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Metal ion metabolism in the moulting crayfish (Austropotamobius pallipes)
0300-9629~‘82,050073-04103.00 0 0 1982Pergamon Pres Ltd
METAL ION METABOLISM IN THE MOULTING CRAYFISH (AUSTROPOTAMOBZUS PALLIPES) ELIZABETH ADAMS, K. Department
of Zoology.
University
(Rvcriwd
SIMKISSand
MARINA
TAYLOR
of Reading. Whiteknights.
14 Srprrrnher
Reading.
U.K.
198 I)
Abstract - 1. The hepatopancreas. antenna1 gland. gills. gonads. muscle. stomach. carapace. gastroliths and blood of 52 crayfish (Atr.st,oporamohius pulliprs) have been analysed for copper. zinc, iron. manganese and calcium throughout the moult cycle. 2. There are significant changes in most of these metals at the time of ecdysis. 3. The results are interpreted in terms of sites of metal storage and mobilization in the hepatopancreas and evidence is presented to suggest that copper and zinc are metabolized along related but independent pathways.
vation. Thus. Alikhan (1972) found that the moult cycle had little effect upon the haemocyanin and hepatopancreas copper systems in Porcellio but that starving resulted in a pronounced loss of haemolymph copper and an increased storage of hepatopancress copper. Since many crustaceans cease to feed during moult these two influences obviously interact to varying degrees according to the life histories of the particular species being studied. The Lellular storage of copper ions is related in some organisms to changes in zinc metabolism. The intracellular protein, metallothionein. has been implicated as a short term storage molecule for both these ions (Cherian & Coyer. 1978) although other workers have suggested that this protein is really part of a detoxification system (Winge & Rajagopalan. 1972). It is interesting to note therefore that zinc concentrations in the hepatopancreas of the crab Concrr vary with the moult cycle and also show positive correlations with the copper content (Martin. 1975). It appears. therefore. that the hepatopancreas of crustaceans may be particularly suitable for studies on the intracellular regulation of a number of metals. We have therefore tried to answer the following questions: What changes in metal concentration occur in the blood and hepatopancreas tissue of the moulting crayfish? Is it possible to interpret any changes that occur in terms of (a) the metabolism of common intracellular ligands or (b) competition for binding to such ligands?
INTRODUCTION
The study of the metabolism of trace elements by invertebrates is currently attracting renewed interest because of the insights that it might provide into the inorganic biochemistry of cells. Of the essential metals that are required in trace amounts by all cells the ones that have been most studied are iron. copper and zinc. Elucidation of the universal role of ferritin. as a molecule that has evolved to meet the requirements of storing iron in a soluble and easily available form in plant and animal cells (Munro & Linder, 1978) was facilitated by studies on vertebrates that have a high requirement for iron in their respiratory pigment. By analogy, therefore, it has been suggested that it might be possible to elucidate the copper and zinc storage systems by studying animals that synthesize the copper-containing respiratory pigment, haemocyanin or which depend upon the zinc-containing enzyme carbonic anhydrase during moult. The crayfish (Austropotamobius pallipes) meets all these requirements. Haemocyanin is synthesized in the digestive gland or hepatopancreas of crustaceans (Senkbeil & Wriston, 1981). In many crustaceans this organ stores large quantities of copper (Zuckerkandl. 1960; Kerkut (‘r t/l.. 1961: Bryan. 1968; Djangmah. 1970; Alikhan. 1972; Martin. 1975) and the cellular sites of these deposits can often be identified as vesicular accumulations of copper ligands (Wieser & Klima. 1969: Walker. 1977; Brown. 1978; Icely & Nott. 1980). Studies on the details of copper metabolism in these animals are. however, complicated by a number of physiological influences. Perhaps the most confusing of these is the influence of the moult cycle upon haemocyanin production. A number of workers have proposed that the moult cycle causes oscillations in the haemocyanin content of the blood with a consequent formation and mobilization of copper deposits in the hepatopancreas (e.g. Zuckerkandl, 1960, with Maia: Martin. 1975. with Cancer). In other studies these variations in blood and hepatopancreas copper levels do not appear to be synchronized (e.g. Kerkut c’t r/l.. 1962 with Ctrrcinus) and at least one additional major influence upon these problems is that of star-
SIATERIALS
AND METHODS
The common crayfish (A~fstroporamohi~r.s polliprs) was collected by diving in a reservoir near Banbury. Oxfordshire. They were kept in large plastic tanks in aerated tap water and fed on a diet of commercial fish pellets (Pond Price. B.P. Nutrition). Animals which had been kept in captivity for several months often had very variable contents of metals and this study has therefore been restricted to specimens that were analysed within a few weeks of capture. The stage of the moult cycle was assessed using the criteria of Passano (1960) summarized in Table 1. A total of 52 animals taken al suitable stages of the moult cycle were 73
ELIZAHIITH ADAMS ~‘t ul.
