Salt and Water Balance of the Spiny Lobster, Panulirus Argus: the Role of the Antennal Glands

Decapod crustaceans are able to regulate total haemolymph osmoconcentration and the concentrations of individual ions at levels different from those in their aquatic environment (Lockwood, 1962, 1967; Potts & Parry, 1963). Further, they actively maintain constant body volume. This clearly is necessary in decapods which are subject to osmotic gain or loss of water, but volume regulation is required even in osmoconformers to replace water lost in urine.

Much attention has been given to the functioning of the gills as sites of active uptake of ions (Koch, 1954; Bielawski, 1964; Lockwood, 1967) and to the antennal glands (Robertson, 1949; Parry, 1955; Gross & Marshall, 1960; Riegel & Lockwood, 1961) in this regulation of salt and water. The antennal glands tend to remove Mg²⁺ and SO₄²⁻ and to conserve K⁺ and Ca²⁺ (Potts & Parry, 1963). But fewer studies have been made of the contribution of the gut, the third major interface between the external medium and haemolymph (Green et al. 1959; Gifford, 1962; Heeg & Cannone, 1966; Dall, 1967, 1970).

This study was undertaken primarily to examine the possible role of the gut in ionic regulation and water balance of the isosmotic marine lobster, Panulirus argus (Latreille).

This paper describes the pattern of ionic regulation demonstrated by this species and the importance of the antennal glands in its maintenance. The following paper (Malley, 1977) reports the ionic analysis of gut fluids, rates of drinking of the medium by lobsters, the movement of water and ions between the proventricular fluid and haemolymph, and assesses the role of the gut in the water and salt economy of this species.

Twenty-three Panulirus argus were obtained from two geographical areas, Florida and Bermuda. Fourteen lobsters of both sexes collected off the Florida Keys were air-shipped by a biological supplier to Ann Arbor, Michigan. Carapace length ranged from 6·3 to 8·8 cm at procurance. Lobsters were maintained up to one year at 19 ± 1·5 °C in a continuously circulating closed sea-water system containing a mixture of natural sea water and artificial sea salts (Instant Ocean, Aquarium Systems, Inc., Wickliffe, Ohio) diluted with distilled water. Average composition of this sea water is given in Table 2. Fresh tap-water was added to adjust salinity to between 35 and 37·5‰ Photoperiod was 12 h light, 12 h dark. This set of conditions is referred to as ‘laboratory maintenance conditions’. Lobsters were fed pieces of thawed squid, generally two to three times per week. Many lobsters moulted during the maintenance period and nearly all moults were successful.

Nine Panulirus argus were obtained directly from the pots of local fishermen in Bermuda and were maintained for 1 week in an open sea-water system at the Bermuda Biological Station under conditions of salinity (35·1 ‰,), temperature (24·0–24·5 °C) and photoperiod (early September) approximating to those in the field. These are referred to as ‘field maintenance conditions’. Lobsters were of both sexes and ranged in carapace length from 7·8 to 10·8 cm and in weight from 416·7 to 999·3 g. They were air-shipped to Ann Arbor 1 week after capture and maintained under the ‘laboratory maintenance conditions’ and fed as described above for over 5 months.

Unless otherwise indicated, all animals had neither recently moulted nor were about to moult when sampled. Lobsters were not fed for at least 2 days prior to sampling.

Before sampling, animals were tied, ventral surface up, to an operating board (see Travis, 1955). Haemolymph was obtained by puncturing the arthrodial membrane proximal to the coxopodite of the fourth or fifth periopod. Serum was obtained following the retraction of the clot in about 1 ml of undiluted haemolymph. Urine was caused to flow by inserting a fine fire-polished glass probe into the nephropore. Samples of the sea-water medium were taken concurrently with body fluid samples. Samples of body fluids and sea water were taken in duplicate or triplicate and were 20 μl in volume, occasionally less, collected and measured in Drummond ‘Microcaps’ micropipettes. These 20 μl samples were diluted immediately with 2·0 ml distilled water, frozen and stored in air-tight polypropylene tubes until analysed. Samples for SO₄²⁻ analysis, however, were 100 μl of fluids (occasionally only 50 μl was available) diluted with 1·0 ml distilled water.

