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Hemocyanin levels in the giant keyhole limpet, Megathura crenulata, from the coast of California
Camp. Biochem. Physiol.Vol. 94A. No. 2, pp. 195-199, 1989
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HEMOCYANIN LEVELS IN THE GIANT KEYHOLE LIMPET, MEGATHURA CRENULATA, FROM THE COAST OF CALIFORNIA N. M. SENOZANand MICHAELBRIGGS Department of Chemistry, California State University, Long Beach, California 90840, USA (Received I March 1989) Abstract-l. The concentration of hemocyanin in the hemolymph of the giant keyhole limpet Megarhura cremdura, has been determined. 2. In 56 specimens of M. crenuluta the hemocyanin concentration ranged from 1.53 to 10.81 g/l with
a mean of 5.45 g/l and a standard deviation of 2.71 g/l. There is no correlation between the hemocyanin concentration and the weight of the animal. The hemolymph volume and the colour of the mantle are not related to the hemocyanin level either. 3. The hemocyanin concentration shows a seasonal variation, the means and the standard deviations for February, April, July, and September being 6.48 k 2.58, 4.44 k 1.72. 2.62 k 1.05, and 7.65 + 1.54 g/l, respectively. 4. The lability of the hemocyanin concentration in Megathuru (7-fold variation) is less pronounced than it is in California Huliotis, but comparable to that of the Australian abalones. 5. It is difficult to reconcile the wide fluctuations observed in the concentration of hemocyanin with an essential respiratory task for this protein.
INTRODUCTION Hemocyanin is a copper containing respiratory protein found in the hemolymph of certain molluscs and arthropods (Senozan, 1976; Bonaventura and Bonaventura, 1983; Ellerton et af., 1983; Preaux and Gielens, 1984; Lamy et al., 1985). Its concentration is believed to be hightly labile and to show large variations, even among the individuals of the same species (Mangum, 1980; van Bruggen, 1980; Ellerton et al., 1983). A good example of such variations has been observed in the abalone from the California coast. In a study that included three species of Huliotis and a total of 140 animals, Wilson found that the concentration of hemocyanin ranged from 0.017 to 20.3 g/l (Pilson, 1965). The abalone belongs to an order of primitive molluscs known as archeogastropods. Another member of this order is the giant keyhole limpet, Meguthura crenulutu, whose hemolymph has supplied much of the hemocyanin used in immunological research (Senozan et al., 1981). M. crenulutu is found only off the coast of California between Monterey and Isla Asuncion in Baja, a region that overlaps with the habitat of the abalones studied by Pilson. The present work was undertaken to see if the wide range of hemocyanin concentrations found in Huliotis is also a characteristic of the Meguthuru and if the weight of the animal and the time of collection had any bearing on the levels of the respiratory pigment in the hemolymph. MATERIALSAND
METHODS
The giant keyhole limpets were collected off the shores of Palos Verdes Peninsula near Los Angeles. The animals were
gathered in February, April, July and September from a depth of about 5-l 5 feet. They were bled within a few hours after being taken from the sea. The blood was obtained by making incisions on the foot and allowing the hemolymph to flow freely into a container. The procedure was similar to that employed by Pilson on abalone (Pilson, 1965). Prior to bleeding, the water in the branchial cavity was emptied and the mantle and the foot of the animal were dried with a towel. The weight, colour and any distinct markings of the animal were recorded together with the volume of the blood collected. Altogether, 56 limpets were used in the study. Samples of hemolymph were digested in nitric acid and the concentration of copper was determined by Atomic Absorption Spectroscopy using the method of Standard Additions (Avinc and Senozan, 1986). The hemocyanin concentration in a given sample was calculated from the copper content assuming that the respiratory molecule is 0.25% by weight copper (Ellerton et al., 1983). Generally most of the copper in blood is associated with the hemocyanin, but some exceptions exist. In the shrimp Paluemon arispersus, for example, as much as 30% of the hemolymph copper may not be bound to hemocyanin (Hagerman and Weber, 1981). For the animals involved in this study a linear relationship between the copper content and the absorbance due to hemocyanin at 345 nm implied that nearly all the copper was associated with hemocyanin. The absorbance at 345 nm was measured on randomly selected samples. To prepare the sample for the spectrophotometric study, the hemolymph was first freed of cell debris and particulate matter by spinning several times in a DuPont RC-5 centrifuge at 10,OOOgfor 30 min and discarding the sedimented material after each spin. The hemocyanin was then pelleted by centrifugation in a Beckman L2-50 preparativk ultracentrifuge ai 95,OOOg (29,000 rpm) for 90 min. The pellet was dissolved in Tris-HCl buffer at pH 8.9, containing 10 mM EDTA and the spectrum was recorded about 345 nm. The free metal levels in the serum after the hemocyanin had been centrifuged out were below 10e4 g/l, the detection limit of the Perkin-Elmer Model 303 Atomic Absorption Spectrophotometer. 195
N. M. SENOZAN and MICHAELBRIGGS
196
Table 1. Concentration
Mean
1.84-10.81 2.79-8.18
6.48 4.44 2.62 7.65 5.46
Sample February? April July September All animals
of hemocyanin in the hemolymph of the giant keyhole limpet
Range (g/U
1.53-5.26 5.83-10.18 1,53-10.81
(g/J)
SD (g/f)
r (con-wt)’
2.58 1.72
0.476 (P z 0.02) 0.126 (P > 0.1)
1.os
0.148fP s-0.1)
1.54 2.7 1
0.226 (P z 0.1) -0.155 (P > 0.1)
r (con-vol)* 0.123 -0.349 0.332 0.401 -0.036
(P (P (P (P
> z > z
0.1) 0.1) O.l} 0.1)
(P > 0.1)
*I (con-wt) is the linear correlation coefficient between the hemocyanin concentration and the animal weight, and P is the probability that such a correlation will be found in the collection even if there were zero
correlation in the population. I (con-vol) is the linear correlation coefficient between the hemocyanin concentration and the hemolymph volume. tThe month of the collection. The number of animals in February, April. July, and September collections are 23. 8, 14. and 1I, respectively.
