J. Exp. Mar. Bid. Ecol., 1983, Vol. 66, pp.

l-10
Elsevier Biomedical Press

UREA AND AMMONIA EXCRETION

BY SOLITARY ASCIDIANS

JOHN A. MARKUS and CHARLES C. LAMBERT

Department of Biological Science, California State University Fullerton, Fullerton, CA 92634, U.S.A

Abstract: The 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 of
nitrogen catabolism. Urea is not excreted by Ciona, but accounts for 4O-50% of the soluble nitrogen
excretion of Styehplicata, S. clava, and S. partita. Because S. montereyensis did not open and function well
under our laboratory conditions, information is lacking for this species. Arginase activity was detected in
homogenates 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 these
ascidians sequester uric acid, xanthine, or hypoxanthine. Weight-specific oxygen consumption was
determined simultaneously with nitrogen excretion; 0 : N ratios of 13-20 were found, demonstrating that
protein forms a major portion of the diet of these ascidians. An hypothesis relating urea production to
habitat is presented.

Most marine invertebrates release their nitrogenous waste in the form of ammonia
which, although toxic, is flushed away by the sea water (reviewed by Campbell &
Bishop, 1970). Ascidians, the most primitive living chordates, are no exception to this
generalization, although they also excrete or sequester other end products of nitrogen
metabolism (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 Ciona
intestinalis,Ascidiella aspersa, and Molgula manhattensisand found that ammonia
accounts for as much as 95% of the total non-protein nitrogen excreted. More recent
evidence of ammonia excretion in the ascidians Botryllusschlosseri,Botrylloidesleachi
(Sabbadin & Tontodonati, 1967), and Styeluplicatu(Fisher, 1975) supports Goodbody’s
hypothesis that ascidians are primarily ammonotelic, rather than purinotelic as
previously reported. However, some ascidians also retain substantial quantities of uric
acid and/or other pukes, such as xanthine (Goodbody, 1965, 1974; Nohi, 1970).
Fisher also reported temperature-dependent excretion of nrea by S. plicatawhere urea
production increased with a decline in temperature, with an apparent shift from
ammonotelism to ureotelism at temperatures < 20 “C.

Reprint requests to CCL.

0022-0981/83/0000-0000/$03.00 0 1983 Elsevier Biomedical Press

2 JOHN A. MARKUS AND CHARLES C. LAMBERT

Since Fisher’s (1975) study on S. plicata, there have been no further reports of
ureotelism in ascidians. In view of the pivotal position of the ascidians in the chordate
evolutionary line and their demonstrated highly varied means of nitrogen excretion, the
question of the extent of ureotelism in the most primitive living chordates is worth
pursuing. Here we report the relationship between soluble nitrogen excretion (ammonia
and urea) and oxygen consumption, the presence of stored nitrogenous waste products,
and characterization of certain urea cycle and purinolytic enzymes in the solitary
ascidians Ciona intestinalis (Linnaeus, 1767), Styela plicata (Lesueur, 1823), S. partita
(Stimpson, 1852), S. montereyensis (Dall, 1872) and S. clava Herdman, 1881, all
abundant 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 similar
Styela clava and S. montereyensis were discriminated by reference to Abbott & Johnson
(1972). Immediately following collection, ascidians were placed in buckets tilled with
sea water from the collection site and transported to the laboratory.
Ascidians collected from Dana Strands and San Diego Bay were acclimated to
Newport Bay water for 4 days.
Prior to incubation, ascidians were thoroughly cleaned, then washed several times
in fresh sea water, submerged in well-aerated bay water, and allowed to recover from
the handling until at least 1.5 h after siphonal extension and “normal” routine activity
had resumed.
Incubation water was collected from the surface of Newport Bay. Incubation vessels
consisted 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 (containing
only incubation water and no ascidian) were set up simultaneously. Duplicate water
samples were taken to determine the concentration of O,, NH,, or urea before the
incubation period. The jars were maintained at 20 “C for 2.5-6 h (mean = 4 h). The
vessels were inverted several times to mix the water, then portions were siphoned off
immediately and fixed for analysis of ammonia, urea, and dissolved oxygen. Ascidians
were then removed from the vessels, gently squeezed, blotted dry, and frozen for later
analysis. At the time of weighing, frozen animals were thawed and the styelids (but not
Ciona) were carefully removed from their tunics. The tunic and the body from each
animal were weighed after drying to constant weight. Analysis of 0, was performed
according to azide modification of the Winkler method (Taras et al., 1971).
Initial and final concentration of ammonia was spectrophotometrically estimated by
 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 to
ammonia (Fawcett & Scott, 1960) by the enzyme urease (Sigma No. U 2125, Type 4)
and the resulting ammonia determined as before. Ammonia values were converted to
urea 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 a
mortar and pestle; a lOO-mg sample was then extracted with 2 ml of the appropriate
buffer. Extracts were also prepared from fresh tissue. All extracts were clarified by
centrifugation 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 the
urate-sequestering ascidian Corellu inJ7atu(Lambert, unpubl.) served as the biological
control. UV absorption spectra were determined for each ascidian with a Beckman-DU
spectrophotometer with and without the addition of uricase. In order to eliminate
spectral contributions due to DNA, RNA, and proteins, ultrafiltrates were prepared
using a Millipore ultrafiltration kit.
Ascending paper and thin-layer chromatography were performed on extracts to
characterize any nonmacromolecular nitrogen-containing compounds. The following
solvent 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 was

