated in amphipod 5 rain.
G. pulex was found to be 20.45 per cent of the wet weight; the haemolymph volume was, therefore, 26 _+5 per cent body wet weight. It should be remembered that this is really a measurement of the total extracellular fluid volume, since the Crustacea have an open circulation. I know of no comparable results from crustaceans of similar size to Garamarus pulex, but this result is close to that recorded by Prosser & Weinstein (1950) for
THE HAEMOLYMPH VOLUME IN G A M M A R U S P U L E X
1125
the crayfish Cambarus virilis in which they found the body fluid constituted 25" 125.6 per cent body wet weight using techniques involving the injection of Evans blue and thiocyanate. Other estimations of crustacean haemolymph volumes have been made on Homarus (Burgher & Smyth, 1953), Maja (Zuckerkandl, 1960), Carcinus maenus (Nagel, 1934) and Eriocheir (Krogh, 1939), in which the haemolymph volume was found to be 17, 8-29, 37 and 35 per cent of the body weight respectively. Since most of these estimations are recorded as percentages of the body weight, the condition of the exoskeleton will undoubtedly give rise to variation within and between species. Haemolymph volume recorded as percentage body weight will also vary with the moulting cycle and this range was shown to be as great as 8-27 per cent body weight in M a j a (Zuckerkandl, 1960). Riegal & Parker (1960) considered the tracer material C 14 inulin well suited for use in the Crustacea, because, as far as is known, it does not enter the cells or combine with proteins and the only space accessible to it, other than the circulating haemolymph, is the lumen and bladder of the kidney. Riegal & Kirschner (1960) showed that the crayfish excreted inulin through its green gland. In the present experiment with Gamrnarus if excretion were considerable, the results would show a higher blood volume than did in fact exist. However, the tracer material was usually left in the amphipods for only 1-2 rain, but when it was left in for 5 min (Fig. 1) the estimated volume of haemolymph indicated that there had been no significant excretion in the experimental period. Acknowledgements--My thanks are due to my supervisor, Dr. D. W. T. Crompton for helpful advice. This work was carried out during the tenure of an S.R.C. studentship.
REFERENCES BURGHERJ. W. & SMYTH C. M. (1953) The general form of circulation in the lobster, Homarus. 07. cell. comp. Physiol. 42, 369-383. CROMPTOND. W. T. (1967) Studies on the haemocytic reaction of Gammarus spp. and its relationship to Polymorphus minutus (Acanthocephala). Parasitology 57, 389--401. HALL T. C. & COCKINGE. C. (1965) High-efficiency liquid-scintillation counting of 14Clabelled material in aqueous solution and determination of specific activity of labelled proteins. Biochem. 07. 96, 626-633. KROGrIA. (1939) Osmotic Regulation in Aquatic Animals, 233 pp. Cambridge University Press, Cambridge. LOCXWOODA. P. M. (1961) 'Ringer' solutions and some notes on the physiological basis of their chemical composition. Comp. Biochem. Physiol. 2, 241-289. NACELH. (1934) Die Aufgaben der Excretionsorgane und der Kiemen bei der Osmoregulation von Carcinus maenus. Z. vergl. Physiol. 21, 468-469. PROSSERC. L. & WEINSTEINS. J. F. (1950) Comparison of blood volume in animals with open and with closed circulatory systems. Physiol. Zool. 23, 113-124. RmCALJ. A. & KIRSCrrNERL. B. (1960) The excretion of inulin and glucose by the crayfish antennal glands. Biol. Bull., Woods Hole 118, 296-307. RmGALJ. A. & PARV,~RR. A. (1960) A comparative study of crayfish blood volumes. Comp. Biochem. Physiol. 1, 302-304. ZUC~RKANDL E. (1960) Y a-t-il plus d'un "espace extracellulaire" chez les crustac6s d~capodes ? Cah. Biol. mar. 1, 25-35.
SHORT COMMUNICATION AN ESTIMATION OF THE HAEMOLYMPH VOLUME IN G A M M A R U S PULEX P E N E L O P E E. B U T T E R W O R T H The Molteno Institute, University of Cambridge
(Received 5 March 1968) Abmtra©t--1. The haemolymph volume of Gammarus pulex was measured by means of isotope dilution using C 14 labelled inulin in a physiological saline. 2. The haemolymph volume was found to be 26 ± 5 per cent wet weight of
G.pu/~. 3. The dry weight of G. pulex was found to be 20"45 per cent of the total wet weight. INTRODUCTION AN ESTIMATIONof the volume of h a e m o l y m p h in Gammarus pulex was required for a physiological study of the parasite Polymorphgs minutus (Goeze, 1782) (Acanthocephala), during its development in the haemocoele of this amphipod. MATERIALS AND METHODS Haemolyph volume of G. pulex was measured by a dilution method involving the use of inulin labelled with C 14. After washing and drying the outer surfaces of the amphipods, they were conf.ned in an apparatus designed to facilitate the introduction of materials into the haemocoele of G. pulex (Crompton, 1967). A physiological saline was prepared for G. pulex; this was derived from Van Harrevald's basic salt solution (Lockwood, 1961), but contained glucose and amino acids. One pl of saline containing a known amount of C!4 inulin, was injected into the haemocoele of G. pule~ with a Hamilton microsyringe inserted through an abdominal intersegmental membrane slightly to the right or left of the mid-dorsal line. The syringe was held in this position for 1 or 2 rain to reduce haemorrhage. It was then removed and the haemolyph taken up by capillarity using 1 pl Drummond micro-pipettes. Small samples of haemolyph (up to 3 pl) were recovered; squeezing the amphipods was avoided as this would have damaged the tissues and introduced impurities. The Drummond pipettes were discharged into counting vials containing 7 ml of scintillation fluid. This consisted of P.P.O.--2,5-diphenyloxazole--and P.O.P.O.P.--1,4-di-[2-(5-phenyloxazolyl)]-benzene--in toluene and ethanol 7 : 3 v/v (Hall & Cocking, 1965). Each pipette was washed three times in this fluid. Standards to estimate the initial radioactivity of the saline were aho prepared. All determinations of radioactivity were made with a Panax A.C. 300/6 water-cooled liquid scintillation counter. The radioactivity in each vial was determined five times for 100 s and the background count was subtracted. The dead time with this instrument was only 20 Vts, which amounts to less than 0"25 per cent of all counts below 2000; therefore, a dead-time correction was made only when the counts exceeded 2000. 1123
