Download the physiology of the antennal gland of carcinus maenas (l.)

J. Exp. Biol. (1969), 51, 41-45
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THE PHYSIOLOGY OF THE ANTENNAL GLAND OF
CARCINUS MAENAS (L.)
V. SOME NITROGENOUS CONSTITUENTS IN THE BLOOD AND URINE
BY R. BINNS
Dept. of Zoology, University of Newcastle upon Tyne*
(Received 11 October 1968)
INTRODUCTION
It is now well established that the antennal glands of Crustacea, both marine and
fresh water, are concerned with regulation of the ionic composition of body fluids
(Robertson, 1957; Shaw, i960; Lockwood, 1962; Potts & Parry, 1964). This activity
is considered by some to be the main function of the antennal gland (Webb, 1940;
Parry, i960). Because it produces urine and is undoubtedly concerned with ionic
regulation, there is a tendency to discuss antennal gland function in terms of the
physiology of the vertebrate kidney. The unqualified inference, often expressed in
general zoology texts, that the antennal gland carries out all the functions of the
vertebrate kidney is unwarranted, since information concerning antennal gland function, other than ionic regulation, is extremely limited. This is particularly so when
considering nitrogen excretion by the antennal gland. A urine-producing 'excretory
organ' is usually thought of as being concerned with, amongst other things, the
elimination of nitrogenous waste products. This function is sometimes ascribed to the
antennal gland, despite the fact that there is remarkably little information on this
aspect of the physiology of the organ. After a survey of nitrogen excretion by Crustacea
and a consideration of analyses of the urine of Maia, Delaunay (1931) concluded that
the antennal gland was not concerned with nitrogen excretion and that its importance
in this respect has been over-estimated by histo-physiologists. With only a little
additional information available, Parry (i960) concluded that the antennal gland was
concerned primarily with ionic regulation. Ramsay (1961) was more cautious and
considered that because information is so limited, and because little is known of
invertebrate biochemistry, the possibility remains that the antennal gland may be
concerned with the removal of some excretory products.
Because the excretion of nitrogen by Crustacea has been little studied, it is considered worthwhile to offer some data resulting from a study of antennal gland function in Carcinus. Measurements of the contribution of urine nitrogen to total nitrogen
excretion by Crustacea have not been made. In this paper, figures obtained from
analyses of some nitrogenous components of blood and urine of Carcinus are presented.
Using this information, the role of the antennal gland of this animal in general
nitrogen excretion can be estimated.
• Present address: Huntingdon Research Centre, Huntingdon.
42
R. BINNS
MATERIALS AND METHODS
Experimental procedure and methods of collecting blood and urine samples have
been described earlier (Binns, 1969a).
Blood and urine were analysed for four components: ammonia, urea, uric acid and
amino acid nitrogen.
Ammonia was estimated by the Conway microdiffusion method (Conway, 1950).
Urea concentrations were determined by the same method, after first determining
ammonia concentrations and then treating body-fluid samples with urease in a
phosphate buffer solution.
a-Amino nitrogen was determined by the method of Folin (1922) as modified by
Danielson (1933). Glycine standards were used for calibration and concentrations of
amino acids in body fluids are expressed as equivalent to glycine concentrations. For
ease of handling, proteins in blood were precipitated using 10% trichloracetic acid.
It was not necessary to treat urine samples with the protein precipitant.
Uric acid was measured by the method of Benedict (1922) as described by Delory
(1949).
For the colorimetric estimation of amino acids and uric acid a Biochem H 810
Absorptiometer, with appropriate filters, was used.
Table 1. Concentrations of some nitrogenous constituents in blood and urine of Carcinus
No. of
Concn., as mg. % observa(mean + s.E.)
tions
Ammonia
Urea
Uric acid
a-Amino
nitrogen
Blood
Urine
Blood
Urine
Blood
Urine
Blood
Urine
Concn. as
mg. N %
I-6I ±0-29
I-l6±O-2O
12
10
5- 3 o±i-2S
1-78 + 0-36
12
I-5O±O-I2
0-82 ±o-12
21
14
1-32
0-96
2-48
0-83
0-50
0-27
57
n-55
24
119
*6i-9±3-3
•6-4 ±i-s
6
Delaunay
(i93i)
(mg. N %)
2-IO
2-7O
0-40
I2-O
Total urine nitrogen due to these four constituents = 3-25 mg. N/100 ml.
• Expressed as equivalent to glycine concentrations.
RESULTS
The results of the analyses of blood and urine from freshly captured crabs are given
in Table 1. Figures obtained by Delaunay (i 931) for Carcinus blood are also included
for comparison.