Na⁺, K⁺, Ca²⁺ and Mg²⁺ were determined in each diluted 20 μl sample by atomic absorption spectrophotometry (Perkin-Elmer Model 290 B). Samples for Ca²⁺ analysis were prepared with 0·5% lanthanum oxide to avoid chemical interferences. For K⁺ analysis, 100 mM-NaCl was added to overcome ionization interferences.

Chloride was measured in the diluted 20μl samples with an Aminco-Cotlove chloride titrator using standard techniques. Salinity of sea water was calculated from the chloride concentration using the formula given by Barnes (1954): salinity (‰) = 0·03 + 1·805 × chlorinity ‰. According to the Instant Ocean artificial sea-water formulation, chlorinity is 1·00048 × chloride concentration.

Sulphate was determined as BaSO₄ by means of a turbidometric method (American Public Health Association, 1965), modified for microsamples. Absorption was measured at 420 nm.

Osmoconcentration of serum and sea water was measured on a Mechrolab Model 301A vapour pressure osmometer using NaCl solutions as reference standards.

Values from the analysis of the duplicate or triplicate samples of a single fluid were averaged and the mean constitutes a determination.

The data from the ion analyses were in molar concentration. Since the content of water varies in the different fluids, the actual ionic gradients between fluids are seen when ion levels are expressed in molal concentration. To determine the water content of body fluids and sea water, samples of known volume and weight were dried to constant weight at 90–100 °C. Serum was substituted here for haemolymph which clots if it is not immediately diluted with water. There is essentially no osmotic difference between haemolymph and serum in several species of decapods examined (Gross, 1964; see also Table 2, serum as % haemolymph). Ion concentrations were converted from molar to molal by multiplying by conversion factors: 1000 g H₂O/ weight in g of water in I 1 of fluid, separately determined for sea water, serum and urine.

Serum (50–1000 μl) was dialysed against the sea water in which the animal was maintained. Dialysis was carried out for 12–24 h at 5 °C in 1 1 of sea water on a shaker or with magnetic stirring.

The body fluids of nine Panulirus argus were sampled at the Bermuda Biological Station within 1-3 days of their capture from the field. Table 1 shows the molar concentrations of Na⁺, K⁺, Ca²⁺, Mg²⁺, Cl⁻and SO₄²⁻ and the water content in haemolymph and urine of these lobsters and in Bermuda sea water. These values are the closest estimates obtained in this study of body fluid concentrations of P. argus under natural conditions. The effective ionic gradients between fluids are indicated by expressing ionic concentrations on a molal basis. The factors for converting the data in Table 1 from molar to molal are for blood, 1·038; urine, 1·016; and sea water, 1019.

Table 2 shows molal concentrations of the major inorganic ions in haemolymph, serum, dialysed serum and sea water as percentages of one another for 23 lobsters from both Bermuda and Florida. These data include the values given in Table 1 as well as determinations made after lobsters were maintained for varying lengths of time in the laboratory. Ionic concentrations are expressed as ratios to allow pooling of data from animals equilibrated with slightly different salinities. Ionic gradients between haemolymph and sea water reflect both active processes of ion regulation and passing factors such as presence of protein and the binding of Ca²⁺ (Robertson, 1949 Dialysis of serum against sea water shows that passive factors account, in P. argus, for some elevation of haemolymph Na⁺, K⁺, Ca²⁺ and Mg²⁺ above sea water levels (Table 2, dialysed serum as % sea water). Measurable amounts of haemolymph Ca²⁺, Mg²⁺ and K⁺ are non-dialysable and thus assumed to be bound. But active ion regulation, seen by comparing serum with dialysed serum, is generally more important than passive factors in accounting for the ionic gradients in this species. Concentrations of Na⁺ and Ca²⁺ in haemolymph are actively regulated above those values in passive equilibrium with sea water, to 109 and 116% respectively. Levels of Mg²⁺ (24%), Cl⁻ (98%) and SO₄²⁻ (54%) in the haemolymph are regulated below sea water concentrations. K⁺ concentration in the haemolymph is variable and may be above or below sea-water concentrations. This ion appears not be regulated at levels different from those in sea water.

