J. Exp. Mar. Bid. Ecol., 1983, Vol. 66, Pp. L-10

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J. Exp. Mar. Bid. Ecol., 1983, Vol. 66, pp. l-10Elsevier Biomedical Press

UREA AND AMMONIA EXCRETIONBY SOLITARY ASCIDIANS

JOHN A. MARKUS and CHARLES C. LAMBERTDepartm ent of Biological Science, California Stat e Univ ersity Fullerton, Fullerton, CA 92634, U.S.A

Abstract:he ascidians Ciona intestinalis Linnaeus, 1767),Styelaplicata (Lesueur, 1823), S. clava Herdman,1881, S. partita (Stimpson, 18521, and S. montereyensis (Dall, 1872) all excrete NH, as one product ofnitrogen catabolism. Urea is not excreted by Ciona, but accounts for 4O-50% of the soluble nitrogenexcretion of Sty ehplicata, S. clava, and S. partita. Because S. montereyensis did not open and function wellunder our laboratory conditions, information is lacking for this species. Arginase activity was detected inhomogenates of S. plicata, S. claw , and S. partitu, implying the possession of a functional ornithine cycle.Urease activity was lacking; therefore, ammonia is not from the degradation of urea. None of theseascidians sequester uric acid, xanthine, or hypoxanthine. Weight-specific oxygen consumption wasdetermined simultaneously with nitrogen excretion; 0 : N ratios of 13-20 were found, demonstrating thatprotein forms a major portion of the diet of these ascidians. An hypothesis relating urea production tohabitat is presented.

Most marine invertebrates release their nitrogenous waste in the form of ammoniawhich, although toxic, is flushed away by the sea water (reviewed by Campbell &Bishop, 1970). Ascidians, the most primitive living chordates, are no exception to thisgeneralization, although they also excrete or sequester other end products of nitrogenmetabolism (Azema, 1937; Das, 1948; Nolfi, 1970; Goodbody, 1974; Fisher, 1975).Early investigators concluded that most ascidians were purinotelic (uricotelic).Goodbody (1957) studied the excretion of soluble nitrogen in the ascidians Cionaintestinalis,Ascidiella aspersa, and Molgula manhatt ensisand found that ammoniaaccounts for as much as 95% of the total non-protein nitrogen excreted. More recentevidence of ammonia excretion in the ascidians Botry llus chlosseri,Botrylloideseachi(Sabbadin & Tontodonati, 1967), and Styeluplicatu Fisher, 1975) supports Goodbodyshypothesis that ascidians are primarily ammonotelic, rather than purinotelic aspreviously reported. However, some ascidians also retain substantial quantities of uricacid and/or other pukes, such as xanthine (Goodbody, 1965, 1974; Nohi, 1970).Fisher also reported temperature-dependent excretion of nrea by S.plicatawhere ureaproduction increased with a decline in temperature, with an apparent shift fromammonotelism to ureotelism at temperatures < 20 C.

Reprint requests to CCL.0022-0981/83/0000-0000/$03.00 0 1983 Elsevier Biomedical Press


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2 J OHN A. MARKUS AND CHARLES C. LAMBERT

Since Fishers (1975) study on S. plicata, there have been no further reports ofureotelism in ascidians. In view of the pivotal position of the ascidians in the chordateevolutionary line and their demonstrated highly varied means of nitrogen excretion, thequestion of the extent of ureotelism in the most primitive living chordates is worthpursuing. Here we report the relationship between soluble nitrogen excretion (ammoniaand urea) and oxygen consumption, the presence of stored nitrogenous waste products,and characterization of certain urea cycle and purinolytic enzymes in the solitaryascidians Ciona intestinalis (Linnaeus, 1767), Sty ela plicata (Lesueur, 1823), S. partita(Stimpson, 1852), S. montereyensis (Dall, 1872) and S. clava Herdman, 1881, allabundant in southern Californian waters.

MATERIALS AND METHODS

FIELD ASSESSMENTS OF AMMONIA AND UREA EXCRETION AND OXYGEN CON-SUMPTION

S. clava, S. plicata, and Ciona were collected from floats in Newport Bay, California,during 1977 and 1978.Sfyela montereyensis were collected subtidally ( % 6 m). S. par&a,S. plicata, and Ciona were collected from San Diego Bay. The superficially similarSty ela clava and S. montereyensis were discriminated by reference to Abbott & Johnson(1972). Immediately following collection, ascidians were placed in buckets tilled withsea water from the collection site and transported to the laboratory.Ascidians collected from Dana Strands and San Diego Bay were acclimated toNewport Bay water for 4 days.