RESULTS
The results are summarized in Figs 1 and 2 and in Table 1. The hemocyanin concentration in the Megathura specimens ranges from 1.53 to 10.8 g/l with a mean of 5.46 and a standard deviation of 2.71 g/l. The distribution of the points in Figs 1 and 2 suggests that there is little correlation between the hemocyanin concentration and the hemolymph volume or the animal weight. Statistical analysis supports this conclusion (Olson, 1987; Remington and Schork, 1985). The correlation coefficients, r, listed in Table 1 correspond to P-values that are greater than 0.1 for all but one of the collections; in other words, the probability of encountering the listed r values in a set of animals collected from a population in which there is zero correlation between the pigment concentration and the animal weight or the hemolymph volume is greater than 10% in all cases except for the weight dependence in the February collection. T?x giant keyhole limpets have either a black or a gray mantle. The coiour of the mantle showed no relation to the hemocyanin content of the hemolymph. There was, however, a seasonal variation in the hemocyanin concentration, the mean values and the standard deviations for Febraury, April, July and September being 6.48 + 2.58, 4.44 f 1.72, 2.62 + 1.05, and 7.65 f 154gi1, respectively. The application of the ANOVA F-test to these means yields F = 16.7 and P < 0.001, indicating that the differences are significant.
The “slit and drip” technique used to collect hemol~ph in this study yields conservative estimates of the blood volume. Nevertheless there is, as expected, an approximately linear correlation between the weight of a keyhole limpet and its hemolymph volume. The hemocyanin concentration, however, is independent of the blood volume. This lack of correlation supports our confidence that a minimum amount of sea-water and intracelhtlar fluid mix with the hemolymph during the bleeding process. DISCUSSION
Hemocyanin levels have been observed to decrease in starving animals, but the changes do not become apparent until after several hours (Hagerman, 1986). The values reported in this study, therefore, can be assumed to reflect the animal’s natural state. The hemocyanin levels in the keyhole limpet display a wide variation, the maximum concentration being 10.8 g/l, the minimum 1.53. As in the California abalone, the concentration of the respiratory pigment in the limpet too is random and does not correlate with the weight or colour of the animal. There are, however, some major differences between the abalone and the keyhole limpet. While in some species of Haliotis the hemocyanin virtually disappears, in none of the 56 ~eg~f~ur~ specimens does the concentration drop below I.5 g/l. On the upper end of the scale, the hemocyanin levels in the abalone reach 20 g/l, but
12-
February April July
0 , 0
f 100
200
Weight of Animal (g)
Fig. 1. Hemocyanin concentration in the hemolymph of M. crenuluta. The specimens collected in different months are represented by different symbols.
Hemocyanin levels in the limpet
197 .=.m
.. .-
.
.
.
l. 5. .
.I.
: ;:: . *. ”
.
l .= .
l*
.
.’
:.9 .. . ::I
2 01
J
0
200
100
Weight
of Animal (g)
in Meguthura the highest concentration found is about half of this value. Comparative details of the occurrence of hemocyanin in archeogastropods are given in Table 2. The range of hemocyanin ~ncentrations in California abalones is still wide when each of the three species is considered alone; even in the case of H. crucherodii, where only seven animals have been used, the spread (lo-fold) is greater than it is for the Megathura. Apparently the large variations found in the California abalones are not typical of all archeogastropods or even of all Haliotis. Working with the Australian species H. roei, H. ruber, and H. laeoigata, Ainslie found the hemocyanin ranges to be 6.1-13.0, 1.0-8.3, and 2.9-6.6 g/l, respectively (Ainslie, 1980). The Australian abalones, so far as the hemocyanin levels are concerned, resemble more the giant keyhole limpet then they do the Huliotis of California. Another difference between the Haliotis and the limpet is in the seasonal variation of the hemocyanin concentration. Pilson (1965) reports that in the abalone “the concentration of hemocyanin was unrelated to . . . the season of the year”. For Megoth~~a, however, we find that the concentration varies with the time of the year; the pigment levels rise in September and February, and drop to a minimum in July. As shown in Fig. 3, not only the means, but the ranges too are effected by the season; the largest variation, for reasons unknown to us, is observed in February. Seasonal changes in the hemocyanin concentration have also been observed for the whelk, Busycon ca~a~icu~atu~, but the pattern is different. In
Table 2. Comparison of the occurrence of hemocyanin in archeogastrooods
M. crenulata (56)’ H. jiulgens (107) H. corrugatrr (26) H. cracberodii (7) H. roei(13) H. ruler (15, H. hmigata (16)
Range (g/U
Mean (g/l) -.