Total haemolymph osmoconcentration is similar to that of full sea water. Osmoconcentrations of serum from four Bermuda lobsters were 1052, 1115, 1086 and 1037 mosmol/1, averaging 99·6% of the laboratory sea water of mean salinity of 36·6‰

Table 3 shows that urinary concentrations of Na⁺, Mg²⁺, Cl⁻ and SO₄²⁻ exceed those in the haemolymph, whereas Ca²⁺ in the urine is lower than in the haemolymph. Concentrations of K+ in the two fluids are not statistically different. These results indicate that the antennal glands help to regulate Mg²⁺, SO₄²⁻ and Cl⁻ levels in the haemolymph. The antennal glands are not effective in maintaining haemolymph concentrations of Na+ and Ca²⁺ above those in sea water since they do not conserve these ions. For example, a portion sufficient to raise dialysed serum Ca²⁺ 14% above sea water is bound and unavailable for filtration (Table 2, dialysed serum as % sea water), but urinary Ca²⁺ concentration is only about 7 % below haemolymph values. The simplest explanation of urine formation in P. argus based on these data (urine as % haemolymph ultrafiltrate) is that the antennal gland forms an ultrafiltrate of the hemolymph from which it resorbs about 5 % of the water and into which it secretes Mg²⁺ and SO₄^2−.

Due to the distance of the laboratory setting from the natural habitat and ready supply of P. argus it was necessary to maintain specimens for considerable periods of time under artificial conditions. The haemolymph of the fourteen lobsters obtained from Florida was sampled initially 7–16 days after arrival in the laboratory and then from one to three times thereafter: between 16 and 28 days, at 3 months and/or at months. The time a lobster was first sampled was termed day 1 for that animal. The ratio of haemolymph to sea-water concentrations for each ion was regressed against time with variation among individuals removed by analysis of covariance. Mean salinity of sea water when lobsters were sampled varied from 35·6 to 36·5‰. Table 4 shows that haemolymph concentrations of Na⁺, K⁺, Mg²⁺, Cl⁻ and initially Ca²⁺ increased significantly with time. Too few SO₄²⁻ determinations were performed on haemolymph for inclusion in this analysis. The 4 % rise in concentrations of Na⁺ over 5 months is probably of negligible importance. Concentration of Ca²⁺ ion in haemolymph sampled between days 9 and 22 was markedly higher than on day 1. But by 5 months it had decreased to values below those on day 1. The increase in Mg²⁺ concentration in the haemolymph was most rapid initially, then levelled off. The increase was significantly more linear with log time than with arithmetic time according to the F test of homogeneity of variance. This ion showed the greatest mean percent change over 5 months. The increase in haemolymph Cl⁻ concentration was also most rapid during the 2–3 weeks after the first sampling and the rate of change decreased thereafter. The increases in concentrations of Mg²⁺, Cl⁻ and the decrease in Ca²⁺ concentrations bring the levels of these ions closer to those in sea water.

The lobsters from Bermuda maintained 13·5 weeks in the laboratory showed the same pattern of ion regulation as did the same individuals sampled in Bermuda within a few days after capture. There were a few statistically significant changes in ion levels after the laboratory maintenance; Mg²⁺ and Cl⁻ haemolymph levels increased by 11·1% (P < 0·01) and 3·7% (P < 0·05), respectively. In another comparison lobsters from Florida maintained 12·5 weeks in the laboratory showed the same pattern of ion regulation as Bermuda lobsters maintained similarly for 13·5 weeks. Florida lobsters, however, regulated haemolymph Cl⁻ significantly lower relative to sea water by 2·7% (P < 0·025) than did Bermuda animals. These comparisons are presented in Malley (1972).