Prior to incubation, ascidians were thoroughly cleaned, then washed several timesin fresh sea water, submerged in well-aerated bay water, and allowed to recover fromthe handling until at least 1.5 h after siphonal extension and normal routine activityhad resumed.

Incubation water was collected from the surface of Newport Bay. Incubation vesselsconsisted of Luminarc canning and storage jars with an average volume of 1.2 + 0.02 1(SE). Ten vessels, each containing one ascidian, and two control vessels (containingonly incubation water and no ascidian) were set up simultaneously. Duplicate watersamples were taken to determine the concentration of O,, NH,, or urea before theincubation period. The jars were maintained at 20 C for 2.5-6 h (mean = 4 h). Thevessels were inverted several times to mix the water, then portions were siphoned offimmediately and fixed for analysis of ammonia, urea, and dissolved oxygen. Ascidianswere then removed from the vessels, gently squeezed, blotted dry, and frozen for lateranalysis. At the time of weighing, frozen animals were thawed and the styelids (but notCiona) were carefully removed from their tunics. The tunic and the body from eachanimal were weighed after drying to constant weight. Analysis of 0, was performedaccording to azide modification of the Winkler method (Taras et al., 1971).

Initial and final concentration of ammonia was spectrophotometrically estimated by


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ASCIDIAN NITROGEN EXCRETION 3

the formation of indophenol blue, using a sensitive modification (Strickland & Parsons,1972) of the phenol-hypochlorite method.

Initial and final urea concentrations were determined from the hydrolysis of urea toammonia (Fawcett & Scott, 1960) by the enzyme urease (Sigma No. U 2125, Type 4)and the resulting ammonia determined as before. Ammonia values were converted tourea nitrogen by subtraction of initial ammonia concentration.STORED EXCRETORY PRODUCTS

Analyses for stored excretory products were performed by UV absorption spectro-photometry and chromatography. Dried tissues were ground to a tine powder with amortar and pestle; a lOO-mg sample was then extracted with 2 ml of the appropriatebuffer. Extracts were also prepared from fresh tissue. All extracts were clarified bycentrifugation at 2000 x g for 20 min in a Sorval Superspeed Centrifuge.

The uric acid assay system was designed after the procedure of Kalckar (1947).Reagent grade uric acid was used as a control to calibrate the assay system, while theurate-sequestering ascidian Corellu inJ7atu (Lambert, unpubl.) served as the biologicalcontrol. UV absorption spectra were determined for each ascidian with a Beckman-DUspectrophotometer with and without the addition of uricase. In order to eliminatespectral contributions due to DNA, RNA, and proteins, ultrafiltrates were preparedusing a Millipore ultrafiltration kit.

Ascending paper and thin-layer chromatography were performed on extracts tocharacterize any nonmacromolecular nitrogen-containing compounds. The followingsolvent systems were used: 1-butanol : ethanol : water (50 : 15 : 35) (Sabbadin &Tontodonati, 1967), water saturated 1-butanol and 15 N ammonium hydroxide(100 : l), and water adjusted to pH 10 with ammonium hydroxide (Beaven et al.,1955). Chromatograms were visualized under short-wave UV light.ENZYME ASSAYS

Enzyme activity of xanthine dehydrogenase, uricase, urease, and arginase wasestimated from fresh whole animal homogenates (excluding tunic). Xanthine dehydro-genase and uricase analysis were adapted from Hult (1969). The arginase assay ofLinton & Campbell (1962) was modified to allow determination of activity in thepresence and absence of the antibiotics penicillin and streptomycin (Needham, 1958).Arginase activity of homogenates was measured by adding L-arginine monohydro-chloride and the cofactor manganese chloride in a glycine buffer. Initial concentrationsof ammonia plus urea were determined from a sample of the reaction mixtureimmediately after adding substrate. This corrected for any contaminating nitrogencompounds. Urease activity was determined by a method similar to that used by Hult(1969).