74
T‘able 1. Stages of the moult cycle (after Passano. Description
‘I,, of cycle
stage
A, Postmoult
16.5
C
21
_
21+
-
D, DI D3 > Dd EI I
Premoult Moult
+
2.5
+ _ -
Intermoult
Characteristics
Gastroliths
A? B, BZ >
17
+ + +
1960)
Soft exoskeleton Branchiostegites Branchiostegites
Fully formed
flexible semi rigid
exoskeleton
Secretion on new spines on telson Branchiostegites flexible Exuvial line apparent Exoskeleton splits
>
weighed. sexed. and a blood sample collected with a hypodermic needle from under the posterior end of the cephalothorax. The animals were killed. dissected and the tissue dried at 90 C before weighing and dissolving the material in 10 ml of boiling concentrated nitric acid and 5 ml concentrated perchloric acid. Reagent blanks were prepared at the same time and the solutions were then made up to 25 ml using 68 mmol.dl- ’ KC1 to suppress ionization effects. Analyses for copper. zinc, iron, manganese and calcium were performed on a Varian 175 atomic absorption spectrophotometer. RESULTS
The hepatopancreas. antenna1 gland. gills. gonads, muscle, stomach. carapace, gastroliths and blood of 52 animals were analysed for copper, zinc. iron, manganese and calcium. Animals were taken at all stages of the moult cycle but to simplify the interpretation of results only significant trends are reported and the moult stages A, and A2. B, and B2 and D1 to DS are grouped together. All results were calculated as pmo1.g dry wt-’ except for blood values which are given as mmol.ml- ‘. Analyses of the calcium content of the carapace and gastroliths confirmed the moult cycle. Gastroliths
C
D
E
Stage
A
Copper Zinc Iron Manganese Calcium
2.77 0.20 2.08 0.17 13.9
+ f & i +
appear at stage D, of premoult and increase in calcium content up to ecdysis (E) and post moult (A) before decreasing in mineral content at A2 and disappearing by stage B,. At moult the shed carapace con-
Ecdysis (E,-E,)
2.35 0.36 3.53 0.19 11.35
* Values are mmol.cm-3
0.86 1.16 2.02 0.005 3.28
+ i_ + + k
0.37 1.97 2.47 0.01 1.29
Table 3. Metal content of the hepatopancreas cycle*
Copper Zinc Iron Manganese Calcium
0.30 1.56 4.44 0.54 17.43
* Values are prno1.g
& + f f f
Postmoult
(A,+21 1.49 0.91 1.2 0.07 9.28
f * + f +
lB,mB2)
0.93 0.38 I.88 0.17 6.94
1.63 0.58 2.29 0.1 8.73
+ k f + +
0.66 0.7 3.87 0.16 4.6
f SD.
Premoult (D,mD,l 0.12 1.13 1.24 0.44 5.44 dry wt-’
Ecdysis (E,-Ez) 0.25 1.50 4.00 0.55 20.06 k SD
C
Fig. I. Calcium concentration of the carapace at various stages of the moult cycle. The shaded region of stage E (ecdysis) represents the shed carapace. Black rectangles represent the stages at which gastroliths are present.
Table 2. Metal content of crayfish blood during moult cycle* Premouh (D, -DJ
B
of moult cycle
* 0.05 +- 1.00 + 1.41 * 0.24 & 7.03
of the crayfish during the moult Postmoult (A,+21 0.71 1.61 4.07 0.33 19.34
f i * f 5
1.48 0.67 1.94 0.29 6.48
(B,-~B,) 1.30 + 2.86 f 7.86 + 0.48 + 33.32 +
1.19 1.68 7.31 0.68 12.87
Metal
Table
4.
Metal
ion metabolism
in the mautting
analyses of the stomach of the Ecdysis
Zinc Iron Manganese Calcium
0.20 1.67 3.75 1.32 734.0
+ & t: f. +
(A,-A,)
0.14 1.04 2.63 1.16 152.0
* Values are pmo1.g dry wt-’
1.11 1.71 3.61 2.51 745.0 f
* * + + +
Copper Zinc Iron Manganese Calcium
0.50 * 0.58 7.37 5 1.06 11.33+ 4.44 26.55 + 14.25
* Values are grno1.g dry wt-’
February 0.35 1.55 4.64 2.73 27.39
+ i 2 f )
Intermoult
(B,m&)
1.58 2.83 6.55 4.38 981.0
0.31 1.26 8.20 2.13 1146.0
* + * + _t
0.22 0.74 8.51 2.33 772.0
(C) 0.60 1.96 4.36 6.42 1910.0
+ & * f _t
0.31 1.51 6.14, 7.23 1198.0
SD.