1124
PENELOPEE. BUTT~WORTH
The volume of haemolymph in each Gammarus was calculated from the equation: X
(1/y).n where x is the number of counts in 1 ~1 of saline injected, y is the volume of haemolymph recovered from the Garamarus in izl and n is the average number of counts for y pl. After removing the haemolymph the amphipods were placed in tin-foil trays, dried at 60°C for 24 hr, and weighed on an electro-microbalance (R.I.I.C. EMB 1). The wet weight and dry weight of ten fresh Gammarus were measured and from these figures the dry weight was calculated as a percentage of the wet weight. RESULTS AND DISCUSSION T h e relationship between haemolymph volume and dry weight of Gammarus pu/ex is shown in Fig. 1. An average haemolymph volume of 1.27 + 0.25 ~I/mg dry weight was found, but it may be less in the smaller shrimps. T h e dry weight of 28
7
o/
24
2o
E
>o
E6
E --~
2
.,r
0
I 4
I I 8 12 Dry w e i g h t ,
I 16
I 20
mg
FIG. 1. Ratio between haemolymph volume and dry weight of Gammarus pulex. O, C 14 inulin circulated in amphipod 1-2 min; l , C x4 inulin circulated in amphipod 5 rain.
G. pulex was found to be 20.45 per cent of the wet weight; the haemolymph volume was, therefore, 26 _+5 per cent body wet weight. It should be remembered that this is really a measurement of the total extracellular fluid volume, since the Crustacea have an open circulation. I know of no comparable results from crustaceans of similar size to Garamarus pulex, but this result is close to that recorded by Prosser & Weinstein (1950) for
THE HAEMOLYMPH VOLUME IN G A M M A R U S P U L E X
1125
the crayfish Cambarus virilis in which they found the body fluid constituted 25" 125.6 per cent body wet weight using techniques involving the injection of Evans blue and thiocyanate. Other estimations of crustacean haemolymph volumes have been made on Homarus (Burgher & Smyth, 1953), Maja (Zuckerkandl, 1960), Carcinus maenus (Nagel, 1934) and Eriocheir (Krogh, 1939), in which the haemolymph volume was found to be 17, 8-29, 37 and 35 per cent of the body weight respectively. Since most of these estimations are recorded as percentages of the body weight, the condition of the exoskeleton will undoubtedly give rise to variation within and between species. Haemolymph volume recorded as percentage body weight will also vary with the moulting cycle and this range was shown to be as great as 8-27 per cent body weight in M a j a (Zuckerkandl, 1960). Riegal & Parker (1960) considered the tracer material C 14 inulin well suited for use in the Crustacea, because, as far as is known, it does not enter the cells or combine with proteins and the only space accessible to it, other than the circulating haemolymph, is the lumen and bladder of the kidney. Riegal & Kirschner (1960) showed that the crayfish excreted inulin through its green gland. In the present experiment with Gamrnarus if excretion were considerable, the results would show a higher blood volume than did in fact exist. However, the tracer material was usually left in the amphipods for only 1-2 rain, but when it was left in for 5 min (Fig. 1) the estimated volume of haemolymph indicated that there had been no significant excretion in the experimental period. Acknowledgements--My thanks are due to my supervisor, Dr. D. W. T. Crompton for helpful advice. This work was carried out during the tenure of an S.R.C. studentship.
REFERENCES BURGHERJ. W. & SMYTH C. M. (1953) The general form of circulation in the lobster, Homarus. 07. cell. comp. Physiol. 42, 369-383. CROMPTOND. W. T. (1967) Studies on the haemocytic reaction of Gammarus spp. and its relationship to Polymorphus minutus (Acanthocephala). Parasitology 57, 389--401. HALL T. C. & COCKINGE. C. (1965) High-efficiency liquid-scintillation counting of 14Clabelled material in aqueous solution and determination of specific activity of labelled proteins. Biochem. 07. 96, 626-633. KROGrIA. (1939) Osmotic Regulation in Aquatic Animals, 233 pp. Cambridge University Press, Cambridge. LOCXWOODA. P. M. (1961) 'Ringer' solutions and some notes on the physiological basis of their chemical composition. Comp. Biochem. Physiol. 2, 241-289. NACELH. (1934) Die Aufgaben der Excretionsorgane und der Kiemen bei der Osmoregulation von Carcinus maenus. Z. vergl. Physiol. 21, 468-469. PROSSERC. L. & WEINSTEINS. J. F. (1950) Comparison of blood volume in animals with open and with closed circulatory systems. Physiol. Zool. 23, 113-124. RmCALJ. A. & KIRSCrrNERL. B. (1960) The excretion of inulin and glucose by the crayfish antennal glands. Biol. Bull., Woods Hole 118, 296-307. RmGALJ. A. & PARV,~RR. A. (1960) A comparative study of crayfish blood volumes. Comp. Biochem. Physiol. 1, 302-304. ZUC~RKANDL E. (1960) Y a-t-il plus d'un "espace extracellulaire" chez les crustac6s d~capodes ? Cah. Biol. mar. 1, 25-35.