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JOHN A. MARKUS AND CHARLES C. LAMBERTRESULTS

SOLUBLE NITROGEN EXCRETION AND OXYGEN CONSUMPTIONThe assessments of ammonia excretion (Table I) revealed that significant quantities

of ammonia were excreted by all of the species of ascidians studied. Ciona intestinalishad the highest rate of ammonia production (86 pg NH,-N *g; a h- ), where g, is thedry mass of the animal including the tunic. Sty ela m ontereyensis did not function wellunder our currentless conditions; the quantities of NH3 produced by the other threestyelids were very similar to each other, but were ~40% of that found for Ciona.

TABLE IWeight specific ammonia excretion for five species of ascidians (a ?r:SEM): only those animals producingfeces during the experimental phase were included in the generation of the mean value.

Number of With tunic Without tunicSpecies individuals (pg NH,-N.g; . h-) (pg NH,-N.g,- h-)Ciona intestinalis 14 86 k 2.5 192 + 5.6*Styela clava 23 29 k 4.0 64 + 6.1Styela montereyensis 5 1 kO.3 4+ 0.7Sty ela partita 11 31 + 6.0 70 f 12.5Sty ela plicata 24 25 k 2.2 76 k 6.0

* Determined using percent dry body/total dry wt = 44.6% calculated from Goodbody (1957) for Cionaintestinalis.

Urea production by Styela clava, S. plicata and S. partita was detected, while Cionaand Sty ela m ontereyensis apparently produced no urea. Urea excretion was determinedsimultaneously with ammonia excretion for only a portion of the individuals reportedin Table I. S. plicata produced significant amounts of urea in every experiment forwhich urea was assessed. S.partita expelled urea in all but one experiment. S. clavafailed to produce detectable amounts of urea on seven occasions; however, for the 12experimental animals reported in Table II, significant quantities of urea were excreted.

TABLE IIWeight-specific urea excretion expressed as nitrogen @g urea-N g-i h-l): total nitrogen excretion andthe percent contribution of urea to the total is also shown.

Number of Urea Total N (U + NH,) % Urea-N/Species individuals (/.q U-N.g;.h-) (pg N.g; h-) total-N~__Styela clava 12 22 * 8.3 49 k 14.8 47 f 8.9Sty ela mont ereyensis 10 0 1 + 0.3 0St yela partit a 11 19 t 3.2 47 + 8.3 42 t 5.0Sty ela plicata 10 39 & 13.1 73 f 18.7 54 * 9.8Ciona intest inalis 14 0 85 k 2.5 0


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ASCIDIAN NITROGEN EXCRETION 5Urea production at 20 C for S. clava, S. partit a and S. plicata accounted for=40-50x of the total soluble nitrogen ~urea-nitrogen plus ~onium-nitrogen) *g- i (total dry wt) * h- excreted. Excretion of total soluble nitrogen by S.pkcata (73 figN . g; * h- ) was similar to that of Ciona (85 pg N *g; * h- ) when urea-nitrogenis included. The rate of excretion of nitrogen for Sty ela plicata slightly exceeded that ofCiona (222 vs. 190 pg N *g; i . h-l), with total nitrogen excretion calculated indepen-dently of tunic weight. Here g, is the dry weight of the animal without tunic. Styelapartita and S. clava excreted z 30% less total nitrogen than S. plicata. In S. mo~tereyensisthe rate of total soluble nitrogen excretion was -C3 % of the rate determined for the otherascidian species, although this datum may be artificial.

TABLE IIIWeight-specific oxygen consumption for five species of ascidians.

Species Number of With tunicindividu~s (ml 0, g&z,, . h- f Without tunic(mlO,~g;&,~ h-l)Ciona intestinolti 16 0.82 i 0.10 1.71 f 0.23Styela cluvu 23 0.30 f 0.03 0.83 f 0.07Styela montereyensis 5 0.09 * 0.01 0.34 + 0.02Styela pamIa 11 0.15 * 0.02 0.48 f 0.04Styela plicata 24 0.46 k 0.04 1.38 f 0.10