estimated from fresh whole animal homogenates (excluding tunic). Xanthine dehydro-
genase and uricase analysis were adapted from Hult (1969). The arginase assay of
Linton & Campbell (1962) was modified to allow determination of activity in the
presence 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 concentrations
of ammonia plus urea were determined from a sample of the reaction mixture
immediately after adding substrate. This corrected for any contaminating nitrogen
compounds. Urease activity was determined by a method similar to that used by Hult
(1969).
 JOHN A. MARKUS AND CHARLES C. LAMBERT

RESULTS

SOLUBLE NITROGEN EXCRETION AND OXYGEN CONSUMPTION

The assessments of ammonia excretion (Table I) revealed that significant quantities

of ammonia were excreted by all of the species of ascidians studied. Ciona intestinalis
had the highest rate of ammonia production (86 pg NH,-N *g; ’ a h- ‘), where g, is the
dry mass of the animal including the tunic. Styela montereyensis did not function well
under our currentless conditions; the quantities of NH3 produced by the other three
styelids were very similar to each other, but were ~40% of that found for Ciona.

TABLE I

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

Number of With tunic Without tunic

Species 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 + 6.1
Styela montereyensis 5 1 kO.3 4+ 0.7
Styela partita 11 31 + 6.0 70 f 12.5
Styela 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 Ciona
intestinalis.

Urea production by Styela clava, S. plicata and S. partita was detected, while Ciona
and Styela montereyensis apparently produced no urea. Urea excretion was determined
simultaneously with ammonia excretion for only a portion of the individuals reported
in Table I. S. plicata produced significant amounts of urea in every experiment for
which urea was assessed. S.partita expelled urea in all but one experiment. S. clava
failed to produce detectable amounts of urea on seven occasions; however, for the 12
experimental animals reported in Table II, significant quantities of urea were excreted.

TABLE II
Weight-specific urea excretion expressed as nitrogen @g urea-N g-i h-l): total nitrogen excretion and
the 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 8.9
Styela montereyensis 10 0 1+ 0.3 0
Styela partita 11 19 t 3.2 47 + 8.3 42 t 5.0
Styela plicata 10 39 & 13.1 73 f 18.7 54 * 9.8
Ciona intestinalis 14 0 85 k 2.5 0
 ASCIDIAN NITROGEN EXCRETION 5

Urea production at 20 “C for S. clava, S. partita 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 fig
N . g; ’ *h- ‘) was similar to that of Ciona (85 pg N *g; ’ *h- ‘) when urea-nitrogen
is included. The rate of excretion of nitrogen for Styela plicata slightly exceeded that of
Ciona (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. Styela
partita and S. clava excreted z 30% less total nitrogen than S. plicata. In S. mo~tereyensis
the rate of total soluble nitrogen excretion was -C3 % of the rate determined for the other
ascidian species, although this datum may be artificial.

TABLE III

Weight-specific oxygen consumption for five species of ascidians.