SD (g/l)
1.53-10.81 0.3-18.9 0.017-15.3 2. t-20.3
6.48 5.4t 1.51 3.H
2.58
7.9
2.2
2-f&d
4.6 4.3
2.1 1.2
%-fold 3.5-fold
6.1-13.0 1J&8.3
2.9-6.6
*Number of animals. tMedian values. fStandard deviations are not reported.
April
July
Sept
Fig. 3. Seasonal variation of the hemocyanin concentration in M. crenuiuta.Each point represents one animal with the
indicated pigment concen~ation.
Fig. 2. The variation of the hemolymph volume in M. crenulcazawith the weight of the animal.
Species
Feb
i $
Variation 7-fold 63-fold 900-fold IO-fold
&sycon the mean hemocyanin concentration remains around 25 g/l from December to April, then surges to 70 g/l in June, and afterwards declines gradually to its winter value (Betzer and Pilson, 1974). The water temperatures near the Palos Verdes Peninsula, where the Megathura samples were collected, vary by about 5°C between February and September; since the hemocyanin levels are high in both September and February, the warmest and the coldest periods, the seasonal variation in the giant keyhole limpet cannot be attributed to the temperature of the surrounding water column. Despite the differences between the abalones and the keyhole limpet archeogastropods are, nevertheless, set apart from the other molluscs and arthropods with respect to the occurrence of hemocyanin in two ways. The mean hemocyanin concentrations are low and the ranges are small in absolute terms. As Fig. 4 shows, in both keyhole limpets and the abalones the lower limits hover around a few grams per litre and the upper limits do not exceed 20 g/l. In most other molluscs and arthropods the difference between the maximum and the minimum concentration is greater than 50 g/l and the averages are well above the values observed for archeogastropods. The iability of hemocyanin levels can also be expressed in terms of R, the ratio of the maximum to the minimum concentration. Since in some abalones the minimum approaches zero, R becomes very large and, therefore, misleading as an index of variability. This is the case for H. corrugata where the concentration range in absolute terms is 15 g/l, but the ratio of the maximum to the minimum is over 900. So long as the minimum concentration does not come very ctose to zero, however, R can be useful as a parameter of lability. In terms of R. cephalopods exhibit the least variability in hemocyanin concentration, the maximum pigment level being about three-halves of the minimum (Johansen, 1965; Senozan et al., 1988). Most animals exhibit much larger values of R and the keyhole limpet is not out of line with the other molluscs or arthropods in this respect. In crabs the hemocyanin concentration diminishes during molting (Zuckerkandl, 1957). In BusJ~on the decrease in the levels of hemocyanin can be tied to the influx of water into the foot muscle of the animal (Mangum, 1979). In Haliotis and the Megathura, however, the fluctuations in the respiratory pigment concentration cannot be correlated with any environmental factors that we
N. M. SENOZANand
198
I
Crustaceans
c
MICHAEL BRIGGS
,
b
k
20 -
T
Species
Fig. 4. Range of hemocyanin concentrations. Bach bar indicates the range of concentrations found in a certain species. The letters identify the species: (a) Blue crab, Caifinectus sapidus. The reported ranges are 6-133 g/i of hemocyanin (258 animals; Horn and Kerr, 1963), 24-63 g/l (16 animals; Horn and Kerr, 1969) and 77-117 g/l (six animals; Mangum and Weiland, 1975). (b) Shore crab, Carcinus maenas; 16 animals (Aglow, 1969b). (c) Shore crab, Carcinus maenas (Boone and Schoffeniels, 1979). (d) Aquatic spider crab; Libinia em&&ata; 86 animals (Burnett, 1979). (e) Crab, Macropipus holsatus; 59 animals (Uelow. 1969b). ff) Snider crab. Maia suuinado: 107 animals (Zuckerkandl. 1957). (PI)Terrestrial ahost crab, dcypode’ &d&a; 57 animals (Burnett; 1979). (h) Shrimp, Palaemon &!$bsus; 16 animals (Hagerman and Weber, 1981). The authors estimate that as much as 30% of the hemolymph copper may not be bound to hemocyanin. If this is the case, the limits shown in the figure should be lowered by 30%. (i) Lobster, ~ornu~ Jericho (Senkbeil and Wriston, 1981). (j) Scorpion, Andra~ton~ uustra~~s; 74 animals (GoyfTon, 1968). (k) Green abalone, HaZiot~ fuigens; 107 animals (Filson, 1965). (1) Abalone, Hal&is corruguta; 26 animals (F&on, 1965). (m) Abalone, Hafiofis cracherodii; seven animals (Pilson, 1965). (n) Abalone, Haliofis ruber; 15 animals (Ainslie, 1980). (0) Abalone, Haliotis lueuigata; 16 animals (Ainslie, 1980). (p) Abalone, Haliotis roei; 13 animals (Ainslie, 1980). (q) Giant keyhole limpet, Magathura crenulata; 56 animals (this work). (r) Whelk, Busycon canaliculafum; 104 animals (Betzer and Filson. 1974). (s) Snail, Helix pomatia; 11 animals (Burton, 1964). (t) Octopus dojeini; 15 animals (Johansen. 1965). (v) Cuttlefish, Sepia offcinalis; 14 animals (Senozan et al., 1988). (w) Octopus oulgaris; five animals (Senozan ef al., 1988).