Mean values of wei~t-specify oxygen consumption were determ~~ for eachspecies (Table III), with Ciona consu~g the greatest (0.82 ml 0, . g; * h-) andSty ela mont ereyensis the least (0.09 ml 0, *g; 1 h- ) quantities of oxygen. The rate ofoxygen consumption by S.plicata was w 50% that of Ciona based on total dry weight.However, on the basis of dry body weight, Sty efa plicata respired at z 80 % the rate ofCiona. The rates of oxygen consumption for Styela clava, S. part ita, and S. montereyensiswere cz 65, 32, and 19.5% of S. plicata, respectively. The relatively high standard errorof the mean of Ciona (x 12%) may be accounted for by the gradual but more rapidutilization of the limited oxygen available in the incubation container by the largeranimals. Therefore, the typical independent relationship of oxygen consumption tooxygen concentration under normal oxygen concentrations becomes dependent uponthe concentration of oxygen once the critical partial pressure of oxygen is reached.ANALYSIS OF STORED EXCRETORY PRODUCTS

UV abso~tion spectra of the tunic and body tissues of Ciona, Styela montereyensis,S. clava, and S. plicata with and without treatment with the enzyme uricase reveal thatthese ascidians do not store or sequester large quantities of uric acid in their soft tissuesor tunics. Fecal pellets from S. plicata and S. clava showed no detectable uric acid.Spectral and enzymatic analyses for the presence of xanthine and hypoxanthine in thetissues and fecal pellets were also negative for all species. The limit of detection for the


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6 JOHN A. MARKUS AND CHARLES C. LAMBERTuric acid analysis of dry tissue (N 10 iufJ g dry wt - ) was determined using reagentgrade uric acid as a standard. Uric acid was detected in the tissues of the controlascidian Coda j~~ata by the characteristic uric acid absorbance peak at 292 nm thatwas abolished by uricase. The relative concentration of uric acid to dry tissue weightof Corella was 2.05 mg * g-. Chromatography of tissue extracts showed no indicationof uric acid or xanthine, except in Corella (uric acid was demonstrated by the same Rfvalue as that obtained for standard uric acid).

Although uric acid, xanthine, and h~ox~thine are not stored byStyela and Clona,there was strong absorbance from 250-270 nm, su~esting that other compounds areincorporated into their tissues. The ascidians studied in this investigation showed acharacteristic UV absorption maximum at 260 nm that was not shifted as a functionof pH. The UV spectral properties of this material appeared to be very similar to thosereported for adenine (Beaven et& 1955). Chromatographic attempts to reveal, separate,and identify the compounds responsible for this strong absorbance were inconclusive.Unfo~unately adenine deaminase, the enzyme that catalyzes the conversion of adenineto hypox~thine, is not commercially available; therefore, enzymatic techniques couldnot be employed to identify adenine as the absorbing compound. Ul~~l~ates anddeproteinized extracts of S. plicaza revealed the same 260 nm absorbance peak, thuseliminating nucleic acids and proteins as a source of this anomalous absorbance.ENZYME ACFIVITIES

Uricase, xanthine oxidase, and urease activities were not detected in any of the freshascidian tissue homogenates, but si~~c~t levels of arginase activity were found forS. pli~ata, S. clava, and S. partita (Table IV). S. plicata showed the highest level of

TABLE IVArginase activity @mol urea - &dt . h- f SE)by whole-body tissue homogenates for urea-excretingascidians.

Without WithSpecies Replicates antibiotics antibiotics_.__ _..... __~_~.__.. -_-.~---__ -

Styeia plicata 8 890 f 30 870 * 28Styela clava 8 530 * 25 455 + 16Styela pariita 4 460 + 28 390 + 30

arginase activity; S. cfava and S. part&a activities were only around 45 to 52% of~.~licata. Arginase activity in these ascidians does not appear to be the resuh ofmicrobial activity since antibiotics only slightly depressed activities.


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ASCIDIANNITROGENEXCRETIONDISCUSSION

These data clearly indicate that Ciona and Styela all excrete ammonia as oneend-product of protein metabolism, thus confirming and extending the findings ofGoodbody (1974) and Fisher (1975). We found that Ciona excreted 192 pgNH,-N . g< *h-i, which compares well with the data of Goodbody (1957) of 120 pgNH,-N . g; l *h- l after conversion to 20 C using a Q,, of 2. Several of Goodbodys(1957) values for ammonia excretion probably reflect a less than normal output dueto the extended incubation period (20-35 h), and the relatively small volume of water(200-250 ml) in which animals were incubated.