Number of With tunic Without tunic

Species individu~s (ml 0, g&z,, . h- ‘f (mlO,~g;&,~ h-l)

Ciona intestinolti 16 0.82 i 0.10 1.71 f 0.23

Styela cluvu 23 0.30 f 0.03 0.83 f 0.07
Styela montereyensis 5 0.09 * 0.01 0.34 + 0.02
Styela pamIa 11 0.15 * 0.02 0.48 f 0.04
Styela plicata 24 0.46 k 0.04 1.38 f 0.10

Mean values of wei~t-specify oxygen consumption were determ~~ for each

species (Table III), with Ciona consu~g the greatest (0.82 ml 0, . g; ’ *h-‘) and
Styela montereyensis the least (0.09 ml 0, *g; ’ 1h- ‘) quantities of oxygen. The rate of
oxygen consumption by S. plicata was w 50% that of Ciona based on total dry weight.
However, on the basis of dry body weight, Styefa plicata respired at z 80 % the rate of
Ciona. The rates of oxygen consumption for Styela clava, S. partita, and S. montereyensis
were cz 65, 32, and 19.5% of S. plicata, respectively. The relatively high standard error
of the mean of Ciona (x 12%) may be accounted for by the gradual but more rapid
utilization of the limited oxygen available in the incubation container by the larger
animals. Therefore, the typical independent relationship of oxygen consumption to
oxygen concentration under normal oxygen concentrations becomes dependent upon
the 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 that
these ascidians do not store or sequester large quantities of uric acid in their soft tissues
or 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 the
tissues and fecal pellets were also negative for all species. The limit of detection for the
6 JOHN A. MARKUS AND CHARLES C. LAMBERT

uric acid analysis of dry tissue (N 10 iufJ* g dry wt - ‘) was determined using reagent
grade uric acid as a standard. Uric acid was detected in the tissues of the control
ascidian Coda j~~ata by the characteristic uric acid absorbance peak at 292 nm that
was abolished by uricase. The relative concentration of uric acid to dry tissue weight
of Corella was 2.05 mg *g-‘. Chromatography of tissue extracts showed no indication
of uric acid or xanthine, except in Corella (uric acid was demonstrated by the same Rf
value as that obtained for standard uric acid).
Although uric acid, xanthine, and h~ox~thine are not stored by Styela and Clona,
there was strong absorbance from 250-270 nm, su~esting that other compounds are
incorporated into their tissues. The ascidians studied in this investigation showed a
characteristic UV absorption maximum at 260 nm that was not shifted as a function
of pH. The UV spectral properties of this material appeared to be very similar to those
reported 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 adenine
to hypox~thine, is not commercially available; therefore, enzymatic techniques could
not be employed to identify adenine as the absorbing compound. Ul~~l~ates and
deproteinized extracts of S. plicaza revealed the same 260 nm absorbance peak, thus
eliminating 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 fresh
ascidian tissue homogenates, but si~~c~t levels of arginase activity were found for
S. pli~ata, S. clava, and S. partita (Table IV). S. plicata showed the highest level of

TABLE IV
Arginase activity @mol urea - &dt . h- ’ f SE) by whole-body tissue homogenates for urea-excreting
ascidians.

Without With
Species Replicates antibiotics antibiotics
_.__ _.....
~ __~_~.__.. -_“-.~---__ -
Styeia plicata 8 890 f 30 870 * 28
Styela clava 8 530 * 25 455 + 16
Styela 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 of
microbial activity since antibiotics only slightly depressed activities.
 ASCIDIANNITROGENEXCRETION

DISCUSSION

These data clearly indicate that Ciona and Styela all excrete ammonia as one
end-product of protein metabolism, thus confirming and extending the findings of
Goodbody (1974) and Fisher (1975). We found that Ciona excreted 192 pg
NH,-N . g< ’ * h-i, which compares well with the data of Goodbody (1957) of 120 pg
NH,-N . g; l * h- l after conversion to 20 ‘C using a Q,, of 2. Several of Goodbody’s
(1957) values for ammonia excretion probably reflect a less than “normal” output due
to 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 from
2-20 pg NH,-N * g; ’ * h- ‘. This upper value is very close to the mean value of
25 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 of
other ascidians, suggesting that ammonia formation is widespread in ascidians although
it 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 weight
specific basis (Lambert, unpubl.). The small ammonium release by Styelu montereyensis
is probably a reflection of that species’ reliance on good current movement to maintain
open siphons and a normal respiratory rate. We suggest that all of the data from
S. 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 species
also 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 arginase
yielding urea and omithine in the urea cycle or the degradation of uric acid. The absence
of detectable uricolytic activity in any of the styelids studied, coupled with the presence
of arginase activity in S. plicutu, S. purtitu, and S. cluvu strongly suggests that urea is
derived from arginine.
None of the ascidians used in this study sequestered uric acid even though this
compound was clearly demonstrated by our methods in the uricotelic ascidian, Corellu
infutu. Storage excretion of purines in ascidians generally does not seem to be as
widespread as suggested by earlier workers (Das, 1948; Goodbody, 1974), who
envisioned the accumulation of purines along with the release of ammonia from proteins
as unique among marine invertebrates. Purine accumulation apparently is not unique
to ascidians; the bay mussel Mytilus edulis has been shown (Ishida, 1956) to contain
0.1-0.2 mg uric acid *g- ’ of soft tissue and 0.9 mg . g- ’ of byssal fibers. Uric acid and
other related purines have been found in oysters and cephalopods (Campbell & Bishop,
1970). Free tissue purines are also of common occurrence in the marine Prosobranchia
8 JOHN A. MARKUS AND CHARLES C. LAMBERT