Table 5. Concentrations of metals in the hepatopancreas of intcrmoult crayfish in autumn and mid winter* October
75
crayfish during the mouft cycle*
Postmoult
(E,-EL) copper
crayfish
0.13 0.52 1.49 1.66 18.8
_t SD.
tains 7.0 $- 4.7 mmol Ca.g dry wt-’ whereas the new exoskeleton contains only 0.52 f 0.43 mmol Ca.g dry wt - ’ and remains poorly calcified until intermoult (Fig. 1). The metal content of the blood throughout this cycle is shown in Table 2 as mmol . ml- ‘. Corresponding data for the concentration of copper, zinc, iron, manganese and calcium in the hepatopancreas are shown in Table 3. Apart from seasonal changes in the gonads there are no clear changes in the metal content of other organs except for the stomach and the analyses of this organ are shown in Table 4.
In an extensive study of the calcium metabolism of the moulting crayfish Greenaway (1974a.b) found that the haemolymph calcium level rose to 16.2 mmol. dl-’ at premoult and then fell to 12.0 mmol.dl-’ at postmoult. These values are similar to the premoult value of 13.9 and postmoult concentration of 9.2 mmoI.dl-’ found in this study. During actual ecdysis. however. the calcium level fell in our study to 3.3 mmol .dl- ’ and this probably reflects the dynamic changes which occur in mineral metabolism at this time. There is a continual loss of calcium from the body during premoult as the exoskeleton is resorbed and this reaches a very high rate in stages D3 -0, immediately before moult. This calcium is presumably transported in the blood and results in the elevated and variable level at this time. Within about 30 minutes of the completion of moult the calcmm balance is reversed and there is a rapid uptake resulting in a positive calcium balance. Thus the blood passes rapidly from a stage where it is involved in transporting calcium for excretion from the body to one where it is involved in the uptake from the environment (Greenaway. 1974a.b). Simultaneous with this change there is a considerable uptake of ucuter a few hours before and after ecdysis via the intestinal tract and possibly the gills (Mykles. 1980).
The changeover from efFlux to influx and the diluting effects of water uptake probably account for the very low values in blood calcium at the time of ecdysis. There are similar. though less extreme changes in the levels of other metals in the blood during this time (Table 2). In all cases there is a fall either during ecdysis (Cu. Mn. Ca) or at stage A of postmoult immediately afterwards (Zn. Fe). These ~uctuations can, variously be ascribed to dilution of the blood. starvation or the formation of body stores during the process of moult. The most obvious of these stores arc the gastroliths of the stomach which form a conspicuous deposit of calcium that is later resorbed during the calcification of the exoskeleton (Fig. I). Despite this, most of the calcium for the formation of the exoskeleton comes via an active uptake from the environmental water although a small amount may also be stored in and resorbed from the hepatopancreas (Table 3) in a way that was demonstrated autoradiographically by Miyawaki & Sasaki (1961). The use of the hepatopan~reas as a store for metal ions during the moult cycle appears to be a common phenomenon, for copper. zinc and iron are all accumulated in this way and of the metals studied in these experiments only manganese is not deposited there (Table 3). The direct physiological cause for this metal uptake by the hepatopancreas is not clear. There are three possibilities. Starvation. induced by the weakening of the exoskeleton during moult. may lead to a metabolism of blood proteins. a loss of haemocyanin and an increased storage of metal in the hepatopancreas. Alternatively the activity of the hepatopancreas cells may be directly influenced by the moult cycle or there may be an increased turnover of these cells. Of these possibilities only starvation may be commented upon at the present time. lntermoult animals collected in October had a slightly higher metal content than similar animals collected in February (Table 5)~ These latter animals were almost certainly starving in that they were extremely lethargic but it is difficult to relate this to their metabolic rate which would be similarly depressed at these low environmental temperatures (5 C). in this respect it is interesting to note that there is a large and apparently transient increase in the metal content of the stomach after ecdysis (Table 4) and prior to the increase in hepatopancreas metal content (Table 3). This could be interpreted as due to the resumption of feeding after the moult but one really needs flux data in order to decide whether the metals are being stored at this site or whether there is an increased uptake of metals from the food at this time. Evidence for this possibility was found by Bryan (1967) who injected solutions into the stomach of crayfish and observed :t
ELIZABETH ADAMS et ul
76
6 Zn /c”
‘&, D* ’
~~~rr~,,cledyr,nr,nr.s -One of us (Elizabeth Adams) wishes to thank the Natural Research Environment Research Council for a studentship. This work was also supported by NERC grant GR3!‘3063A and SRC grant GR/A 87746. ‘C*\
4
\
\
\
A*
2
REFERENCES \ 8\*,
Zn + Cu (mmo1.g dry wt?