Fisher (1976) reported ammonia excretion rates for Styela plicafu ranging from2-20 pg NH,-N * g; * h- . This upper value is very close to the mean value of25 pg NH,-N * g; * h- determined for S. plicatu in this investigation.The ammonium excretion rates for S. cluvu and S. purtifu fall within the range ofother ascidians, suggesting that ammonia formation is widespread in ascidians althoughit is not always excreted. Corellu utilizes surplus ammonia for egg flotation (Lambert& Lambert, 1978) and thus releases less into the ambient water than Cionu on a weightspecific basis (Lambert, unpubl.). The small ammonium release by Sty elu m ontereyensisis probably a reflection of that species reliance on good current movement to maintainopen siphons and a normal respiratory rate. We suggest that all of the data fromS. montereyensis is a tremendous underestimate of its activities in its normal habitat,with surge and currents aiding in water flow through the branch&al sac.The west coast S. plicutu excrete about half their soluble nitrogen in the form of urea,which is in agreement with the value determined on the Atlantic coast (Fisher, 1975);S. cluvu and S. part&z also release substantial amounts of urea, but Cionu does not.Preliminary studies with Corellu injlutu (Lambert, unpubl.) indicate that this speciesalso does not release urea, suggesting that urea is not universally released by ascidians.

Excretory urea may be from two sources: the hydrolysis of arginine by arginaseyielding urea and omithine in the urea cycle or the degradation of uric acid. The absenceof detectable uricolytic activity in any of the styelids studied, coupled with the presenceof arginase activity in S. plicutu, S. purtitu, and S. cluvu strongly suggests that urea isderived from arginine.

None of the ascidians used in this study sequestered uric acid even though thiscompound was clearly demonstrated by our methods in the uricotelic ascidian, Corelluinfutu. Storage excretion of purines in ascidians generally does not seem to be aswidespread as suggested by earlier workers (Das, 1948; Goodbody, 1974), whoenvisioned the accumulation of purines along with the release of ammonia from proteinsas unique among marine invertebrates. Purine accumulation apparently is not uniqueto ascidians; the bay mussel My tilus edulis has been shown (Ishida, 1956) to contain0.1-0.2 mg uric acid * g- of soft tissue and 0.9 mg . g- of byssal fibers. Uric acid andother related purines have been found in oysters and cephalopods (Campbell & Bishop,1970). Free tissue purines are also of common occurrence in the marine Prosobranchia


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8 JOHN A. MARKUS AND CHARLES C. LAMBERT

(Duerr, 1969), varying from 0.37-4.29 mg * g- oftissue. Needham (1970) has proposedthat polychaetes excrete their purines as allantoin and allantoic acid, although uric acidhas been detected by Hult (1969) in the tissue of some species. Hartenstein (1970)suggested that marine crustaceans have a complete series of uricolytic enzymes, butsome forms may retain small quantities of guanine in their tissues.

Oxygen consumption rates reported here for Styela and Ciona (Table III) are similarto those reported previously for ascidians and other invertebrates. Jorgensen (1952)reported similar rates of oxygen consumption for the ascidians Molgzda manhattensisand Ciona intestinalis and the bivalve Ostrea virginica; however, they were reported asrelative values and not as weight-specific rates. We found a two-fold excess oxygenconsumption rate for Styela plicata relative to Fisher (1976). This may reflect thedifference between laboratory-stressed animals and thoseunder semi-natural conditions.The availability of food has a positive correlation with the rate of oxygen consumptionin Mytilus after a feeding initiation threshold is reached (Thompson & Bayne, 1974).Thus a significant portion of the variability in oxygen consumption rates for Styelaplicata may be due to the use of filtered and unfiltered sea water for incubation, whilethe higher rate of oxygen consumption of Ciona relative to the species of Styelainvestigated may indicate a real difference in metabolic rate.

Snow & Williams (197 1) demonstrated that the atomic ratio of oxygen consumed toammonia-nitrogen excreted (0 : N) by marine organisms is more conveniently andprecisely determined with current analytical procedures than the respiratory quotient.Oxidation of equivalent weights of protein and lipid yields an 0 : N ratio of x 24 (Ikeda,1974). Thus, some statements are now possible regarding the metabolic fuel of theascidians investigated here. Mean values of 0 : N ratios for Styela and Ciona werecalculated (Table V). If only ammonia nitrogen excretion is considered, it appears thatprotein and lipid are important nutritional constituents in all the bay species (i.e.,excluding S. montereyensis). Lipid and carbohydrate superficially appear to contributemore to the metabolic fuel of Styela and Ciona; however, urea is another source ofexcretory nitrogen in Styela and not in Ciona. Since we find no evidence of uricolyticenzyme activity in Styela and have no reason to suppose that urea is derived from anyprocess but the degradation of arginine in the urea cycle, the urea is either excretoryor a byproduct of the arginine pathway. When urea-nitrogen and ammonia-nitrogen

TABLE VAtomic ratio of oxygen consumption to nitrogen excreted for five species of ascidians (X + SEM).