(Duerr, 1969), varying from 0.37-4.29 mg *g- ’ oftissue. Needham (1970) has proposed
that polychaetes excrete their purines as allantoin and allantoic acid, although uric acid
has been detected by Hult (1969) in the tissue of some species. Hartenstein (1970)
suggested that marine crustaceans have a complete series of uricolytic enzymes, but
some forms may retain small quantities of guanine in their tissues.
Oxygen consumption rates reported here for Styela and Ciona (Table III) are similar
to those reported previously for ascidians and other invertebrates. Jorgensen (1952)
reported similar rates of oxygen consumption for the ascidians Molgzda manhattensis
and Ciona intestinalis and the bivalve Ostrea virginica; however, they were reported as
relative values and not as weight-specific rates. We found a two-fold excess oxygen
consumption rate for Styela plicata relative to Fisher (1976). This may reflect the
difference between laboratory-stressed animals and thoseunder semi-natural conditions.
The availability of food has a positive correlation with the rate of oxygen consumption
in Mytilus after a feeding initiation threshold is reached (Thompson & Bayne, 1974).
Thus a significant portion of the variability in oxygen consumption rates for Styela
plicata may be due to the use of filtered and unfiltered sea water for incubation, while
the higher rate of oxygen consumption of Ciona relative to the species of Styela
investigated may indicate a real difference in metabolic rate.
Snow & Williams (197 1) demonstrated that the atomic ratio of oxygen consumed to
ammonia-nitrogen excreted (0 : N) by marine organisms is more conveniently and
precisely 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 the
ascidians investigated here. Mean values of 0 : N ratios for Styela and Ciona were
calculated (Table V). If only ammonia nitrogen excretion is considered, it appears that
protein and lipid are important nutritional constituents in all the bay species (i.e.,
excluding S. montereyensis). Lipid and carbohydrate superficially appear to contribute
more to the metabolic fuel of Styela and Ciona; however, urea is another source of
excretory nitrogen in Styela and not in Ciona. Since we find no evidence of uricolytic
enzyme activity in Styela and have no reason to suppose that urea is derived from any
process but the degradation of arginine in the urea cycle, the urea is either excretory
or a byproduct of the arginine pathway. When urea-nitrogen and ammonia-nitrogen

TABLE V

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

Species 0 : NH,-N 0 : N,,,,,

Ciona intestinal& 20+ 1.5 (14) 20* 1.5 (14)

Styela clava 32 + 3.0 (23) 17+ 2.3 (12)
Styela montereyensis 181 f 25.5 (10) 181 f 25.5 (10)
Styela partita 24 + 2.6 (11) 14* 1.4 (11)
Styela plicata 42+ 4.2 (24) 13* 1.0 (IO)
 ASCIDIAN NITROGEN EXCRETION 9

are 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% reduction
ofthe 0 : N value at 20 “C and the lability of urea excretion between different temperature
regimes. He suggested that 29-50% of the catabolic fuel of S. plicatu was protein. The
4ts presented here for S. plicata agree with those of Fisher. S. clava, S. partita and
Ciona appear to be utilizing the same nutritional resource as Sty& plicata based on
0 : N values.
The finding of fairly widespread urea production by ascidians presents the problem
of why energetically more “expensive” urea is released rather than ammonia which is
less “costly” to form and non-toxic enough that some ascidians release all their soluble
nitrogen in this form. One possibility might be that the urea is used to help maintain
animals subjected to hyposaline or other stressful conditions. Indeed we find that
Ciona, which produces no urea, is the first ascidian to undergo local extinctions during
the rainy season, while Styelu plicatu, which produces the most urea, is rarely killed by
dilute water during our winters. Possibly the styelids are able to close up tightly during
adverse 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 Health
Department Field Laboratories where part of this study was conducted. L. McClanahan
and D. Bailey made helpful suggestions on the work in progress. We are indebted to
M. Horn, G. Lambert, R. Seapy, and an anonymous reviewer for valuable suggestions
on this manuscript.

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