Fig. 2. The ratio of Zn/Cu in the hepatopancreas at various stages of the moult cycle (A -+ E). Note that the ratio of the two metals is normally correlated with their total metal content, shown for convenience by the broken line. Immediately post moult (stage A) this relationship no longer holds indicating the preferential storage of copper in the hepatopancreas at this time.
subsequent increase in the level of this metal in the hepatopancreas. The fact that the copper and possibly zinc content of the hepatopancreas increase during stage A of the postmoult whereas iron and calcium increase later during stage B (Table 3) could be interpreted as demonstrating two separate systems. Support for this concept is available histologically since it is claimed that copper and possibly zinc are metabolized by R cells (Ogura. 1959; Lyon, unpublished) whereas iron and calcium are concentrated in the F cell (Ogura, 1959: Miyawaki and Sasaki. 1961). Plotting all the data for copper and zinc concentrations in the hepatopancreas throughout the moult cycle gives a highly significant positive correlation (P < 0.001). The two metals are related by the equation [Cu] = 0.58 [Zn] - 0.56(r = 0.81) 01
PI [al]
1
xpz=j’
In more physiological terms this indicates how copper is preferentially mobilized before moult and then stored postmoult in the hepatopancreas. This phenomenon could be due to either competition between the two metals for a limited number of common ligands or to a separate synthesis and metabolism of two metalloproteins. These possibilities are tested in Fig. 2 where the ratio of Zn/Cu is plotted against the total content of these two metals throughout the moult cycle. It is clear that immediately postmoult (stage A), copper is preferentially stored relative to zinc and that the situation is then reversed at stage B. It is concluded. therefore. that although the content of the two metals is strongly correlated (Fig. 2. broken line) there is no evidence for a simple competition between copper and zinc for a limited number of protein ligands but that each metal is probably metabolized along separate biochemical pathways.
ALIKHAN M. A. (1972) Haemolymph and hepatopancreas copper in Porwllio Itrcvis Latreille (Porcellionidae. Peracarda). Camp. Bioc,kem. P/1!,.+/. 42A. 823-832. BROWN B. E. (1978) Lead detoxification by a copper-tolerant isopod. Nutuw. Lmd. 276, 388-390. BRYAN G. W. (1967) Zinc regulation in the freshwater crayfish (including some comparative copper-analyses). J. r.up. Biol. 46. 281-296. BRYAN G. W. (19681 Concentrations of zinc and copper in the tissues of decapod crustaceans. J. ,ncrr. Biol. A5.s. U.K. 48, 303-321. CHFKIAN M. G. & GOYEK R. A. (1978) Metallothioneins and their role in the metabolism and toxicity of metals. /& Sci. 23. I-10. DJANC;MAHJ. S. (1970) The efTects of feeding and starvation on copper in the blood and hepatopancreas and on blood proteins of C,unyon cw/guri.\. Camp. Biochmt. P/I!,siol. 32, 709-731. GREENAWAY P. (1974a) Calcium balance at the premoult stage of the freshwater crayfish .Illst,oportrnt~jhilr\ pullipes (Lereboullet), J. r.yp. Biol. 61. 27-34. GREENAWAY P. (1974b) Calcium balance at the postmoult stage of the freshwater crayfish .4I,.st,opottrnlohilrs ptrlliprs (Lereboullet). J. zup. Biol. 61. 35 -45. ICELY J. D. & NOTT J. A. (1980) Accumulation of copper within “hepatopancreatic” caecae of Corophium whrtutot (Crustacea: Amphipoda). &fur. Eiol. 57, 193-199. KERKCT G. A.. M~RITZ P. M. & MLXDAY K. A. (1961) Variations of copper concentrations in Curcinw mkww. Ctrh. Biol. ,hftrr. 2, 399-408. MARTIN J-L. M. (1975) Le cuivre et le zmc chez Cuncer irroratus (Crustace: Decapode): Metabolisme compare au tours du cycle d’intermue. Camp. Biochem. Physiol. SlA, 777-784. MIYAWAI(I M. & SASAU N. (1961) A preliminary report on uptake of CaS’ by the hepatopancreas of crayfish Promnhm~s c,lurkii. Kwmmot0 J. Sci. 5, l70- 172. MUNRO H. N. & LINDER M. N. (1978) Ferritin: structure. biosynthesis and role m iron metabolism. Phnir~l. Rcr. 58. 3 177396.
MYKLES D. L. (1980) The mechanism of fluid absorption at ecdysis in the American lobster Uorntrr~~.s cmrrictrnus. .I. c’.xp. Biol. 84, 89-101. OGURA K. (1959) Midgut gland cells accumulatmg iron or copper in the crayfish P,oconhtrtx.\ c/wLii. Amot. Zoo/. Jopun 32, 133. 14’. PASSANO L. M. (1960) Moulting and its control. In Physioloyy of Crustucru. Vol. 1 (Edited by WATERMAN T. H.). pp. 473-536. Academic Press. New York. SENKBEIL E. G. & WRISTON J. C. (1981) Hemocyanin synthesis in the American lobster Horntrr~h mwkmu. Camp. Bi~hm. PIl~siol. 638. 163- 17 1. WALKER G. (1977) Copper granules in the barnacle &I/~UIU~ hilomoidt~s.
Mw.
Biol. 39, 343 -349.