Species 0 : NH,-N 0 : N,,,,,Ciona intestinal&Styela clavaStyela montereyensisSty ela partitaSty ela plicata

20+ 1.5 (14) 20* 1.5 (14)32 + 3.0 (23) 17+ 2.3 (12)

181 f 25.5 (10) 181 f 25.5 (10)24 + 2.6 (11) 14* 1.4 (11)42+ 4.2 (24) 13* 1.0 (IO)


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ASCIDIAN NITROGEN EXCRETION 9are summed, the oxygen to nitrogen ratios for the bay species are similar. Fisher (1975)reported an 0 : N value of 25 for S.plicatu but only ammonia-nitrogen was considered.

Fisher excluded urea-nitrogen from his computations because of the 50% reductionofthe 0 : N value at 20 C and the lability of urea excretion between different temperatureregimes. He suggested that 29-50% of the catabolic fuel of S.plicatu was protein. The4ts presented here for S. plicata agree with those of Fisher. S. clava, S . partita andCiona appear to be utilizing the same nutritional resource as Sty& plicata based on0 : N values.

The finding of fairly widespread urea production by ascidians presents the problemof why energetically more expensive urea is released rather than ammonia which isless costly to form and non-toxic enough that some ascidians release all their solublenitrogen in this form. One possibility might be that the urea is used to help maintainanimals subjected to hyposaline or other stressful conditions. Indeed we find thatCiona, which produces no urea, is the first ascidian to undergo local extinctions duringthe rainy season, while S tyelu plicatu, which produces the most urea, is rarely killed bydilute water during our winters. Possibly the styelids are able to close up tightly duringadverse conditions and accumulate urea, which is much less toxic than ammonia.Further work on blood urea levels will be necessary to substantiate this possibility.

ACKNOWLEDGEMENTS

We are grateful to M. Wehner and H. Schroth for use of the Orange County HealthDepartment Field Laboratories where part of this study was conducted. L. McClanahanand D. Bailey made helpful suggestions on the work in progress. We are indebted toM. Horn, G. Lambert, R. Seapy, and an anonymous reviewer for valuable suggestionson this manuscript.

REFERENCESABBOTT,D. P. &J. V. JOHNSON,1972.The ascidians Sty ela barnharti, S. plicata, S. clava and S. montereyensisin Californian waters. Bull. South. Calif Acad. Sci., Vol. 71, pp. 95-105.AZI?MA,M., 1937. Recherches sur le sang et lexcretion chez les Ascides. Ann. Inst. Oceanogr. Monaco,Vol. 17, pp. l-150.BEAVEN,G.H., E.R. HOLIDAY& E.A. JOHNSON, 1955. Optical properties of nucleic acids and theircomponents. In, The nucleic acids. Vol. I. Chemistry and biology , edited by E. Chargaff & J. N. Davidson,Academic Press, New York, pp. 502-508.CAMPBELL, .W. & S.H. BISHOP, 1970. Nitrogen metabolism in mollusks. In, Comparative biochemistry of

nitrogen metabolism, edited by J. W. Campbell, Academic Press, New York, pp. 103-206.DAS, S. M., 1948. The physiology of excretion in Molgula (Tunicata, Ascidiacea). Biol. Bull. (W oods Hole,

Mass.), Vol. 95, pp. 307-319.DUERR, F. G., 1969.The uric acid content of several species of prosobranch and pulmonate snails as relatedto nitrogen excretion. Comp. Biochem. Physiol., Vol. 26, pp. 1051-1059.FAWCETT, . K. & J. E. SCOTT, 1960. A rapid and precise method for the determination of urea. J. Clin.Pathol., Vol. 13, pp. 156-159.FISHER, T. R., 1975. Bioenergetics, growth, and reproduction of the solitary tunicate Sryela plicata. Ph.D.thesis, Duke University. 182 pp, Abstract No. 76-18949.