These ' normal' values for nitrogenous constituents in the blood and urine of crabs
direct from the field are generally of the same order as those found by Delaunay for
Carcinus and for Maia.
A feature of these estimations is that the concentrations of presumed excretory
nitrogenous compounds are greater in the blood than in the urine. Ammonia tends
to be slightly higher in the blood than in the urine, the blood concentration of uric
acid is almost double and the blood urea treble the respective urine concentrations of
these molecules.
The antennal gland of Carcinus maenas (L.). V
43
DISCUSSION
It now appears that the urine of Carcinus is produced by a so-called filtration
process. Urine/blood (U/B) ratios of 1 for both inulin and sorbitol in crabs living in
100% sea water (Binns, 1969 a) and a tendency for glucose U/B ratios to approach
unity after treatment with phloridzin (Binns, 1969 c) indicate that the primary urine
is produced by a non-selective movement of a protein-free filtrate of the blood into
the antennal gland.
In view of this it is perhaps surprising that the blood concentrations of uric acid
and of highly diffusible, small molecules such as urea and ammonia are apparently
greater than the respective urine concentrations of these substances. Comparable
results have been found for another crustacean but have not been commented upon.
Delaunay (1931) found that in Maia the recorded blood concentrations of ammonia
and urea tended to be higher than those in the urine.
A possible explanation of these anomalies is that the procedures or analytical
methods used gave erroneously high blood concentrations for uric acid, ammonia and
urea. For instance, it is likely that Benedict's arsenophosphotungstic acid reagent is
not entirely specific for uric acid. Another reactive material present in the blood but
not in the urine (say, some large molecule not filtered into the antennal gland) would
tend to give a high blood 'uric acid' concentration. The presence of bacteria or of
naturally occurring enzymes in blood samples may have resulted in the production
of additional ammonia and urea during the time blood samples were standing prior
to being analysed. These discrepancies are pointed out, but they are taken to indicate
technical difficulties rather than a real physiological difference between the concentrations of these nitrogenous compounds in blood and urine. More, carefully controlled,
estimates would be needed to demonstrate conclusively that any of these familiar
excretory products are, in fact, more concentrated in the blood than in the urine.
Large numbers of both marine and freshwater Crustacea have been shown to be
predominantly ammonotelic (Delaunay, 1934; Dresel & Moyle, 1950), this method of
eliminating waste nitrogen being associated with the aquatic habit. More than 80% of
the total nitrogen excreted by Carcinus is in the form of ammonia (Needham, 1955).
Eriocheir in distilled water loses ammonia at the same rate regardless of whether the
openings of the antennal glands are blocked or unblocked (Krogh, 1938). Urine
ammonia represents only 4-12% of the total nitrogen content of the urine of Maia
squinado, and is not likely to be an important fraction of the total ammonia lost
(Delaunay, 1931). Since the urine of Carcinus does not contain large amounts of
ammonia, or of the other nitrogenous compounds estimated, and the antennal gland
does not concentrate any of them, the possible importance of this organ in the excretion of nitrogen needs to be examined.
The contribution of the urinary nitrogen to general nitrogen excretion can be
calculated from a knowledge of the urine concentrations of the four nitrogenous
compounds estimated (this paper), taking the rate of urine production in normal sea
water to be 4*35% body weight per day (Binns, 1969b) and the rate of nitrogen
elimination by Carcinus to be I*I mg. N/25 g. crab/day (Needham, 1955). These
calculations show that the elimination of nitrogen as ammonia, urea, uric acid and
a-amino nitrogen in the urine accounts for 3*2% of the total nitrogen excreted by
44
R- BINNS
Carcinus. Ammonia, the animal's main excretory product, enters the external medium
almost entirely by diffusion. Only 1-04% of the total nitrogen loss can be accounted
for by ammonia nitrogen in the urine. Nitrogen excreted by the kidney of Salmo
gairdneri, a freshwater teleost, has been measured and it is interesting to note that, in
this animal also, the excretory organ contributes only 3 % of the total nitrogen loss
(Fromm, 1963).
Because of the extremely small amounts of nitrogen eliminated in the urine of
Carcinus the antennal gland must be regarded as being unimportant from the point
of view of general nitrogen excretion.