W~ESER W. & KUMA J. (1969) Compartmentalization of copper in the hepatopancreas of isopods. Mikroskopir 22. 1-9.
WINCE D. R. & RAJAGOPALAN H. V. (19771 Purification and some properties of cadmium-bindmg protein from rat liver. A&s Biochrnl. Biophj~s. 153. 755 762. ZLCKEKKANDL E. (1960) Hemocyanine et cuivre chez un rustace decapode dans leurs rapports avec le cycle d’intermue. Am. Inst. Owur~o~~r.. .$I onm o 38. I - 122.
METAL ION METABOLISM IN THE MOULTING CRAYFISH (AUSTROPOTAMOBZUS PALLIPES) ELIZABETH ADAMS, K. Department
of Zoology.
University
(Rvcriwd
SIMKISSand
MARINA
TAYLOR
of Reading. Whiteknights.
14 Srprrrnher
Reading.
U.K.
198 I)
Abstract - 1. The hepatopancreas. antenna1 gland. gills. gonads. muscle. stomach. carapace. gastroliths and blood of 52 crayfish (Atr.st,oporamohius pulliprs) have been analysed for copper. zinc, iron. manganese and calcium throughout the moult cycle. 2. There are significant changes in most of these metals at the time of ecdysis. 3. The results are interpreted in terms of sites of metal storage and mobilization in the hepatopancreas and evidence is presented to suggest that copper and zinc are metabolized along related but independent pathways.
vation. Thus. Alikhan (1972) found that the moult cycle had little effect upon the haemocyanin and hepatopancreas copper systems in Porcellio but that starving resulted in a pronounced loss of haemolymph copper and an increased storage of hepatopancress copper. Since many crustaceans cease to feed during moult these two influences obviously interact to varying degrees according to the life histories of the particular species being studied. The Lellular storage of copper ions is related in some organisms to changes in zinc metabolism. The intracellular protein, metallothionein. has been implicated as a short term storage molecule for both these ions (Cherian & Coyer. 1978) although other workers have suggested that this protein is really part of a detoxification system (Winge & Rajagopalan. 1972). It is interesting to note therefore that zinc concentrations in the hepatopancreas of the crab Concrr vary with the moult cycle and also show positive correlations with the copper content (Martin. 1975). It appears. therefore. that the hepatopancreas of crustaceans may be particularly suitable for studies on the intracellular regulation of a number of metals. We have therefore tried to answer the following questions: What changes in metal concentration occur in the blood and hepatopancreas tissue of the moulting crayfish? Is it possible to interpret any changes that occur in terms of (a) the metabolism of common intracellular ligands or (b) competition for binding to such ligands?
INTRODUCTION
The study of the metabolism of trace elements by invertebrates is currently attracting renewed interest because of the insights that it might provide into the inorganic biochemistry of cells. Of the essential metals that are required in trace amounts by all cells the ones that have been most studied are iron. copper and zinc. Elucidation of the universal role of ferritin. as a molecule that has evolved to meet the requirements of storing iron in a soluble and easily available form in plant and animal cells (Munro & Linder, 1978) was facilitated by studies on vertebrates that have a high requirement for iron in their respiratory pigment. By analogy, therefore, it has been suggested that it might be possible to elucidate the copper and zinc storage systems by studying animals that synthesize the copper-containing respiratory pigment, haemocyanin or which depend upon the zinc-containing enzyme carbonic anhydrase during moult. The crayfish (Austropotamobius pallipes) meets all these requirements. Haemocyanin is synthesized in the digestive gland or hepatopancreas of crustaceans (Senkbeil & Wriston, 1981). In many crustaceans this organ stores large quantities of copper (Zuckerkandl. 1960; Kerkut (‘r t/l.. 1961: Bryan. 1968; Djangmah. 1970; Alikhan. 1972; Martin. 1975) and the cellular sites of these deposits can often be identified as vesicular accumulations of copper ligands (Wieser & Klima. 1969: Walker. 1977; Brown. 1978; Icely & Nott. 1980). Studies on the details of copper metabolism in these animals are. however, complicated by a number of physiological influences. Perhaps the most confusing of these is the influence of the moult cycle upon haemocyanin production. A number of workers have proposed that the moult cycle causes oscillations in the haemocyanin content of the blood with a consequent formation and mobilization of copper deposits in the hepatopancreas (e.g. Zuckerkandl, 1960, with Maia: Martin. 1975. with Cancer). In other studies these variations in blood and hepatopancreas copper levels do not appear to be synchronized (e.g. Kerkut c’t r/l.. 1962 with Ctrrcinus) and at least one additional major influence upon these problems is that of star-
SIATERIALS
AND METHODS
The common crayfish (A~fstroporamohi~r.s polliprs) was collected by diving in a reservoir near Banbury. Oxfordshire. They were kept in large plastic tanks in aerated tap water and fed on a diet of commercial fish pellets (Pond Price. B.P. Nutrition). Animals which had been kept in captivity for several months often had very variable contents of metals and this study has therefore been restricted to specimens that were analysed within a few weeks of capture. The stage of the moult cycle was assessed using the criteria of Passano (1960) summarized in Table 1. A total of 52 animals taken al suitable stages of the moult cycle were 73
ELIZAHIITH ADAMS ~‘t ul.