can ascertain. While one limpet has as much as 10 g/I of hemocyanin, another individual living in the same environment and presumably subject to the same stresses has one-seventh as much. We have watched the giant keyhole limpets in their natural habitat in daylight hours and their motor activities-or the lack of them-did not seem to differ from each other even though the hemocyanin contents varied widely. The role of hemocyanin as a transporter of oxygen has been demonstrated in the Australian species of Haliotis and many other molluscs and arthropods (Ainshe, 1980; Mangum, 1980). The copper protein is undoubtedly essential for the respiration of cephalopods and active crustaceans, but questions have been raised about its significance in sedentary animals such as the keyhole limpet. Prosser has remarked that in sluggish life “animals that contain hemocyanin could probably get along without it” (Prosser, 1973). Fox has noted that “the enormous ranges in hemocyanin concentration in Haliotis plasma appear to lack compatibility with any physiological role thus far suggested” and has put forward the idea that hemocyanin may be a detoxifying agent
for copper (Fox, 1979). Others have mentioned the possibility of hemocyanin acting as a vehicle for the transport and storage of amino acids and Pilson has suggested that it might simply be a relic of an ancient molecule no longer serving a purpose (Wieser. 1965; Uglow, 1969a; Boone and Schoffeniels, 1979; Senkbeil and Wriston, 1981). Whatever role is eventually assigned to the hemocyanin of the keyho’te limpet, it must be consistent with the wide concentration fluctuations observed. Presently, it is difbcult to reconcile these fluctuations with an essential respiratory task. Acknowledgements-We wish to thank Erica Schneider and Chuck Kristensen for their assistance in collecting the keyhole limpets and in helping with the measurements. This work was in part supported by the California State University, Long Beach.
REFERENCES Ainslie R. C. (1980) The quantitative role of haemocyanin in the respiration of abalone (genus Hulioris). J. exp. Zool. 211, 87-99.
Hemocyanin lev,els in the limpet Avinc A. and Senozan N. (1986) The Determination of hemocyanin in Sepia oficinalis. Doga (Turkish Journal of Chemistry) 10, 97-100. Betzer S. B. and Pilson M. E. Q. (1974) The seasonal cycle of copper concentration in Busycon canaliculatum L. Biol. Bull. 146, 165-175. Bonaventura C. and Bonaventura J. (1983) Respiratory pigments: Structure and function. In The Mollusca (Edited by Wilbur K. M. and Hochachka P. W.), Vol. 2, pp. I-50. Academic Press, New York. Boone W. R. and Schoffeniels E. (1979) Hemocyanin synthesis during hypo-osmotic stress in the shore crab Car&us maenas (L.). Comp. Biochem. Physiol. 63B, 207-214.
Burnett L. E. (1979) The effect of environmental oxygen levels on the respiratory function of hemocyanin in the crabs, Libinia emarginata and Ocypode quadrata. J. exp. 2001. 210, 289-299.
Burton R. F. (1964) Variations in the volume and concentration of the blood of the snail, Helix pomafia L., in relation to the water content of the body. Can. J. Zool. 42, 1085-1097. Ellerton H. D., Ellerton N. F. and Robinson H. A. (1983) Hemocyanin-A current perspective. Prog. Biophvs. molec. Biol. 41, 143-248.
_
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Fox D. L. (19791Biochromv. D. 195. Universitv ofcalifornia Press, Berkeley, California’. Goyffon M. (1968) La cupremie du Scorpion Androctonus australis (L.) Hector C. L. Koch. 1839. C. R. Seances Sot. Biol. Ses Fil. 162, 1123-l 128. Hagerman L. (1986) Haemocyanin concentration in the shrimp Crangon crangon (L.) after exposure to moderate hypoxia. Comp. Biochem. Physiol. MA, 721-724. Hagerman L. and Weber R. E. (1981) Respiratory rate. haemolymph oxygen tension and haemocyanin level in the shrimp Palaemon adspersus Rathke. J. exp. mar. Biol. Ecol. 54, 13-20. Horn E. C. and Kerr M. S. (1963) Hemolymph protein and copper concentrations of adult blue crabs (Callinectes sapidus kathbun). Biol. Bull. 125, 4999507. Horn E. C. and Kerr M. S. (1969) The hemolvmnh nroteins of the Blue Crab, Callinectes ‘sapidus-I.-Hdmdcyanins and certain major protein constituents. Comp. Biochem. Physiol. 29, 493-508.
Johansen K. (1965) Cardiac output in the large cephalopod Octopus dofleini. J. exp. Biol. 42, 475480.