Despite this conclusion, it is possible that the antennal gland may be concerned
with the excretion of waste products from the blood. Total reducing substances
(T.R.S.) equivalent to 14-5 mg. glucose % are present in the urine of Carcinus in 100%
sea water, but the urine contains less than 1 mg. glucose % at normal blood glucose
concentrations (Binns, 1969c). The reducing substances in the urine, other than glucose, have not been identified. It has been suggested that substances such as uric
acid, ascorbic acid, amino sugars, tyrosine, dihydroxyphenylalanine and other phenols,
or sulphydryl compounds may all contribute to the T.R.S. value if they are present
in body fluids (Wyatt, 1961). Secretory activity, indicated by the concentration of
injected dyes, has been demonstrated in the antennal gland (Cuenot, 1893; Lison,
1942) and it is possible that complex, indiffusible molecules, which cannot otherwise
be disposed of by metabolism, may be secreted and contribute to the relatively high
concentration of T.R.S. in the urine. Until more is known about the composition of the
urine, the question of whether the antennal gland is of importance in clearing any
waste products of metabolism from the blood cannot be decided.
This work constitutes part of a Ph.D. thesis submitted to the University of Newcastle upon Tyne. It is a pleasure to thank Professor J. Shaw for his continual interest,
and for invaluable help given during the course of the work. I am also grateful to
S.R.C. for providing a postgraduate studentship.
REFERENCES
BENEDICT, S. R. (1922). The determination of uric acid in blood, jf. biol. Chem. 51, 187-207.
BINNS, R. (1969a). The physiology of the antennal gland of Carcinus maenas (L.). I. The mechanism of
urine production. J. exp. Biol. 51, 1-10.
BINNS, R. (19696). The physiology of the antennal gland of Carcinus maenas (L.). II. Urine production
rates. J. exp. Biol. 51, 11-16.
BINNS, R. (1969c). The physiology of the antennal gland of Carcinus maenas (L.). III. Glucose reabsorption. J. exp. Biol. 51, 17-27.
CONWAY, E. J. (1950). Microdiffusion Analysis and Volumetric Error. 3rd rev. edition. London: Crosby
Lockwood.
CUENOT, L. (1893). Etudes physiologiques sur les Crustaces Decapodes. Archs. Biol. 13, 254—303.
DANIELSON, I. S. (1933). Amino acid nitrogen in blood and its determination. J. biol. Chem. 101,
SOS-22.
DELAUNAY, H. (193I). L'excr£tion axotee des invertebres. Biol. Rev. 6, 265—301.
DELAUNAY, H. (1934). Le metabolisme de l'ammonique d'apres les recherches relatives aux Invertebres
Ann. physiol. physiochim. biol. i o , 695—729.
DELORY, G. E. (1949). Photoelectric Methods in Clinical Biochemistry. London: Hilger and Watts, Ltd.
DRESEL, E. I. B. & MOYLE, V. (1950). Nitrogenous excretion in amphipods and isopods. J. exp. Biol. 27,
210-25.
FOLIN, O. (1922). A system of blood analysis. III. A new colorimetric method for the determination of
amino acid nitrogen in blood. J. biol. Chem. 61, 377-91.
The antennal gland of Carcinus maenas (L.). V
45
FROMM, P. O. (1963). Studies on renal and extra-renal excretion in a freshwater teleost Salmo gairdneri.
Comp. Biochem. Physiol. i o , 121—28.
KROGH, A. (1938). The active absorption of ions in some freshwater animals. Z. vergl. physiol. 25,
335-5°LISON, L. (1942). Recherches sur l'histophysiologie comparee de l'excretion chez les arthropods. Acad.
roy. Belg. Classe sci. Mem 2 erne serie. 19, 1-107.
LOCKWOOD, A. P. M. (1962). The osmoregulation of Crustacea. Biol. Rev. 37, 257-305.
NEEDHAM, A. E. (1955). Factors affecting nitrogen excretion in Crustacea. Physiologia comp. Oecol. 4,
209-39.
PARRY, G. (i960). Excretion. The Physiology of Crustacea, (ed. T. H. Waterman), vol. 1, pp. 341-66.
New York: Academic Press.
POTTS, W. T. W. & PARRY, T. (1964). Osmotic and Ionic Regulation in Animals, 423 pp. Oxford: Pergamon Press.
RAMSAY, J. A. (1961). The comparative physiology of renal function in invertebrates. The Cell and the
Organism (ed. J. A. Ramsay and V. B. Wigglesworth), pp. 158-74. Cambridge University Press.
ROBERTSON, J. D. (1957). Osmotic and ionic regulation in aquatic invertebrates. Recent Advances in
Invertebrate Physiology, pp. 229-46. Eugene: University of Oregon Publication.
SHAW, J. (i960). The mechanisms of osmoregulation. Comparative Biochemistry (ed. M. Florkin and
H. S. Mason), vol. 2, pp. 471-518. New York: Academic Press.
WEBB, D. A. (1940). Ionic regulation in Carcinus maenas. Proc. R. Soc. B 129, 107—36.
WYATT, G. R. (1961). The biochemistry of insect haemolymph. A. Rev. Ent. 6, 75-102.