Panulirus argus is isosmotic with sea water but shows pronounced regulation of Na+, Ca²⁺, Mg^24-, SO₄²⁻ and Cl⁻ in the haemolymph at levels different from those in sea water. Most marine decapods are isosmotic with their environment and regulate Na⁺, K⁺ and Ca²⁺ above, and Mg²⁺ and SO₄²⁻ below, sea-water values. Chloride is generally in passive equilibrium between haemolymph and sea water (Robertson, 1960). Panulirus argus thus differs from the majority of marine decapods in regulating haemolymph Cl⁻ levels and in not regulating those of K⁺. Otherwise this species is typical in its isosmoticity with sea water and its regulation of Na⁺, Ca^24-, Mg²⁴⁻and SO₄²⁻

Osmotic and ionic regulation has been documented in the tropical rock lobster Panulirus longipes in a study by Dall (1974) published after the present study was completed. In P. longipes in ambient sea water of 36%,, molar concentrations of ions in the haemolymph as functions of molar concentrations in the sea water were Na⁺, 106·8%; K⁺, 104·1%; Ca²⁺, 122·7%, Mg²⁴⁺, 22·8% and Cl⁻, 91·4%. Sulphate was not measured. Thus P. longipes shows precisely the same pattern of ionic regulation as P. argus, also maintaining haemolymph Cl⁻ below sea water levels (P < 0·001) and not maintaining haemolymph K⁺ different from full sea water concentration (N.S.). Interestingly, P. longipes does regulate K⁺ in other salinities, at supra sea-water concentrations when the lobster is in lower salinities down to 20‰ and at sub sea water concentrations when immersed in higher salinities up to 45 ‰. Ion concentrations in the serum of the related Palinurus elephas are similar to the haemolymph levels reported here for P. argus except that P. elephas does not regulate Cl⁻ (Robertson, 1949).

Panulirus argus has haemolymph Mg²⁺ and SO₄²⁻ levels, relative to sea water, amongst the lowest in marine decapods (Robertson, 1953; Prosser, 1973) although relatively lower Mg²⁴⁺ values have been reported in Homaridae (Robertson, 1949) and marine pandalid shrimp (Mackay & Prosser, 1970). A number of species which can invade brackish water (Parry, 1955) and fresh water (Prosser, 1973) also have low haemolymph Mg²⁴⁺ levels. Low Mg²⁴⁺ levels are correlated in the palinurids, as in the related homarids, with a relatively high level of activity. Magnesium ion is noted for its anaesthetic effect (Heilbrurnn, 1952). The tendency for low haemolymph Mg²⁴⁺ levels to be accompanied by slightly raised Na⁺ levels (Robertson, 1949) can be seen in P. argus as well.

The proportionately large changes in concentrations of Ca²⁴⁺ and Mg²⁴⁺ in the haemolymph which occur with time may indicate that significant changes in some aspects of ion metabolism occur during the time that animals are commonly maintained in the laboratory. Nevertheless, although the magnitude of the ionic gradients between haemolymph and sea water decreases for Ca²⁺, Mg²⁴⁺ and Cl⁻, these ions continue to be regulated. The species of ions regulated in the haemolymph and the role of the antennal gland in this regulation remain constant between the Florida and Bermuda populations of this species, when lobsters are transferred from field to laboratory maintenance conditions and when they are maintained in the laboratory for up to 5 months. In this study data from the two geographically separated populations have been pooled.

Panulirus argus demonstrates little conservation of water, resorbing only an estimated 5% of the volume of the untrafiltrate. Burger (1957) found that the marine lobster Homarus americanus also shows little or no tendency to resorb water from the urine as evidenced by urine/haemolymph (U/H) inulin ratios of 1·0–1·1. In contrast, the shore crab Carcinus maenas in 100% sea water absorbs considerable water from the urine resulting in U/H inulin ratios of about 2·0 (Riegel & Lockwood, 1961). Water conservation in C. maenas is presumably an adaptation to littoral life. Exposure of the latter species to water-saturated air for 4 days results in enhanced water resorption from the urine with U/H inulin values rising to 2·4 (Riegel & Lockwood, 1961). The small degree of water resorption from the urine by P. argus correlates with its being marine and strictly non-intertidal, like H. americanus, and relatively stenohaline.