Lamy J.. Truchot J.-P. and Gilles R. (1985) Respiratory Pigments in Animals. Springer, Berlin. Mangum C. P. (1979) A note on blood and water mixing in
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389-391.
Mangum C. P. (1980) Respiratory function of the hemocyanin. Am. Zool. 20, 19-38. Mangum C. P. and Weiland A. L. (1975) The function of hemocyanin in respiration of the Blue Crab Callinectes sapidus. J. exp. Zool. 193, 257-263.
Olson C. L. (1987) Statistics, pp. 434437 and 512-522. Allyn and Bacon, Boston. Pilson M. E. Q. (1965) Variation of hemocyanin concentration in the blood of four species of Haliotis. Biol. Bull. 128, 459472.
Preaux G. and Gielens C. (1984) Hemocyanins. In Copper Proteins and Copper Enzymes (Edited by Lontie R.), pp. 159-205. CRC Press, Boca Raton, Florida. Prosser C. L. (1973) Comparative Animal Physiology, 3rd edn, p. 352. W. B. Saunders, Philadelphia. Remington R. D. and Schork M. A. (1985) Statistics with Applications To The Biological and Health Sciences, 2nd edn. .DD. . 252-254 and 262-268. Prentice-Hall. Enalewood _ Cliffs, New Jersey. Senkbeil E. G. and Wriston J. C. Jr (1981) Hemocyanin synthesis in the American Lobster, Homarus americanus. Comp. Biochem. Physiol. 68B, 163-l Il. Senozan N. M. (1976) Hemocyanin: the copper blood. J. Chem. Ed. 53, 684688.
Senozan N. M., Landrum J., Bonaventura J. and Bonaventura C. (1981) Hemocyanin of the giant keyhole limpet, Megathura crenulata. In Inoerrebrare Oxygen-Binding Proteins (Edited by Lamy J. and Lamy J.), pp. 703-723. Marcel Dekker, New York. Senozan N. M.. Avinc A. and Unver Z. 11988) Hemocvanin levels in Octopus vulgaris and the cuitlefish Sepia ojicinalis from the Aegean Sea. Comp. Biochem. Physiol. 9lA, 581-585.
Uglow R. F. (1969a) Haemolymph protein concentrations in portunid crabs--I. Studies on adult Carcinus maenas. Comp. Biochem. Physiol. 30, 1083-1090.
Uglow R. F. (1969b) Haemolymph protein concentrations in portunid crabs-III. The effect of Sacculina. Comp. Biochem. Phvsiol. 31, 969-973.
Van Bruggen E. F. J. (1980) Hemocyanin: The mystery of blue blood. Trends biochem. Sci. j, 185-188. _ _ Wieser W. (1965) Electronhoretic studies on blood oroteins in an ecological series of isopod and amphipod species. 1
I
J. Mar. Biol. Ass. UK 45, 507-523.
Zuckerkandl E. (1957) La teneur en cuivre de l’hbmolymphe de Maia squinado aux divers stades d’intermue. C. R. Seances Sot. Biol. Ses Fil. 151, 460-463.
0300-9629/89 $3.00+ 0.00 0 I989PergamonPressplc
Printedin Great Britain
HEMOCYANIN LEVELS IN THE GIANT KEYHOLE LIMPET, MEGATHURA CRENULATA, FROM THE COAST OF CALIFORNIA N. M. SENOZANand MICHAELBRIGGS Department of Chemistry, California State University, Long Beach, California 90840, USA (Received I March 1989) Abstract-l. The concentration of hemocyanin in the hemolymph of the giant keyhole limpet Megarhura cremdura, has been determined. 2. In 56 specimens of M. crenuluta the hemocyanin concentration ranged from 1.53 to 10.81 g/l with
a mean of 5.45 g/l and a standard deviation of 2.71 g/l. There is no correlation between the hemocyanin concentration and the weight of the animal. The hemolymph volume and the colour of the mantle are not related to the hemocyanin level either. 3. The hemocyanin concentration shows a seasonal variation, the means and the standard deviations for February, April, July, and September being 6.48 k 2.58, 4.44 k 1.72. 2.62 k 1.05, and 7.65 + 1.54 g/l, respectively. 4. The lability of the hemocyanin concentration in Megathuru (7-fold variation) is less pronounced than it is in California Huliotis, but comparable to that of the Australian abalones. 5. It is difficult to reconcile the wide fluctuations observed in the concentration of hemocyanin with an essential respiratory task for this protein.