74
T‘able 1. Stages of the moult cycle (after Passano. Description
‘I,, of cycle
stage
A, Postmoult
16.5
C
21
_
21+
-
D, DI D3 > Dd EI I
Premoult Moult
+
2.5
+ _ -
Intermoult
Characteristics
Gastroliths
A? B, BZ >
17
+ + +
1960)
Soft exoskeleton Branchiostegites Branchiostegites
Fully formed
flexible semi rigid
exoskeleton
Secretion on new spines on telson Branchiostegites flexible Exuvial line apparent Exoskeleton splits
>
weighed. sexed. and a blood sample collected with a hypodermic needle from under the posterior end of the cephalothorax. The animals were killed. dissected and the tissue dried at 90 C before weighing and dissolving the material in 10 ml of boiling concentrated nitric acid and 5 ml concentrated perchloric acid. Reagent blanks were prepared at the same time and the solutions were then made up to 25 ml using 68 mmol.dl- ’ KC1 to suppress ionization effects. Analyses for copper. zinc, iron, manganese and calcium were performed on a Varian 175 atomic absorption spectrophotometer. RESULTS
The hepatopancreas. antenna1 gland. gills. gonads, muscle, stomach. carapace, gastroliths and blood of 52 animals were analysed for copper, zinc. iron, manganese and calcium. Animals were taken at all stages of the moult cycle but to simplify the interpretation of results only significant trends are reported and the moult stages A, and A2. B, and B2 and D1 to DS are grouped together. All results were calculated as pmo1.g dry wt-’ except for blood values which are given as mmol.ml- ‘. Analyses of the calcium content of the carapace and gastroliths confirmed the moult cycle. Gastroliths
C
D
E
Stage
A
Copper Zinc Iron Manganese Calcium
2.77 0.20 2.08 0.17 13.9
+ f & i +
appear at stage D, of premoult and increase in calcium content up to ecdysis (E) and post moult (A) before decreasing in mineral content at A2 and disappearing by stage B,. At moult the shed carapace con-
Ecdysis (E,-E,)
2.35 0.36 3.53 0.19 11.35
* Values are mmol.cm-3
0.86 1.16 2.02 0.005 3.28
+ i_ + + k
0.37 1.97 2.47 0.01 1.29
Table 3. Metal content of the hepatopancreas cycle*
Copper Zinc Iron Manganese Calcium
0.30 1.56 4.44 0.54 17.43
* Values are prno1.g
& + f f f
Postmoult
(A,+21 1.49 0.91 1.2 0.07 9.28
f * + f +
lB,mB2)
0.93 0.38 I.88 0.17 6.94
1.63 0.58 2.29 0.1 8.73
+ k f + +
0.66 0.7 3.87 0.16 4.6
f SD.
Premoult (D,mD,l 0.12 1.13 1.24 0.44 5.44 dry wt-’
Ecdysis (E,-Ez) 0.25 1.50 4.00 0.55 20.06 k SD
C
Fig. I. Calcium concentration of the carapace at various stages of the moult cycle. The shaded region of stage E (ecdysis) represents the shed carapace. Black rectangles represent the stages at which gastroliths are present.
Table 2. Metal content of crayfish blood during moult cycle* Premouh (D, -DJ
B
of moult cycle
* 0.05 +- 1.00 + 1.41 * 0.24 & 7.03
of the crayfish during the moult Postmoult (A,+21 0.71 1.61 4.07 0.33 19.34
f i * f 5
1.48 0.67 1.94 0.29 6.48
(B,-~B,) 1.30 + 2.86 f 7.86 + 0.48 + 33.32 +
1.19 1.68 7.31 0.68 12.87
Metal
Table
4.
Metal
ion metabolism
in the mautting
analyses of the stomach of the Ecdysis
Zinc Iron Manganese Calcium
0.20 1.67 3.75 1.32 734.0
+ & t: f. +
(A,-A,)
0.14 1.04 2.63 1.16 152.0
* Values are pmo1.g dry wt-’
1.11 1.71 3.61 2.51 745.0 f
* * + + +
Copper Zinc Iron Manganese Calcium
0.50 * 0.58 7.37 5 1.06 11.33+ 4.44 26.55 + 14.25
* Values are grno1.g dry wt-’
February 0.35 1.55 4.64 2.73 27.39
+ i 2 f )
Intermoult
(B,m&)
1.58 2.83 6.55 4.38 981.0
0.31 1.26 8.20 2.13 1146.0
* + * + _t
0.22 0.74 8.51 2.33 772.0
(C) 0.60 1.96 4.36 6.42 1910.0
+ & * f _t
0.31 1.51 6.14, 7.23 1198.0
SD.