The antennal glands of P. argus are effective in the hyporegulation of Mg²⁴⁺, SO₄²⁻ and Cl⁻ in the haemolymph. Although the haemolymph levels of Mg²⁺ and SO₄^2−.are among the lowest reported in decapod crustaceans, the antennal gland does not concentrate these ions to the extent seen in certain other decapods. In the spiny lobster, Mg²⁺ and SO₄^2−.U/H values are 1-3 (Table 3). This contrasts with U/H values reported for Mg²⁺ in Carcinus maenas in 100 % sea water of about 4 (Riegel & Lockwood, 1961) or about 8 (Lockwood & Riegel, 1969). These authors do not report results for SO₄^2−. Elevation of Mg²⁴⁺ in the urine of C. maenas is in part associated with the resorption of water from the presumptive urine. Sodium, K⁺, Ca²⁺ and possibly Cl^−.are also resorbed (Riegel & Lockwood, 1961). Magnesium is therefore passively concentrated in the urine. In addition, Mg²⁺ is secreted into the urine and this is paralleled by a withdrawal of Na⁺ from the urine against a concentration gradient. For Na⁺, U/H values in 100% sea water are about 0·75 (Lockwood & Riegel, 1969). Reciprocal urinary Mg²⁺ and Na⁺ concentrations are observed as well in the lined shore crab, Pachygrapsus crassipes (Gross & Capen, 1966). The exchange between Na⁺ and Mg²⁺ is apparently not 1:1. In contrast to these results, in P. argus elevated urinary Mg²⁺ concentrations are not associated with reduced Na⁺, and U/H of Na⁺ is close to 1·o. Panulirus argus concentrates urinary SO₄²⁻ to the same extent as Mg²⁴⁺ and would appear to maintain electrical balance in this way, rather than by exchanging Mg²⁴⁺ for Na⁺.

Handling of SO₄²⁻ by the antennal gland has been less thoroughly studied than for Mg²⁴⁺. Antennal glands of Carcinus maenas concentrate SO₄²⁻ to 224 % of haemolymph values (Webb, 1940); of the spider crab Maia squinado to 214% (Robertson, 1953); of the crab Cancer pagurus to 133% (Robertson, 1939); and of the lobster Homarus vulgaris to 159% (Robertson, 1949). The lobster Nephrops norvegicus concentrates only to 106% and Palinurus vulgaris not at all (Robertson, 1949). As for Mg²⁺, argus maintains haemolymph SO₄²⁻ low relative to sea water, about 50% compater with 60% in C. maenas (Webb, 1940). But the antennal glands of the spiny lobster do not concentrate this ion to the extent that C. maenas does and U/H is 1·3 in the lobster compared with the 2·2 in the crab as mentioned above.

The concentration of Cl⁻ by the antennal gland is modest (U/H, 1·05), in keeping with the small hyporegulation of the haemolymph Cl⁻ (serum as % dialysed serum, 98%)

In summary, Panulirus argus shows little tendency to conserve water by resorption from the urine. There appears to be little tendency after ultra-filtration for ions to exchange between haemolymph and the presumptive urine except for Mg²⁺ and SO₄²⁻ which appear to be secreted into the urine and in more or less equal quantities. The data suggest that the Mg²⁺ secreted into the urine is not exchanged for Na⁺, or at least not to the extent seen in several species of shore crabs. Urinary concentrations of Mg²⁺, SO₄²⁻ and Cl⁻exceed those in the haemolymph, indicating a role of the antennal glands in the regulation of these ions.

This paper is based on a dissertation submitted to the Department of Zoology, University of Michigan, in partial fulfilment of the requirements for the Ph.D. degree. The research at the Bermuda Biological Station for Research, St George’s West, Bermuda, was supported by a Horace H. Rackham Dissertation Grant. I wish to thank the director and staff of the Bermuda Biological Station for assistance in obtaining lobsters. My thanks to Dr. Wong Tat Meng of the University of Science of Malaysia and Prof. R. Freeman of the University of Otago, New Zealand, for valuable suggestions on an early stage of the manuscript.