INTRODUCTION Hemocyanin is a copper containing respiratory protein found in the hemolymph of certain molluscs and arthropods (Senozan, 1976; Bonaventura and Bonaventura, 1983; Ellerton et af., 1983; Preaux and Gielens, 1984; Lamy et al., 1985). Its concentration is believed to be hightly labile and to show large variations, even among the individuals of the same species (Mangum, 1980; van Bruggen, 1980; Ellerton et al., 1983). A good example of such variations has been observed in the abalone from the California coast. In a study that included three species of Huliotis and a total of 140 animals, Wilson found that the concentration of hemocyanin ranged from 0.017 to 20.3 g/l (Pilson, 1965). The abalone belongs to an order of primitive molluscs known as archeogastropods. Another member of this order is the giant keyhole limpet, Meguthura crenulutu, whose hemolymph has supplied much of the hemocyanin used in immunological research (Senozan et al., 1981). M. crenulutu is found only off the coast of California between Monterey and Isla Asuncion in Baja, a region that overlaps with the habitat of the abalones studied by Pilson. The present work was undertaken to see if the wide range of hemocyanin concentrations found in Huliotis is also a characteristic of the Meguthuru and if the weight of the animal and the time of collection had any bearing on the levels of the respiratory pigment in the hemolymph. MATERIALSAND
METHODS
The giant keyhole limpets were collected off the shores of Palos Verdes Peninsula near Los Angeles. The animals were
gathered in February, April, July and September from a depth of about 5-l 5 feet. They were bled within a few hours after being taken from the sea. The blood was obtained by making incisions on the foot and allowing the hemolymph to flow freely into a container. The procedure was similar to that employed by Pilson on abalone (Pilson, 1965). Prior to bleeding, the water in the branchial cavity was emptied and the mantle and the foot of the animal were dried with a towel. The weight, colour and any distinct markings of the animal were recorded together with the volume of the blood collected. Altogether, 56 limpets were used in the study. Samples of hemolymph were digested in nitric acid and the concentration of copper was determined by Atomic Absorption Spectroscopy using the method of Standard Additions (Avinc and Senozan, 1986). The hemocyanin concentration in a given sample was calculated from the copper content assuming that the respiratory molecule is 0.25% by weight copper (Ellerton et al., 1983). Generally most of the copper in blood is associated with the hemocyanin, but some exceptions exist. In the shrimp Paluemon arispersus, for example, as much as 30% of the hemolymph copper may not be bound to hemocyanin (Hagerman and Weber, 1981). For the animals involved in this study a linear relationship between the copper content and the absorbance due to hemocyanin at 345 nm implied that nearly all the copper was associated with hemocyanin. The absorbance at 345 nm was measured on randomly selected samples. To prepare the sample for the spectrophotometric study, the hemolymph was first freed of cell debris and particulate matter by spinning several times in a DuPont RC-5 centrifuge at 10,OOOgfor 30 min and discarding the sedimented material after each spin. The hemocyanin was then pelleted by centrifugation in a Beckman L2-50 preparativk ultracentrifuge ai 95,OOOg (29,000 rpm) for 90 min. The pellet was dissolved in Tris-HCl buffer at pH 8.9, containing 10 mM EDTA and the spectrum was recorded about 345 nm. The free metal levels in the serum after the hemocyanin had been centrifuged out were below 10e4 g/l, the detection limit of the Perkin-Elmer Model 303 Atomic Absorption Spectrophotometer. 195
N. M. SENOZAN and MICHAELBRIGGS
196
Table 1. Concentration
Mean
1.84-10.81 2.79-8.18
6.48 4.44 2.62 7.65 5.46
Sample February? April July September All animals
of hemocyanin in the hemolymph of the giant keyhole limpet
Range (g/U
1.53-5.26 5.83-10.18 1,53-10.81
(g/J)
SD (g/f)
r (con-wt)’
2.58 1.72
0.476 (P z 0.02) 0.126 (P > 0.1)
1.os
0.148fP s-0.1)
1.54 2.7 1
0.226 (P z 0.1) -0.155 (P > 0.1)
r (con-vol)* 0.123 -0.349 0.332 0.401 -0.036
(P (P (P (P
> z > z
0.1) 0.1) O.l} 0.1)
(P > 0.1)
*I (con-wt) is the linear correlation coefficient between the hemocyanin concentration and the animal weight, and P is the probability that such a correlation will be found in the collection even if there were zero
correlation in the population. I (con-vol) is the linear correlation coefficient between the hemocyanin concentration and the hemolymph volume. tThe month of the collection. The number of animals in February, April. July, and September collections are 23. 8, 14. and 1I, respectively.
RESULTS
The results are summarized in Figs 1 and 2 and in Table 1. The hemocyanin concentration in the Megathura specimens ranges from 1.53 to 10.8 g/l with a mean of 5.46 and a standard deviation of 2.71 g/l. The distribution of the points in Figs 1 and 2 suggests that there is little correlation between the hemocyanin concentration and the hemolymph volume or the animal weight. Statistical analysis supports this conclusion (Olson, 1987; Remington and Schork, 1985). The correlation coefficients, r, listed in Table 1 correspond to P-values that are greater than 0.1 for all but one of the collections; in other words, the probability of encountering the listed r values in a set of animals collected from a population in which there is zero correlation between the pigment concentration and the animal weight or the hemolymph volume is greater than 10% in all cases except for the weight dependence in the February collection. T?x giant keyhole limpets have either a black or a gray mantle. The coiour of the mantle showed no relation to the hemocyanin content of the hemolymph. There was, however, a seasonal variation in the hemocyanin concentration, the mean values and the standard deviations for Febraury, April, July and September being 6.48 + 2.58, 4.44 f 1.72, 2.62 + 1.05, and 7.65 f 154gi1, respectively. The application of the ANOVA F-test to these means yields F = 16.7 and P < 0.001, indicating that the differences are significant.