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10 JOHN A. MARKUS AND CHARLES C. LAMBERTFISHER, T. R., 1976. Oxygen uptake of the solitary tunicate, Sty ela plicata. Bio!. Bull. (Woods Hole, Mass .),Vol. 151, pp.297-305.GOODBODY, ., 1957. Nitrogen excretion in Ascidacea. I. Excretion of ammonia and total non-proteinnitrogen. J. Exp. Bioi., Vol. 31, pp.297-305.GOODBODY, ., 1965. Nitrogen excretion in Ascidiacea. II. Storage excretion and the uricolytic enzymesystem. J. Exp. BioL, Vol. 42, pp. 299-305.GOODBODY, ., 1974. The physiology of ascidians. Adv. Mar. Biol., Vol. 12, pp. l-149.HARTENSTEIN,R., 1970. Nitrogen metabolism in non-insect arthropods. In, Comparative biochemistry of

nitrogen metabolism, edited by J. W. Campbell, Academic Press, New York, pp. 299-385.HULT, J. E., 1969. Nitrogenous waste products and excretory enzymes in the marine polychaete ~irr~rrnjaspirabrancha. Comp. Biochem. PhysioL, Vol. 3 1, pp. 1S-24.IKEDA, T., 1974. Nutritional ecology of marine zooplankton. Mem. Fat. Fish. Hokkaido Univ., Vol. 22,pp. l-97.ISHIDA, S., 1956. The purine content of My tilus eduhs tissues and byssus. Bull. Mar. Biol. Sm. Asamushi,TGhoku Un i v . ,Vol. 8, pp. 9-18.JBRGENSEN,C. B., 1952. On the relation between water transport and food requirements in some marinefilter feeding invertebrates. Bioi. Bull. (W oods Hol e, Mass .), Vol. 103, pp. 356363.KALCKAR,H. M., 1947. Differential s~ctrophotometry of purine compounds by means of specific enzymes.I. Determination of hydroxypurine compounds. J. Biol. Gem., Vol. 167, p. 429.LAMBERT,C. C. & G. LAMBERT, 978. Tunicate eggs utilize ammonium ions for flotation. Science, Vol. 200,pp. 64-65.LINTON, S.N. & J. W. CAMPBELL,1962. Studies on the urea cycle enzymes in the terrestrial snail, Otolaiactea. Arch. Biochem. Biophys., Vol. 97, pp. 360-369.NEEDHAM,A. E., 1958.The arginase activity of the tissue of the earthworms ~arnbr~e~ terresttis and Eisenia

_roetida. J. Exp. Biol., Vol. 37, pp. 776782.NEEDHAM,A. E., 1970.Nitrogen metabolism in Annelida. In, Comparative b~ocbem~t ~ofnitrogen metabolism,edited by J. W. Campbell, Academic Press, New York, pp. 207-297.NOLFI, J. R., 1970. Biosynthesis of uric acid in the tunicate Molguia manhatt ensis with a general scheme forthe functions of stored purines in animals. Comp. B&hem. Physi ol., Vol. 35, pp. 827-842.SABBADIN,A. & A. TONTODONATI, 967. Nitrogenous excretion in the compound ascidians Botryllusschlosseri (Pallas) and Bot~~~o~ des eacht(Savigny). Monit. Zool. Ital. @VS.), Vol. 1, pp. 185-190.

SNOW,N. B. & P. J.WILLIAMS,1971. A simple method to determine the 0 : N ratio of small marine animals.J. M ar. BioL As soc. U. K., Vol. 51, pp. 105-l 10.STRICKLAND,. D. & T. PARSONS,1972. A practical handbook of seawater analysis. Bull. Fish. Res. BoardCan., No. 167, 310 pp.TARAS, M.J., A.E. GREENBERG,R.D. HOAK & M.C. RAND (editors), 1971. Standard methods for t heexamination of water and waste water. American Public Health Association, p. 874.THOMPSON,R. J. & B. L. BAYNE, 1974. Some relationships between growth, metabolism and food in themussel My tilas edufis. Mar. Bi ai., Vol. 27, pp. 317-326.

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J. Exp. Mar. Bid. Ecol., 1983, Vol. 66, Pp. L-10