Table 5. Concentrations of metals in the hepatopancreas of intcrmoult crayfish in autumn and mid winter* October
75
crayfish during the mouft cycle*
Postmoult
(E,-EL) copper
crayfish
0.13 0.52 1.49 1.66 18.8
_t SD.
tains 7.0 $- 4.7 mmol Ca.g dry wt-’ whereas the new exoskeleton contains only 0.52 f 0.43 mmol Ca.g dry wt - ’ and remains poorly calcified until intermoult (Fig. 1). The metal content of the blood throughout this cycle is shown in Table 2 as mmol . ml- ‘. Corresponding data for the concentration of copper, zinc, iron, manganese and calcium in the hepatopancreas are shown in Table 3. Apart from seasonal changes in the gonads there are no clear changes in the metal content of other organs except for the stomach and the analyses of this organ are shown in Table 4.
In an extensive study of the calcium metabolism of the moulting crayfish Greenaway (1974a.b) found that the haemolymph calcium level rose to 16.2 mmol. dl-’ at premoult and then fell to 12.0 mmol.dl-’ at postmoult. These values are similar to the premoult value of 13.9 and postmoult concentration of 9.2 mmoI.dl-’ found in this study. During actual ecdysis. however. the calcium level fell in our study to 3.3 mmol .dl- ’ and this probably reflects the dynamic changes which occur in mineral metabolism at this time. There is a continual loss of calcium from the body during premoult as the exoskeleton is resorbed and this reaches a very high rate in stages D3 -0, immediately before moult. This calcium is presumably transported in the blood and results in the elevated and variable level at this time. Within about 30 minutes of the completion of moult the calcmm balance is reversed and there is a rapid uptake resulting in a positive calcium balance. Thus the blood passes rapidly from a stage where it is involved in transporting calcium for excretion from the body to one where it is involved in the uptake from the environment (Greenaway. 1974a.b). Simultaneous with this change there is a considerable uptake of ucuter a few hours before and after ecdysis via the intestinal tract and possibly the gills (Mykles. 1980).
The changeover from efFlux to influx and the diluting effects of water uptake probably account for the very low values in blood calcium at the time of ecdysis. There are similar. though less extreme changes in the levels of other metals in the blood during this time (Table 2). In all cases there is a fall either during ecdysis (Cu. Mn. Ca) or at stage A of postmoult immediately afterwards (Zn. Fe). These ~uctuations can, variously be ascribed to dilution of the blood. starvation or the formation of body stores during the process of moult. The most obvious of these stores arc the gastroliths of the stomach which form a conspicuous deposit of calcium that is later resorbed during the calcification of the exoskeleton (Fig. I). Despite this, most of the calcium for the formation of the exoskeleton comes via an active uptake from the environmental water although a small amount may also be stored in and resorbed from the hepatopancreas (Table 3) in a way that was demonstrated autoradiographically by Miyawaki & Sasaki (1961). The use of the hepatopan~reas as a store for metal ions during the moult cycle appears to be a common phenomenon, for copper. zinc and iron are all accumulated in this way and of the metals studied in these experiments only manganese is not deposited there (Table 3). The direct physiological cause for this metal uptake by the hepatopancreas is not clear. There are three possibilities. Starvation. induced by the weakening of the exoskeleton during moult. may lead to a metabolism of blood proteins. a loss of haemocyanin and an increased storage of metal in the hepatopancreas. Alternatively the activity of the hepatopancreas cells may be directly influenced by the moult cycle or there may be an increased turnover of these cells. Of these possibilities only starvation may be commented upon at the present time. lntermoult animals collected in October had a slightly higher metal content than similar animals collected in February (Table 5)~ These latter animals were almost certainly starving in that they were extremely lethargic but it is difficult to relate this to their metabolic rate which would be similarly depressed at these low environmental temperatures (5 C). in this respect it is interesting to note that there is a large and apparently transient increase in the metal content of the stomach after ecdysis (Table 4) and prior to the increase in hepatopancreas metal content (Table 3). This could be interpreted as due to the resumption of feeding after the moult but one really needs flux data in order to decide whether the metals are being stored at this site or whether there is an increased uptake of metals from the food at this time. Evidence for this possibility was found by Bryan (1967) who injected solutions into the stomach of crayfish and observed :t
ELIZABETH ADAMS et ul
76
6 Zn /c”
‘&, D* ’
~~~rr~,,cledyr,nr,nr.s -One of us (Elizabeth Adams) wishes to thank the Natural Research Environment Research Council for a studentship. This work was also supported by NERC grant GR3!‘3063A and SRC grant GR/A 87746. ‘C*\
4
\
\
\
A*
2
REFERENCES \ 8\*,
Zn + Cu (mmo1.g dry wt?