The “slit and drip” technique used to collect hemol~ph in this study yields conservative estimates of the blood volume. Nevertheless there is, as expected, an approximately linear correlation between the weight of a keyhole limpet and its hemolymph volume. The hemocyanin concentration, however, is independent of the blood volume. This lack of correlation supports our confidence that a minimum amount of sea-water and intracelhtlar fluid mix with the hemolymph during the bleeding process. DISCUSSION
Hemocyanin levels have been observed to decrease in starving animals, but the changes do not become apparent until after several hours (Hagerman, 1986). The values reported in this study, therefore, can be assumed to reflect the animal’s natural state. The hemocyanin levels in the keyhole limpet display a wide variation, the maximum concentration being 10.8 g/l, the minimum 1.53. As in the California abalone, the concentration of the respiratory pigment in the limpet too is random and does not correlate with the weight or colour of the animal. There are, however, some major differences between the abalone and the keyhole limpet. While in some species of Haliotis the hemocyanin virtually disappears, in none of the 56 ~eg~f~ur~ specimens does the concentration drop below I.5 g/l. On the upper end of the scale, the hemocyanin levels in the abalone reach 20 g/l, but
12-
February April July
0 , 0
f 100
200
Weight of Animal (g)
Fig. 1. Hemocyanin concentration in the hemolymph of M. crenuluta. The specimens collected in different months are represented by different symbols.
Hemocyanin levels in the limpet
197 .=.m
.. .-
.
.
.
l. 5. .
.I.
: ;:: . *. ”
.
l .= .
l*
.
.’
:.9 .. . ::I
2 01
J
0
200
100
Weight
of Animal (g)
in Meguthura the highest concentration found is about half of this value. Comparative details of the occurrence of hemocyanin in archeogastropods are given in Table 2. The range of hemocyanin ~ncentrations in California abalones is still wide when each of the three species is considered alone; even in the case of H. crucherodii, where only seven animals have been used, the spread (lo-fold) is greater than it is for the Megathura. Apparently the large variations found in the California abalones are not typical of all archeogastropods or even of all Haliotis. Working with the Australian species H. roei, H. ruber, and H. laeoigata, Ainslie found the hemocyanin ranges to be 6.1-13.0, 1.0-8.3, and 2.9-6.6 g/l, respectively (Ainslie, 1980). The Australian abalones, so far as the hemocyanin levels are concerned, resemble more the giant keyhole limpet then they do the Huliotis of California. Another difference between the Haliotis and the limpet is in the seasonal variation of the hemocyanin concentration. Pilson (1965) reports that in the abalone “the concentration of hemocyanin was unrelated to . . . the season of the year”. For Megoth~~a, however, we find that the concentration varies with the time of the year; the pigment levels rise in September and February, and drop to a minimum in July. As shown in Fig. 3, not only the means, but the ranges too are effected by the season; the largest variation, for reasons unknown to us, is observed in February. Seasonal changes in the hemocyanin concentration have also been observed for the whelk, Busycon ca~a~icu~atu~, but the pattern is different. In
Table 2. Comparison of the occurrence of hemocyanin in archeogastrooods
M. crenulata (56)’ H. jiulgens (107) H. corrugatrr (26) H. cracberodii (7) H. roei(13) H. ruler (15, H. hmigata (16)
Range (g/U
Mean (g/l) -.
SD (g/l)
1.53-10.81 0.3-18.9 0.017-15.3 2. t-20.3
6.48 5.4t 1.51 3.H
2.58
7.9
2.2
2-f&d
4.6 4.3
2.1 1.2
%-fold 3.5-fold
6.1-13.0 1J&8.3
2.9-6.6
*Number of animals. tMedian values. fStandard deviations are not reported.
April
July
Sept
Fig. 3. Seasonal variation of the hemocyanin concentration in M. crenuiuta.Each point represents one animal with the
indicated pigment concen~ation.
Fig. 2. The variation of the hemolymph volume in M. crenulcazawith the weight of the animal.
Species
Feb
i $
Variation 7-fold 63-fold 900-fold IO-fold
&sycon the mean hemocyanin concentration remains around 25 g/l from December to April, then surges to 70 g/l in June, and afterwards declines gradually to its winter value (Betzer and Pilson, 1974). The water temperatures near the Palos Verdes Peninsula, where the Megathura samples were collected, vary by about 5°C between February and September; since the hemocyanin levels are high in both September and February, the warmest and the coldest periods, the seasonal variation in the giant keyhole limpet cannot be attributed to the temperature of the surrounding water column. Despite the differences between the abalones and the keyhole limpet archeogastropods are, nevertheless, set apart from the other molluscs and arthropods with respect to the occurrence of hemocyanin in two ways. The mean hemocyanin concentrations are low and the ranges are small in absolute terms. As Fig. 4 shows, in both keyhole limpets and the abalones the lower limits hover around a few grams per litre and the upper limits do not exceed 20 g/l. In most other molluscs and arthropods the difference between the maximum and the minimum concentration is greater than 50 g/l and the averages are well above the values observed for archeogastropods. The iability of hemocyanin levels can also be expressed in terms of R, the ratio of the maximum to the minimum concentration. Since in some abalones the minimum approaches zero, R becomes very large and, therefore, misleading as an index of variability. This is the case for H. corrugata where the concentration range in absolute terms is 15 g/l, but the ratio of the maximum to the minimum is over 900. So long as the minimum concentration does not come very ctose to zero, however, R can be useful as a parameter of lability. In terms of R. cephalopods exhibit the least variability in hemocyanin concentration, the maximum pigment level being about three-halves of the minimum (Johansen, 1965; Senozan et al., 1988). Most animals exhibit much larger values of R and the keyhole limpet is not out of line with the other molluscs or arthropods in this respect. In crabs the hemocyanin concentration diminishes during molting (Zuckerkandl, 1957). In BusJ~on the decrease in the levels of hemocyanin can be tied to the influx of water into the foot muscle of the animal (Mangum, 1979). In Haliotis and the Megathura, however, the fluctuations in the respiratory pigment concentration cannot be correlated with any environmental factors that we
N. M. SENOZANand
198
I
Crustaceans
c
MICHAEL BRIGGS
,
b
k
20 -
T
Species
Fig. 4. Range of hemocyanin concentrations. Bach bar indicates the range of concentrations found in a certain species. The letters identify the species: (a) Blue crab, Caifinectus sapidus. The reported ranges are 6-133 g/i of hemocyanin (258 animals; Horn and Kerr, 1963), 24-63 g/l (16 animals; Horn and Kerr, 1969) and 77-117 g/l (six animals; Mangum and Weiland, 1975). (b) Shore crab, Carcinus maenas; 16 animals (Aglow, 1969b). (c) Shore crab, Carcinus maenas (Boone and Schoffeniels, 1979). (d) Aquatic spider crab; Libinia em&&ata; 86 animals (Burnett, 1979). (e) Crab, Macropipus holsatus; 59 animals (Uelow. 1969b). ff) Snider crab. Maia suuinado: 107 animals (Zuckerkandl. 1957). (PI)Terrestrial ahost crab, dcypode’ &d&a; 57 animals (Burnett; 1979). (h) Shrimp, Palaemon &!$bsus; 16 animals (Hagerman and Weber, 1981). The authors estimate that as much as 30% of the hemolymph copper may not be bound to hemocyanin. If this is the case, the limits shown in the figure should be lowered by 30%. (i) Lobster, ~ornu~ Jericho (Senkbeil and Wriston, 1981). (j) Scorpion, Andra~ton~ uustra~~s; 74 animals (GoyfTon, 1968). (k) Green abalone, HaZiot~ fuigens; 107 animals (Filson, 1965). (1) Abalone, Hal&is corruguta; 26 animals (F&on, 1965). (m) Abalone, Hafiofis cracherodii; seven animals (Pilson, 1965). (n) Abalone, Haliofis ruber; 15 animals (Ainslie, 1980). (0) Abalone, Haliotis lueuigata; 16 animals (Ainslie, 1980). (p) Abalone, Haliotis roei; 13 animals (Ainslie, 1980). (q) Giant keyhole limpet, Magathura crenulata; 56 animals (this work). (r) Whelk, Busycon canaliculafum; 104 animals (Betzer and Filson. 1974). (s) Snail, Helix pomatia; 11 animals (Burton, 1964). (t) Octopus dojeini; 15 animals (Johansen. 1965). (v) Cuttlefish, Sepia offcinalis; 14 animals (Senozan et al., 1988). (w) Octopus oulgaris; five animals (Senozan ef al., 1988).
can ascertain. While one limpet has as much as 10 g/I of hemocyanin, another individual living in the same environment and presumably subject to the same stresses has one-seventh as much. We have watched the giant keyhole limpets in their natural habitat in daylight hours and their motor activities-or the lack of them-did not seem to differ from each other even though the hemocyanin contents varied widely. The role of hemocyanin as a transporter of oxygen has been demonstrated in the Australian species of Haliotis and many other molluscs and arthropods (Ainshe, 1980; Mangum, 1980). The copper protein is undoubtedly essential for the respiration of cephalopods and active crustaceans, but questions have been raised about its significance in sedentary animals such as the keyhole limpet. Prosser has remarked that in sluggish life “animals that contain hemocyanin could probably get along without it” (Prosser, 1973). Fox has noted that “the enormous ranges in hemocyanin concentration in Haliotis plasma appear to lack compatibility with any physiological role thus far suggested” and has put forward the idea that hemocyanin may be a detoxifying agent
for copper (Fox, 1979). Others have mentioned the possibility of hemocyanin acting as a vehicle for the transport and storage of amino acids and Pilson has suggested that it might simply be a relic of an ancient molecule no longer serving a purpose (Wieser. 1965; Uglow, 1969a; Boone and Schoffeniels, 1979; Senkbeil and Wriston, 1981). Whatever role is eventually assigned to the hemocyanin of the keyho’te limpet, it must be consistent with the wide concentration fluctuations observed. Presently, it is difbcult to reconcile these fluctuations with an essential respiratory task. Acknowledgements-We wish to thank Erica Schneider and Chuck Kristensen for their assistance in collecting the keyhole limpets and in helping with the measurements. This work was in part supported by the California State University, Long Beach.
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