Fig. 2. The ratio of Zn/Cu in the hepatopancreas at various stages of the moult cycle (A -+ E). Note that the ratio of the two metals is normally correlated with their total metal content, shown for convenience by the broken line. Immediately post moult (stage A) this relationship no longer holds indicating the preferential storage of copper in the hepatopancreas at this time.
subsequent increase in the level of this metal in the hepatopancreas. The fact that the copper and possibly zinc content of the hepatopancreas increase during stage A of the postmoult whereas iron and calcium increase later during stage B (Table 3) could be interpreted as demonstrating two separate systems. Support for this concept is available histologically since it is claimed that copper and possibly zinc are metabolized by R cells (Ogura. 1959; Lyon, unpublished) whereas iron and calcium are concentrated in the F cell (Ogura, 1959: Miyawaki and Sasaki. 1961). Plotting all the data for copper and zinc concentrations in the hepatopancreas throughout the moult cycle gives a highly significant positive correlation (P < 0.001). The two metals are related by the equation [Cu] = 0.58 [Zn] - 0.56(r = 0.81) 01
PI [al]
1
xpz=j’
In more physiological terms this indicates how copper is preferentially mobilized before moult and then stored postmoult in the hepatopancreas. This phenomenon could be due to either competition between the two metals for a limited number of common ligands or to a separate synthesis and metabolism of two metalloproteins. These possibilities are tested in Fig. 2 where the ratio of Zn/Cu is plotted against the total content of these two metals throughout the moult cycle. It is clear that immediately postmoult (stage A), copper is preferentially stored relative to zinc and that the situation is then reversed at stage B. It is concluded. therefore. that although the content of the two metals is strongly correlated (Fig. 2. broken line) there is no evidence for a simple competition between copper and zinc for a limited number of protein ligands but that each metal is probably metabolized along separate biochemical pathways.
ALIKHAN M. A. (1972) Haemolymph and hepatopancreas copper in Porwllio Itrcvis Latreille (Porcellionidae. Peracarda). Camp. Bioc,kem. P/1!,.+/. 42A. 823-832. BROWN B. E. (1978) Lead detoxification by a copper-tolerant isopod. Nutuw. Lmd. 276, 388-390. BRYAN G. W. (1967) Zinc regulation in the freshwater crayfish (including some comparative copper-analyses). J. r.up. Biol. 46. 281-296. BRYAN G. W. (19681 Concentrations of zinc and copper in the tissues of decapod crustaceans. J. ,ncrr. Biol. A5.s. U.K. 48, 303-321. CHFKIAN M. G. & GOYEK R. A. (1978) Metallothioneins and their role in the metabolism and toxicity of metals. /& Sci. 23. I-10. DJANC;MAHJ. S. (1970) The efTects of feeding and starvation on copper in the blood and hepatopancreas and on blood proteins of C,unyon cw/guri.\. Camp. Biochmt. P/I!,siol. 32, 709-731. GREENAWAY P. (1974a) Calcium balance at the premoult stage of the freshwater crayfish .Illst,oportrnt~jhilr\ pullipes (Lereboullet), J. r.yp. Biol. 61. 27-34. GREENAWAY P. (1974b) Calcium balance at the postmoult stage of the freshwater crayfish .4I,.st,opottrnlohilrs ptrlliprs (Lereboullet). J. zup. Biol. 61. 35 -45. ICELY J. D. & NOTT J. A. (1980) Accumulation of copper within “hepatopancreatic” caecae of Corophium whrtutot (Crustacea: Amphipoda). &fur. Eiol. 57, 193-199. KERKCT G. A.. M~RITZ P. M. & MLXDAY K. A. (1961) Variations of copper concentrations in Curcinw mkww. Ctrh. Biol. ,hftrr. 2, 399-408. MARTIN J-L. M. (1975) Le cuivre et le zmc chez Cuncer irroratus (Crustace: Decapode): Metabolisme compare au tours du cycle d’intermue. Camp. Biochem. Physiol. SlA, 777-784. MIYAWAI(I M. & SASAU N. (1961) A preliminary report on uptake of CaS’ by the hepatopancreas of crayfish Promnhm~s c,lurkii. Kwmmot0 J. Sci. 5, l70- 172. MUNRO H. N. & LINDER M. N. (1978) Ferritin: structure. biosynthesis and role m iron metabolism. Phnir~l. Rcr. 58. 3 177396.
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Mw.
Biol. 39, 343 -349.
W~ESER W. & KUMA J. (1969) Compartmentalization of copper in the hepatopancreas of isopods. Mikroskopir 22. 1-9.
WINCE D. R. & RAJAGOPALAN H. V. (19771 Purification and some properties of cadmium-bindmg protein from rat liver. A&s Biochrnl. Biophj~s. 153. 755 762. ZLCKEKKANDL E. (1960) Hemocyanine et cuivre chez un rustace decapode dans leurs rapports avec le cycle d’intermue. Am. Inst. Owur~o~~r.. .$I onm o 38. I - 122.